CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING LEBER'S CONGENITAL AMAUROSIS 10 (LCA10)

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application No.

61/950,733, filed March 10, 2014, and U.S. Provisional Application No. 62/036,576, filed August 12, 2014, the contents of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to CRISPR/CAS-related methods and components for editing of a target nucleic acid sequence, and applications thereof in connection with Leber's Congenital Amaurosis 10 (LCA10).

BACKGROUND

Leber's congenital amaurosis (LCA) is the most severe form of inherited retinal dystrophy, with an onset of disease symptoms in the first years of life (Leber, T., Archiv fur Ophthalmologie (in German). 15 (3): 1-25, 1869) and an estimated prevalence of approximately 1 in 50,000 worldwide (Koenekoop et al., Clin Experiment Ophthalmol. 35(5): 473-485, 2007; Stone, Am J Ophthalmol. 144(6): 791-811, 2007). Genetically, LCA is a heterogeneous disease. To date, fifteen genes have been identified with mutations that result in LCA (den Hollander et al., Prog Retin Eye Res. 27(4): 391-419, 2008; Estrada-Cuzcano et al., Invest Ophthalmol Vis Sci. 52(2): 834-9, 2011). The CEP290 gene is the most frequently mutated LCA gene accounting for approximately 15% of all cases (Stone, Am J Ophthalmol. 144(6): 791-811, 2007; den Hollander et al., Prog Retin Eye Res. 27(4): 391-419, 2008; den Hollander et al., Am J Hum Genet. 79(3): 556-561, 2006; Perrault et al., Hum Mutat. 28(4):4 16, 2007). Severe mutations in CEP290 have also been reported to cause systemic diseases that are characterized by brain defects, kidney malformations, Polydactyly and/or obesity (Baal et al., Am J Hum Genet. 81, 170-179, 2007; den Hollander et al., Prog Retin Eye Res. 27(4): 391-419, 2008; Helou et al., J Med Genet. 44: 657-663, 2007; Valente et al., Nat Genet. 38: 623-625, 2006). Patients with LCA and early-onset retinal dystrophy often carry hypomorphic CEP290 alleles (Stone, Am J Ophthalmol. 144(6): 791-811, 2007; den Hollander et al., Am J Hum Genet. 79(3): 556-561, 2006; Perrault et al, Hum Mutat. 28(4):4 16, 2007; Coppieters et al, Hum Mutat 31, E1709- E1766. 2010; Littink et al, Invest Ophthalmol Vis Sci 51, 3646-3652, 2010).

LCA, and other retinal dystrophies such as Retinitis Pigmentosa (RP), have long been considered incurable diseases. However, the first phase I II clinical trials using gene

augmentation therapy have led to promising results in a selected group of adult LCA/RP patients with mutations in the RPE65 gene (Bainbridge et al., N Engl J Med. 358, 2231-2239, 2008; Cideciyan et al., Proc Natl Acad Sci U S A. 105, 15112-15117, 2008; Hauswirth et al., N Engl J Med. 358, 2240-2248, 2008; Maguire et al., N Engl J Med. 358: 2240-2248, 2008). Unilateral subretinal injections of adeno-associated virus particles carrying constructs encoding the wild- type RPE65 cDNA were shown to be safe and moderately effective in some patients, without causing any adverse effects. In a follow-up study including adults and children, visual improvements were more sustained, especially in the children all of whom gained ambulatory vision (Maguire et al., Lancet. 374, 1597-1605, 2009). Although these studies demonstrated the potential to treat LCA using gene augmentation therapy and increased the development of therapeutic strategies for other genetic subtypes of retinal dystrophies (den Hollander et al., J Clin Invest 120: 3042-3053, 2010), it is hard to control the expression levels of the therapeutic genes when using gene augmentation therapy.

Leber's congenital amaurosis 10 (LCA 10), one type of LCA, is is an inherited (autosomal recessive) retinal degenerative disease characterized by severe loss of vision at birth. All subjects having LCA10 have had at least one C.2991+1655A to G (adenine to guanine) mutation in the CEP290 gene. Heterozygous nonsense, frameshift, and splice-site mutations have been identified on the remaining allele. A C.2991+1655A to G mutation in the CEP290 gene give rise to a cryptic splice donor cite in intron 26 which results in the inclusion of an aberrant exon of 128 bp in the mutant CEP290 mRNA, and inserts a premature stop codon (P.C998X). The sequence of the cryptic exon contains part of an Alu repeat. There are currently no approved therapeutics for LCA 10.

Despite advances that have been made using gene therapy, there remains a need for therapeutics to treat retinal dystrophies, including LCA10. SUMMARY OF THE INVENTION

Methods and compositions discussed herein, provide for treating or delaying the onset or progression of diseases of the eye, e.g., disorders that affect retinal cells, e.g., photoreceptor cells.

Methods and compositions discussed herein, provide for treating or delaying the onset or progression of Leber's Congenital Amaurosis 10 (LCAIO), an inherited retinal degenerative disease characterized by severe loss of vision at birth. LCAIO is caused by a mutation in the CEP290 gene, e.g., a C.2991+1655A to G (adenine to guanine) mutation in the CEP290 gene which gives rise to a cryptic splice site in intron 26. This is a mutation at nucleotide 1655 of intron 26 of CEP290, e.g., an A to G mutation. CEP290 is also known as: CT87; MKS4; POC3; rdl6; BBS 14; JBTS5; LCAIO; NPHP6; SLSN6; and 3HllAg.

Methods and compositions discussed herein, provide for treating or delaying the onset or progression of LCAIO by gene editing, e.g., using CRISPR-Cas9 mediated methods to alter a LCAIO target position, as disclosed below.

"LCAIO target position", as used herein, refers to nucleotide 1655 of intron 26 of the

CEP290 gene, and the mutation at that site that gives rise to a cryptic splice donor site in intron 26 which results in the inclusion of an aberrant exon of 128bp (c.2991+1523 to c.2991+1650) in the mutant CEP290 mRNA, and inserts a premature stop codon (p.C998X). The sequence of the cryptic exon contains part of an Alu repeat region. The Alu repeats span from c.2991+1162 to c.2991+1638. In an embodiment, the LCAIO target position is occupied by an adenine (A) to guanine (G) mutation (c.2991+1655A to G).

In one aspect, methods and compositions discussed herein, provide for altering a LCAIO target position in the CEP290 gene. The methods and compositions described herein introduce one or more breaks near the site of the LCA target position (e.g., C.2991+1655A to G) in at least one allele of the CEP290 gene. Altering the LCAIO target position refers to (1) break-induced introduction of an indel (also referred to herein as NHEJ-mediated introduction of an indel) in close proximity to or including a LCAIO target position (e.g., C.2991+1655A to G), or (2) break- induced deletion (also referred to herein as NHEJ-mediated deletion) of genomic sequence including the mutation at a LCAIO target position (e.g., C.2991+1655A to G). Both approaches give rise to the loss or destruction of the cryptic splice site resulting from the mutation at the LCAIO target position (e.g., C.2991+1655A to G). In an embodiment, a single strand break is introduced in close proximity to or at the LCAIO target position (e.g., C.2991+1655A to G) in the CEP290 gene. While not wishing to be bound by theory, it is believed that break-induced indels (e.g., indels created following NHEJ) destroy the cryptic splice site. In an embodiment, the single strand break will be accompanied by an additional single strand break, positioned by a second gRNA molecule.

In an embodiment, a double strand break is introduced in close proximity to or at the LCAIO target position (e.g., C.2991+1655A to G) in the CEP290 gene. While not wishing to be bound by theory, it is believed that break-induced indels (e.g., indels created following NHEJ) destroy the cryptic splice site. In an embodiment, a double strand break will be accompanied by an additional single strand break may be positioned by a second gRNA molecule. In an embodiment, a double strand break will be accompanied by two additional single strand breaks positioned by a second gRNA molecule and a third gRNA molecule.

In an embodiment, a pair of single strand breaks is introduced in close proximity to or at the LCAIO target position (e.g., C.2991+1655A to G) in the CEP290 gene. While not wishing to be bound by theory, it is believed that break- induced indels destroy the cryptic splice site. In an embodiment, the pair of single strand breaks will be accompanied by an additional double strand break, positioned by a third gRNA molecule. In an embodiment, the pair of single strand breaks will be accompanied by an additional pair of single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule.

In an embodiment, two double strand breaks are introduced to flank the LCAIO target position in the CEP290 gene (one 5' and the other one 3' to the mutation at the LCAIO target position, e.g., C.2991+1655A to G) to remove (e.g., delete) the genomic sequence including the mutation at the LCAIO target position. It is contemplated herein that in an embodiment the break- induced deletion of the genomic sequence including the mutation at the LCAIO target position is mediated by NHEJ. In an embodiment, the breaks (i.e., the two double strand breaks) are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat. The breaks, i.e., two double strand breaks, can be positioned upstream and downstream of the LCAIO target position, as discussed herein.

In an embodiment, one double strand break (either 5' or 3' to the mutation at the LCAIO target position, e.g., C.2991+1655A to G) and two single strand breaks (on the other side of the mutation at the LCAIO target position from the double strand break) are introduced to flank the LCA10 target position in the CEP290 gene to remove (e.g., delete) the genomic sequence including the mutation at the LCA10 target position. It is contemplated herein that in an embodiment the break-induced deletion of the genomic sequence including the mutation at the LCA10 target position is mediated by NHEJ. In an embodiment, the breaks (i.e., the double strand break and the two single strand breaks) are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat. The breaks, e.g., one double strand break and two single strand breaks, can be positioned upstream and downstream of the LCA10 target position, as discussed herein.

In an embodiment, two pairs of single strand breaks (two 5' and the other two 3' to the mutation at the LCA10 target position, e.g., C.2991+1655A to G) are introduced to flank the LCA10 target position in the CEP290 gene to remove (e.g., delete) the genomic sequence including the mutation at the LCA10 target position. It is contemplated herein that in an embodiment the break-induced deletion of the genomic sequence including the mutation at the LCA10 target position is mediated by NHEJ. In an embodiment, the breaks (e.g., two pairs of single strand breaks) are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat. The breaks, e.g., two pairs of single strand breaks, can be positioned upstream or downstream of the LCA10 target position, as discussed herein.

The LCA10 target position may be targeted by cleaving with either a single nuclease or dual nickases, e.g., to induce break-induced indel in close proximity to or including the LCA10 target position or break-induced deletion of genomic sequence including the mutation at the

LCA10 target position in the CEP290 gene. The method can include acquiring knowledge of the mutation carried by the subject, e.g., by sequencing the appropriate portion of the CEP290 gene.

In one aspect, disclosed herein is a gRNA molecule, e.g., an isolated or non-naturally occurring gRNA molecule, comprising a targeting domain which is complementary with a target domain from the CEP290 gene.

When two or more gRNAs are used to position two or more cleavage events, e.g., double strand or single strand breaks, in a target nucleic acid, it is contemplated that in an embodiment the two or more cleavage events may be made by the same or different Cas9 proteins. For example, when two gRNAs are used to position two double strand breaks, a single Cas9 nuclease may be used to create both double strand breaks. When two or more gRNAs are used to position two or more single stranded breaks (single strand breaks), a single Cas9 nickase may be used to create the two or more single strand breaks. When two or more gRNAs are used to position at least one double strand break and at least one single strand break, two Cas9 proteins may be used, e.g., one Cas9 nuclease and one Cas9 nickase. It is contemplated that in an embodiment when two or more Cas9 proteins are used that the two or more Cas9 proteins may be delivered sequentially to control specificity of a double strand versus a single strand break at the desired position in the target nucleic acid.

In some embodiments, the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecule hybridize to the target domain from the target nucleic acid molecule (i.e., the CEP290 gene) through complementary base pairing to opposite strands of the target nucleic acid molecule. In some embodiments, the first gRNA molecule and the second gRNA molecule are configured such that the PAMs are oriented outward.

In an embodiment, the targeting domain of a gRNA molecule is configured to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat, or the endogenous CEP290 splice sites, in the target domain. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule.

In an embodiment, the targeting domain of a gRNA molecule is configured to position a cleavage event sufficiently far from a preselected nucleotide, e.g., the nucleotide of a coding region, such that the nucleotide is not altered. In an embodiment, the targeting domain of a gRNA molecule is configured to position an intronic cleavage event sufficiently far from an intron/exon border, or naturally occurring splice signal, to avoid alteration of the exonic sequence or unwanted splicing events. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 10. In some embodiments, the targeting domain is selected from those in Table 10. For example, in certain embodiments, the targeting domain is:

GACACTGCCAATAGGGATAGGT;

GTC A A A AGCTACCGGTT ACCTG ;

GTTCTGTCCTC AGTA A A AGGT A ;

G A AT AGTTTGTTCTGGGT AC ;

GAG A A AGGG ATGGGC ACTTA ;

GATGC AG A ACTAGTGTAG AC ; GTC AC ATGGG AGTC AC AGGG ; or

GAGT ATCTCCTGTTTGGC A .

In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Table 10. In an embodiment, the two or more gRNAs or targeting domains are selected from one or more of the pairs of gRNAs or targeting domains described herein, e.g., as indicated in Table 10. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Table 10.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 1A-1D. In some embodiments, the targeting domain is selected from those in Table 1A-1D. For example, in certain embodiments, the targeting domain is:

GAG AU ACUC AC A AUU AC A AC ; or

GAUACUCACAAUUACAACUG. In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 1A-1D. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 1A-1D.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 2A-2C. In some embodiments, the targeting domain is selected from those in Tables 2A-2C. For example, in certain embodiments, the targeting domain is:

G AG AU ACUC AC A AUU AC A AC ; or

GAU ACUC AC A AUU AC A A . In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 2A-2C. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 2A-2C.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 6A-6D. In some embodiments, the targeting domain is selected from those in Tables 6A-6D. For example, in certain embodiments, the targeting domain is:

GCACCUGGCCCCAGUUGUAAUU. In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 6A-6D. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 6A-6D.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 3A-3D. In some embodiments, the targeting domain is selected from those in Tables 3A-3D. For example, in certain embodiments, the targeting domain is:

GCUACCGGUUACCUGAA;

GC AG A ACU AGUGU AG AC ;

GUUGAGUAUCUCCUGUU;

GAUGC AG A ACU AGUGU AG AC ; or

GCUUGAACUCUGUGCCAAAC.

In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 3A-3D. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 3A-3D.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 7A-7D. In some embodiments, the targeting domain is selected from those in Tables 7A-7D. For example, in certain embodiments, the targeting domain is:

GA A AG AUG A A A A AU ACUCUU ;

GA A AU AG AUGU AG AUUG ;

GAAAUAUUAAGGGCUCUUCC;

GA AC A A A AGCC AGGG ACC AU ;

GAACUCUAUACCUUUUACUG;

GAAGAAUGGAAUAGAUAAUA;

GAAUAGUUUGUUCUGGGUAC;

GA AUGG A AU AG AU A AU A ;

GA AUUU AC AG AGUGC AUCC A ;

GAGAAAAAGGAGCAUGAAAC;

GAG AGCC AC AGUGC AUG ;

GAGGU AG A AUC A AG A AG ;

GAGUGCAUCCAUGGUCC;

GAU A ACU AC A A AGGGUC ;

GAU AG AG AC AGG A AU A A ;

GAUG A A A A AU ACUCUUU ;

GAUGACAUGAGGUAAGU;

GAUGC AG A ACU AGUGU AG AC ;

GC AG A ACU AGUGU AG AC ;

GC AUGUGGUGUC A A AU A ;

GCCUG A AC A AGUUUUG A A AC ;

GCUACCGGUUACCUGAA;

GCUCUUUUCUAUAUAUA;

GCUUGAACUCUGUGCCAAAC;

GCUUUUGACAGUUUUUAAGG;

GCUUUUGUUCCUUGGAA;

GG A AC A A A AGCC AGGG ACC A ;

GGACUUGACUUUUACCCUUC;

GG AG A AU AGUUUGUUCU ;

GG AGUC AC AUGGG AGUC AC A ;

GGAU AGG AC AG AGG AC A ;

GGCUGU A AG AU A ACU AC A A A ;

GGG AG A AU AGUUUGUUC ;

GGG AGUC AC AUGGG AGUC AC ;

GGGCUCUUCCUGGACCA;

GGGU AC AGGGGU A AG AG A A A ;

GGUCCCUGGCUUUUGUUCCU;

GU A A AGGUUC AUG AG ACU AG ;

GUAACAUAAUCACCUCUCUU;

GU A AG ACUGG AG AU AG AG AC ;

GU AC AGGGGU A AG AG A A ; GUAGCUUUUGACAGUUUUUA;

GUCACAUGGGAGUCACA;

GUGG AG AGCC AC AGUGC AUG ;

GUU AC A AUCUGUG A AU A ;

GUUCUGUCCUCAGUAAA;

GUUGAGUAUCUCCUGUU;

GUUUAGAAUGAUCAUUCUUG;

GUUUGUUCUGGGUACAG;

UA A A A ACUGUC A A A AGCU AC ;

UA A A AGGU AU AG AGUUC A AG ;

UA A AUC AUGC A AGUG ACCU A ; or

UA AG AU A ACU AC A A AGGGUC .

In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 7A-7D. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 7A-7D.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 4A-4D. In some embodiments, the targeting domain is selected from those in Table 4A-4D. For example, in certain embodiments, the targeting domain is:

G A AUCCUG A A AGCU ACU .

In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 4A-4D. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 4A-4D.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 8A-8E. In some embodiments, the targeting domain is selected from those in Tables 8A-8E. For example, in certain embodiments, the targeting domain is:

GCU A A AUC AUGC A AGUG ACCU A AG ; GGUCACUUGCAUGAUUUAG;

GUCACUUGCAUGAUUUAG;

GCCUAGGACUUUCUAAUGCUGGA;

GGACUUUCUAAUGCUGGA;

GGGACCAUGGGAGAAUAGUUUGUU;

GG ACC AUGGG AG A AU AGUUUGUU ;

GACC AUGGG AG A AU AGUUUGUU ;

GGUCCCUGGCUUUUGUUCCUUGGA;

GUCCCUGGCUUUUGUUCCUUGGA;

GAAAACGUUGUUCUGAGUAGCUUU;

GUUGUUCUGAGUAGCUUU;

GGUCCCUGGCUUUUGUUCCU;

GUCCCUGGCUUUUGUUCCU;

GAC AUCUUGUGG AU A AUGU AUC A ;

GUCCU AGGC A AG AG AC AUCUU ;

GCC AGC A A A AGCUUUUG AGCU A A ;

GC A A A AGCUUUUG AGCU A A ;

GAUCUUAUUCUACUCCUGUGA;

GCUUUCAGGAUUCCUACUAAAUU;

GUUCUGUCCUCAGUAAAAGGUA;

GAACAACGUUUUCAUUUA;

GU AG A AU AUC AU A AGUU AC A AUCU ;

GA AU AUC AU A AGUU AC A AUCU ;

GUGGCUGUAAGAUAACUACA;

GGCUGU A AG AU A ACU AC A ;

GUUUAACGUUAUCAUUUUCCCA;

GUAAGAGAAAGGGAUGGGCACUUA;

GAG A A AGGG AUGGGC ACUU A ;

GA A AGGG AUGGGC ACUU A ;

GU A A AUG A A A ACGUUGUU ;

GAU A A AC AUG ACUC AU A AUUU AGU ;

GG A AC A A A AGCC AGGG ACC AUGG ;

GA AC A A A AGCC AGGG ACC AUGG ;

GGG AG A AU AGUUUGUUCUGGGU AC ;

GG AG A AU AGUUUGUUCUGGGU AC ;

GAG A AU AGUUUGUUCUGGGU AC ;

GAAUAGUUUGUUCUGGGUAC;

GA A AU AG AGGCUU AUGG AUU ;

GUUCUGGGUACAGGGGUAAGAGAA;

GGGU AC AGGGGU A AG AG A A ;

GGU AC AGGGGU A AG AG A A ;

GUAAAUUCUCAUCAUUUUUUAUUG;

GGAGAGGAUAGGACAGAGGACAUG;

GAGAGGAUAGGACAGAGGACAUG; GAGGAUAGGACAGAGGACAUG;

GG AU AGG AC AG AGG AC AUG ;

GAUAGGACAGAGGACAUG;

GA AU A A AUGU AG A AUUUU A AUG ;

GUCAAAAGCUACCGGUUACCUG;

GUUUUU A AGGCGGGG AGUC AC AU ;

GUCUUACAUCCUCCUUACUGCCAC;

GAGUC AC AGGGU AGG AUUC AUGUU ;

GUC AC AGGGU AGG AUUC AUGUU ;

GGC AC AG AGUUC A AGCU A AU AC AU ;

GC AC AG AGUUC A AGCU A AU AC AU ;

GAGUUC A AGCU A AU AC AU ;

GAUGC AG A ACU AGUGU AG AC ;

GUGUUGAGUAUCUCCUGUUUGGCA;

GUUGAGUAUCUCCUGUUUGGCA;

GAGUAUCUCCUGUUUGGCA;

GA A A AUC AG AUUUC AUGUGUG ;

GCC AC A AG A AUG AUC AUUCU A A AC ;

GGCGGGG AGUC AC AUGGG AGUC A ;

GCGGGG AGUC AC AUGGG AGUC A ;

GGGG AGUC AC AUGGG AGUC A ;

GGG AGUC AC AUGGG AGUC A ;

GG AGUC AC AUGGG AGUC A ;

GCUUUUGACAGUUUUUAAGGCG;

GAUC AUUCUUGUGGC AGU A AG ;

GAGCAAGAGAUGAACUAG;

GCCUG A AC A AGUUUUG A A AC ;

GU AG AUUG AGGU AG A AUC A AG A A ;

GAUUG AGGU AG A AUC A AG A A ;

GG AUGU A AG ACUGG AG AU AG AG AC ;

GAUGU A AG ACUGG AG AU AG AG AC ;

GU A AG ACUGG AG AU AG AG AC ;

GGG AGUC AC AUGGG AGUC AC AGGG ;

GG AGUC AC AUGGG AGUC AC AGGG ;

GAGUC AC AUGGG AGUC AC AGGG ;

GUC AC AUGGG AGUC AC AGGG ;

GUUUACAUAUCUGUCUUCCUUAA; or

GAUUUC AUGUGUG A AG A A .

In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 8A-8E. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 8A-8E.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 5A-5B. In some embodiments, the targeting domain is selected from those in Tables 5A-5B. For example, in certain embodiments, the targeting domain is:

GAGUUC A AGCU A AU AC AUG A ;

GUUGUUCUGAGUAGCUU;

GGC A A A AGC AGC AG A A AGC A ;

GUUGUUCUGAGUAGCUU; or

GGC A A A AGC AGC AG A A AGC A .

In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 5A-5B. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 5A-5B.

In an embodiment, the LCA10 target position in the CEP290 gene is targeted. In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Tables 9A-9B. In some embodiments, the targeting domain is selected from those in Tables 9A-9B. For example, in certain embodiments, the targeting domain is:

GGC A A A AGC AGC AG A A AGC A ;

GUGGCUGAAUGACUUCU;

GUUGUUCUGAGUAGCUU;

GACU AG AGGUC ACG A A A ; or

GAGUUC A AGCU A AU AC AUG A . In an embodiment, when two or more gRNAs are used to position two or more breaks, e.g., two or more single stranded breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 9A-9B. In an embodiment, when two or more gRNAs are used to position four breaks, e.g., four single strand breaks in the target nucleic acid sequence, each guide RNA is independently selected from one of Tables 9A-9B. In an embodiment, the gRNA, e.g., a gRNA comprising a targeting domain, which is complementary with a target domain from the CEP290 gene, is a modular gRNA. In other embodiments, the gRNA is a chimeric gRNA.

In an embodiment, when two gRNAs are used to position two breaks, e.g., two single strand breaks, in the target nucleic acid sequence, each guide RNA is independently selected from one or more of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A- 5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

In an embodiment, the targeting domain which is complementary with a target domain from the CEP290 gene comprises 16 or more nucleotides in length. In an embodiment, the targeting domain which is complementary with a target domain from the CEP290 gene is 16 nucleotides or more in length. In an embodiment, the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In an embodiment, the targeting domain is 18 nucleotides in length. In an embodiment, the targeting domain is 19 nucleotides in length. In an embodiment, the targeting domain is 20 nucleotides in length. In an embodiment, the targeting domain is 21 nucleotides in length. In an embodiment, the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain is 23 nucleotides in length. In an embodiment, the targeting domain is 24 nucleotides in length. In an embodiment, the targeting domain is 25 nucleotides in length. In an embodiment, the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides. A gRNA as described herein may comprise from 5' to 3': a targeting domain

(comprising a "core domain", and optionally a "secondary domain"); a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain. In some embodiments, the proximal domain and tail domain are taken together as a single domain.

In an embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 20 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In another embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In another embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In another embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 40 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

A cleavage event, e.g., a double strand or single strand break, is generated by a Cas9 molecule. The Cas9 molecule may be an enzymatically active Cas9 (eaCas9) molecule, e.g., an eaCas9 molecule that forms a double strand break in a target nucleic acid or an eaCas9 molecule forms a single strand break in a target nucleic acid (e.g., a nickase molecule).

In an embodiment, the eaCas9 molecule catalyzes a double strand break.

In some embodiments, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In this case, the eaCas9 molecule is an HNH-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at D10, e.g., D10A. In other embodiments, the eaCas9 molecule comprises N- terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity. In an embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at H840, e.g., H840A. In an

embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at H863, e.g., H863A.

In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which the targeting domain of said gRNA is complementary.

In another aspect, disclosed herein is a nucleic acid, e.g., an isolated or non-naturally occurring nucleic acid, e.g., DNA, that comprises (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in CEP290 gene as disclosed herein.

In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. In an embodiment, the nucleic acid encodes a gRNA molecule comprising a targeting domain that is selected from those in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

In an embodiment, the nucleic acid encodes a modular gRNA, e.g., one or more nucleic acids encode a modular gRNA. In other embodiments, the nucleic acid encodes a chimeric gRNA. The nucleic acid may encode a gRNA, e.g., the first gRNA molecule, comprising a targeting domain comprising 16 nucleotides or more in length. In one embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 16 nucleotides in length. In other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 17 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 18 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 19 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 20 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 21 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 22 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 23 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 24 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 25 nucleotides in length. In still other embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA comprising from 5' to 3' : a targeting domain (comprising a "core domain", and optionally a "secondary domain"); a first

complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain. In some embodiments, the proximal domain and tail domain are taken together as a single domain.

In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNA molecule, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 20 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNA molecule, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNA molecule, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA comprising e.g., the first gRNA molecule, a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 40 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid comprises (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CEP290 gene as disclosed herein, and further comprising (b) a sequence that encodes a Cas9 molecule.

The Cas9 molecule may be a nickase molecule, a enzymatically activating Cas9 (eaCas9) molecule, e.g., an eaCas9 molecule that forms a double strand break in a target nucleic acid and an eaCas9 molecule forms a single strand break in a target nucleic acid. In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which the targeting domain of said gRNA is complementary.

In an embodiment, the eaCas9 molecule catalyzes a double strand break.

In some embodiments, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In other embodiments, the said eaCas9 molecule is an HNH-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at D10, e.g., D10A. In other embodiments, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH- like domain cleavage activity. In another embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at H840, e.g., H840A. In another embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at H863, e.g., H863A.

A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CEP290 gene as disclosed herein; and (b) a sequence that encodes a Cas9 molecule.

A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CEP290 gene as disclosed herein; (b) a sequence that encodes a Cas9 molecule; and further comprises (c)(i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain of the CEP290 gene, and optionally, (ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the CEP290 gene; and optionally, (iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the CEP290 gene.

In an embodiment, a nucleic acid encodes a second gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a LCAIO target position in the CEP290 gene to allow alteration, e.g., alteration associated with NHEJ, of the LCAIO target position, either alone or in combination with the break positioned by said first gRNA molecule.

In an embodiment, a nucleic acid encodes a third gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a LCAIO target position in the CEP290 gene to allow alteration, e.g., alteration associated with NHEJ, either alone or in combination with the break positioned by the first and/or second gRNA molecule.

In an embodiment, a nucleic acid encodes a fourth gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a LCAIO target position in the CEP290 gene to allow alteration, e.g., alteration associated with NHEJ, either alone or in combination with the break positioned by the first gRNA molecule, the second gRNA molecule and/or the third gRNA molecule.

In an embodiment, a nucleic acid encodes a second gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, in combination with the break position by said first gRNA molecule, sufficiently close to a LCAIO target position in the CEP290 gene to allow alteration, e.g., alteration associated with NHEJ, of the a LCAIO target position in the CEP290 gene, either alone or in combination with the break positioned by said first gRNA molecule.

In an embodiment, a nucleic acid encodes a third gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, in combination with the break position by said first and/or second gRNA molecule sufficiently close to a LCAIO target position in the CEP290 gene to allow alteration, e.g., alteration associated with NHEJ, either alone or in combination with the break positioned by the first and/or second gRNA molecule.

In an embodiment, a nucleic acid encodes a fourth gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, in combination with the break positioned by the first gRNA molecule, the second gRNA molecule and/or the third gRNA molecule, sufficiently close to a LCAIO target position in the CEP290 gene to allow alteration, e.g., alteration associated with NHEJ, either alone or in combination with the break positioned by the first gRNA molecule, the second gRNA molecule and/or the third gRNA molecule.

In an embodiment, the nucleic acid encodes a second gRNA molecule. The second gRNA is selected to target the LCAIO target position. Optionally, the nucleic acid may encode a third gRNA, and further optionally, the nucleic acid may encode a fourth gRNA molecule.

In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain selected from those in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. In an embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. In a further embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain selected from those in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

In an embodiment, the nucleic acid encodes a second gRNA which is a modular gRNA, e.g., wherein one or more nucleic acid molecules encode a modular gRNA. In other

embodiments, the nucleic acid encoding a second gRNA is a chimeric gRNA. In other embodiments, when a nucleic acid encodes a third or fourth gRNA, the third and fourth gRNA may be a modular gRNA or a chimeric gRNA. When multiple gRNAs are used, any

combination of modular or chimeric gRNAs may be used.

A nucleic acid may encode a second, a third, and/or a fourth gRNA, each independently, comprising a targeting domain comprising 16 nucleotides or more in length. In an embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 16 nucleotides in length. In other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 17 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 18 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 19 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 20 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 21 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 22 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 23 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 24 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 25 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides. In an embodiment, the targeting domain comprises 26 nucleotides.

In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA, each independently, comprising from 5' to 3': a targeting domain (comprising a "core domain", and optionally a "secondary domain"); a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain. In some embodiments, the proximal domain and tail domain are taken together as a single domain.

In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA, each independently, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 20 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA, each independently, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA, each independently, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA, each independently, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 40 nucleotides in length; and a targeting domain of equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In some embodiments, when the CEP290 gene is altered, e.g., by NHEJ, the nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CEP290 gene as disclosed herein; (b) a sequence that encodes a Cas9 molecule; optionally, (c)(i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain of the CEP290 gene, and further optionally, (ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the CEP290 gene; and still further optionally, (iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the CEP290 gene.

As described above, a nucleic acid may comprise (a) a sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CEP290, and (b) a sequence encoding a Cas9 molecule. In some embodiments, (a) and (b) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno-associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector, e.g., an AAV vector described herein. Exemplary AAV vectors that may be used in any of the described compositions and methods include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rhlO vector, a modified AAV.rhlO vector, an

AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified

AAV.rh43 vector, an AAV.rh64Rl vector, and a modified AAV.rh64Rl vector.

In other embodiments, (a) is present on a first nucleic acid molecule, e.g. a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) is present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecules may be AAV vectors, e.g., the AAV vectors described herein.

In other embodiments, the nucleic acid may further comprise (c)(i) a sequence that encodes a second gRNA molecule as described herein. In some embodiments, the nucleic acid comprises (a), (b) and (c)(i). Each of (a) and (c)(i) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno-associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector, e.g., an AAV vectors described herein.

In other embodiments, (a) and (c)(i) are on different vectors. For example, (a) may be present on a first nucleic acid molecule, e.g. a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (c)(i) may be present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. In an embodiment, the first and second nucleic acid molecules are AAV vectors, e.g., the AAV vectors described herein. In another embodiment, each of (a), (b), and (c)(i) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, one of (a), (b), and (c)(i) is encoded on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and a second and third of (a), (b), and (c)(i) is encoded on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors, e.g., the AAV vectors described herein.

In an embodiment, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, a first AAV vector; and (b) and (c)(i) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors, e.g., the AAV vectors described herein.

In other embodiments, (b) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (a) and (c)(i) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors, e.g., the AAV vectors described herein.

In other embodiments, (c)(i) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) and (a) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors, e.g., the AAV vectors described herein.

In another embodiment, each of (a), (b) and (c)(i) are present on different nucleic acid molecules, e.g., different vectors, e.g., different viral vectors, e.g., different AAV vector. For example, (a) may be on a first nucleic acid molecule, (b) on a second nucleic acid molecule, and (c)(i) on a third nucleic acid molecule. The first, second and third nucleic acid molecule may be AAV vectors, e.g., the AAV vectors described herein.

In another embodiment, when a third and/or fourth gRNA molecule are present, each of (a), (b), (c)(i), (c) (ii) and (c)(iii) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector, e.g., an AAV vector. In an alternate embodiment, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In further embodiments, each of (a), (b), (c)(i), (c) (ii) and (c)(iii) may be present on more than one nucleic acid molecule, but fewer than five nucleic acid molecules, e.g., AAV vectors, e.g., the AAV vectors described herein.

The nucleic acids described herein may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a), e.g., a promoter described herein, e.g., a promoter described in Table 19. The nucleic acid may further comprise a second promoter operably linked to the sequence that encodes the second, third and/or fourth gRNA molecule of (c), e.g., a promoter described herein. The promoter and second promoter differ from one another. In some embodiments, the promoter and second promoter are the same.

The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the Cas9 molecule of (b), e.g., a promoter described herein, e.g., a promoter described in Table 19.

In another aspect, disclosed herein is a composition comprising (a) a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CEP290 gene, as described herein. The composition of (a) may further comprise (b) a Cas9 molecule, e.g., a Cas9 molecule as described herein. A composition of (a) and (b) may further comprise (c) a second, third and/or fourth gRNA molecule, e.g., a second, third and/or fourth gRNA molecule described herein.

In another aspect, methods and compositions discussed herein, provide for treating or delaying the onset or progression of LCA10 by altering the LCA10 target position in the

CEP290 gene.

In another aspect, disclosed herein is a method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising contacting said cell with: (a) a gRNA that targets the CEP290 gene, e.g., a gRNA as described herein; (b) a Cas9 molecule, e.g., a Cas9 molecule as described herein; and optionally, (c) a second, third and/or fourth gRNA that targets CEP290 gene, e.g., a gRNA as described herein.

In some embodiments, the method comprises contacting said cell with (a) and (b).

In some embodiments, the method comprises contacting said cell with (a), (b), and (c). The gRNA of (a) may be selected from any of Tables 1A-1D, Tables 2A-2C, Tables

3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, or a gRNA that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. The gRNA of (c) may be selected from any of Tables 1A-1D, Tables 2A-2C, Tables 3A- 3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A- 9B, or Table 10, or a gRNA that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

In some embodiments, the method comprises contacting a cell from a subject suffering from or likely to develop LCAIO. The cell may be from a subject having a mutation at a LCAIO target position.

In some embodiments, the cell being contacted in the disclosed method is a photoreceptor cell. The contacting may be performed ex vivo and the contacted cell may be returned to the subject's body after the contacting step. In other embodiments, the contacting step may be performed in vivo.

In some embodiments, the method of altering a cell as described herein comprises acquiring knowledge of the presence of a LCAIO target position in said cell, prior to the contacting step. Acquiring knowledge of the presence of a LCAIO target position in the cell may be by sequencing the CEP290 gene, or a portion of the CEP290 gene.

In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV vector described herein, that expresses at least one of (a), (b), and (c). In some embodiments, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses each of (a), (b), and (c). In another embodiment, the contacting step of the method comprises delivering to the cell a Cas9 molecule of (b) and a nucleic acid which encodes a gRNA (a) and optionally, a second gRNA (c)(i) (and further optionally, a third gRNA (c)(iv) and/or fourth gRNA (c)(iii)).

In an embodiment, contacting comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rhlO vector, a modified AAV.rhlO vector, an AAV.rh32/33 vector, a modified

AAV.rh32/33 vector, an AAV.rh43vector, a modified AAV.rh43vector, an AAV.rh64Rl vector, and a modified AAV.rh64Rlvector, e.g., an AAV vector described herein.

In an embodiment, contacting comprises delivering to said cell said Cas9 molecule of (b), as a protein or an mRNA, and a nucleic acid which encodes and (a) and optionally (c).

In an embodiment, contacting comprises delivering to said cell said Cas9 molecule of (b), as a protein or an mRNA, said gRNA of (a), as an RNA, and optionally said second gRNA of (c), as an RNA.

In an embodiment, contacting comprises delivering to said cell said gRNA of (a) as an RNA, optionally said second gRNA of (c) as an RNA, and a nucleic acid that encodes the Cas9 molecule of (b).

In another aspect, disclosed herein is a method of treating, or preventing a subject suffering from developing, LCA10, e.g., by altering the structure, e.g., sequence, of a target nucleic acid of the subject, comprising contacting the subject (or a cell from the subject) with:

(a) a gRNA that targets the CEP290 gene, e.g., a gRNA disclosed herein;

(b) a Cas9 molecule, e.g., a Cas9 molecule disclosed herein; and

optionally, (c)(i) a second gRNA that targets the CEP290 gene, e.g., a second gRNA disclosed herein, and

further optionally, (c)(ii) a third gRNA, and still further optionally, (c)(iii) a fourth gRNA that target the CEP290, e.g., a third and fourth gRNA disclosed herein.

In some embodiments, contacting comprises contacting with (a) and (b).

In some embodiments, contacting comprises contacting with (a), (b), and (c)(i).

In some embodiments, contacting comprises contacting with (a), (b), (c)(i) and (c)(ii).

In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and

(c)(iii).

The gRNA of (a) or (c) (e.g., (c)(i), (c)(ii), or (c)(iii)) may be independently selected from any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, or a gRNA that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

In an embodiment, said subject is suffering from, or likely to develop LCAIO. In an embodiment, said subject has a mutation at a LCAIO target position.

In an embodiment, the method comprises acquiring knowledge of the presence of a mutation at a LCAIO target position in said subject.

In an embodiment, the method comprises acquiring knowledge of the presence of a mutation a LCAIO target position in said subject by sequencing the CEP290 gene or a portion of the CEP290 gene.

In an embodiment, the method comprises altering the LCAIO target position in the

CEP290 gene.

In an embodiment, a cell of said subject is contacted ex vivo with (a), (b) and optionally (c). In an embodiment, said cell is returned to the subject's body.

In an embodiment, the method comprises introducing a cell into said subject's body, wherein said cell subject was contacted ex vivo with (a), (b) and optionally (c).

In an embodiment, the method comprises said contacting is performed in vivo. In an embodiment, the method comprises sub-retinal delivery. In an embodiment, contacting comprises sub-retinal injection. In an embodiment, contacting comprises intra-vitreal injection.

In an embodiment, contacting comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector described herein, e.g., a nucleic acid that encodes at least one of (a), (b), and optionally (c).

In an embodiment, contacting comprises delivering to said subject said Cas9 molecule of

(b) , as a protein or mRNA, and a nucleic acid which encodes and (a) and optionally (c).

In an embodiment, contacting comprises delivering to said subject said Cas9 molecule of (b), as a protein or mRNA, said gRNA of (a), as an RNA, and optionally said second gRNA of

(c) , as an RNA.

In an embodiment, contacting comprises delivering to said subject said gRNA of (a), as an RNA, optionally said second gRNA of (c), as an RNA, and a nucleic acid that encodes the Cas9 molecule of (b). In another aspect, disclosed herein is a reaction mixture comprising a gRNA, a nucleic acid, or a composition described herein, and a cell, e.g., a cell from a subject having, or likely to develop LCAIO, or a subject having a mutation at a LCAIO target position.

In another aspect, disclosed herein is a kit comprising, (a) a gRNA molecule described herein, or a nucleic acid that encodes said gRNA, and one or more of the following:

(b) a Cas9 molecule, e.g., a Cas9 molecule described herein, or a nucleic acid or mRNA that encodes the Cas9;

(c) (i) a second gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(i);

(c)(ii) a third gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(ii); or

(c)(iii) a fourth gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(iii).

In an embodiment, the kit comprises nucleic acid, e.g., an AAV vector, e.g., an AAV vector described herein, that encodes one or more of (a), (b), (c)(i), (c)(ii), and (c)(iii). In an embodiment, the kit further comprises a governing gRNA molecule, or a nucleic acid that encodes a governing gRNA molecule.

In yet another aspect, disclosed herein is a gRNA molecule, e.g., a gRNA molecule described herein, for use in treating LCAIO in a subject, e.g., in accordance with a method of treating LCAIO as described herein.

In an embodiment, the gRNA molecule in used in combination with a Cas9 molecule, e.g., a Cas9 molecule described herein. Additionaly or alternatively, in an embodiment, the gRNA molecule is used in combination with a second, third and/or fouth gRNA molecule, e.g., a second, third and/or fouth gRNA molecule described herein.

In still another aspect, disclosed herein is use of a gRNA molecule, e.g., a gRNA molecule described herein, in the manufacture of a medicament for treating LCAIO in a subject, e.g., in accordance with a method of treating LCAIO as described herein.

In an embodiment, the medicament comprises a Cas9 molecule, e.g., a Cas9 molecule described herein. Additionaly or alternatively, in an embodiment, the medicament comprises a second, third and/or fouth gRNA molecule, e.g., a second, third and/or fouth gRNA molecule described herein. In one aspect, disclosed herein is a recombinant adenovirus-associated virus (AAV) genome comprising the following components:

-ter NLSHpoly(A) signalHspacer 3Hright ITR|

wherein the left ITR component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of the left ITR nucleotide sequences disclosed in Table 24, or any of the nucleotide sequences of SEQ ID NOS: 407-415;

wherein the spacer 1 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 416;

wherein the PHI promoter component comprises, or consists of, an RNA polymerase III promoter sequence;

wherein the gRNA component comprises a targeting domain and a scaffold domain, wherein the targeting domain is 16-26 nucleotides in length, and comprises, or consists of, a targeting domain sequence disclosed herein, e.g., in any of Tables 1A-1D,

Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10; and

wherein the scaffold domain (also referred to as a tracr domain in Figs. 19A-24F) comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%,

98%, or 99% homology with, a nucleotide sequence of SEQ ID NO: 418;

wherein the spacer 2 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length e.g., SEQ ID NO: 419;

wherein the PII promoter component comprises, or consists of, a polymerase II promoter sequence, e.g., a constitutive or tissue specific promoter, e.g., a promoter disclosed in Table 19;

wherein the N-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 420 or a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 434;

wherein the Cas9 component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

421 or a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 435;

wherein the C-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

422 or a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 434;

wherein the poly(A) signal component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of the nucleotide sequences disclosed in Table 26, or any of the nucleotide sequences of SEQ ID NOS: 424, 455 or 456;

wherein the spacer 3 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 425; and

wherein the right ITR component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of the right ITR nucleotide sequences disclosed in Table 24, or any of the nucleotide sequences of SEQ ID NOS: 436-444.

In an embodiment, the left ITR component comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences of SEQ ID NOS: 407-415.

In an embodiment, the spacer 1 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 416.

In an embodiment, the PHI promoter component is a U6 promoter component.

In an embodiment, the U6 promoter component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 417;

In an embodiment, the U6 promoter component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 417.

In an embodiment, the PHI promoter component is an HI promoter component that comprises an HI promoter sequence. In an embodiment, the PHI promoter component is a tRNA promoter component that comprises a tRNA promoter sequence.

In an embodiment, the targeting domain comprises, or consists of, a nucleotide sequence that is the same as a nucleotide sequence selected from Table 10.

In an embodiment, the gRNA scaffold domain comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 418.

In an embodiment, the spacer 2 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 419;

In an embodiment, the PII promoter component is a CMV promoter component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 401. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 401.

In an embodiment, the PII promoter component is an EFS promoter component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 402. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 402.

In an embodiment, the PII promoter component is a GRK1 promoter (e.g., a human GRK1 promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 403. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 403.

In an embodiment, the PII promoter component is a CRX promoter (e.g., a human CRX promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 404. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 404.

In an embodiment, the PII promoter component is an NRL promoter (e.g., a human NRL promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

405. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 405.

In an embodiment, the PII promoter component is an RCVRN promoter (e.g., a human RCVRN promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 406. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 406.

In an embodiment, the N-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 420 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 434.

In an embodiment, the Cas9 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 421 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 435.

In an embodiment, the C-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 422 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 434.

In an embodiment, the poly(A) signal component comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences disclosed in Table 26, or any of the nucleotide sequences of SEQ ID NOS: 424, 455 or 456. In an embodiment, the poly(A) signal component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 424.

In an embodiment, the spacer 3 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 425. In an embodiment, the right ITR component comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences of SEQ ID NOS: 436-444.

In an embodiment, the recombinant AAV genome further comprises a second gRNA component comprising a targeting domain and a scaffold domain,

wherein the targeting domain consists of a targeting domain sequence disclosed herein, in any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10; and

wherein the scaffold domain (also referred to as a tracr domain in Figs. 19A-24F) comprises, or consists of, a nucleotide sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 418.

In an embodiment, the targeting domain of the second gRNA component comprises, or consists of, a nucleotide sequence that is the same as a nucleotide sequence selected from Table 10. In an embodiment, the second gRNA component is between the first gRNA component and the spacer 2 component.

In an embodiment, the second gRNA component has the same nucleotide sequence as the first gRNA component. In another embodiment, the second gRNA component has a nucleotide sequence that is different from the second gRNA component.

In an embodiment, the recombinant AAV genome further comprises a second PHI promoter component that comprises, or consists of, an RNA polymerase III promoter sequence;

In an embodiment, the recombinant AAV genome further comprises a second PHI promoter component (e.g., a second U6 promoter component) between the first gRNA

component and the second gRNA component.

In an embodiment, the second PHI promoter component (e.g., the second U6 promoter component) has the same nucleotide sequence as the first PHI promoter component (e.g., the first U6 promoter component). In another embodiment, the second PHI promoter component (e.g., the second U6 promoter component) has a nucleotide sequence that is different from the first PHI promoter component (e.g. the first U6 promoter component).

In an embodiment, the PHI promoter component is an HI promoter component that comprises an HI promoter sequence. In an embodiment, the PHI promoter component is a tRNA promoter component that comprises a tRNA promoter sequence.

In an embodiment, the recombinant AAV genome further comprises a spacer 4 component between the first gRNA component and the second PHI promoter component (e.g., the second U6 promoter component). In an embodiment, the spacer 4 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 427. In an embodiment, the spacer 4 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 427.

In an embodiment, the recombinant AAV genome comprises the following components:

[left ITR|-|spacer 1|— |PIII promoter|-|gRNA|-|spacer 4|-|second PHI promoter|-|second gRNA| spacer 2ΗΡΠ promoterHN-ter NLSHCas9HC-ter NLSHpoly(A) signalHspacer 3Hright ITR|

In an embodiment, the recombinant AAV genome further comprises an affinity tag component (e.g., 3xFLAG component), wherein the affinity tag component (e.g., 3xFLAG component) comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotides sequence of SEQ ID NO: 423, or a nucleotide sequence encoding any of the amino acid sequences disclosed in Table 25 or any of the amino acid sequences of SEQ ID NO: 435 or 451-454.

In an embodiment, the affinity tag component (e.g., 3xFLAG component) is between the C-ter NLS component and the poly(A) signal component. In an embodiment, the an affinity tag component (e.g., 3xFLAG component) comprises, or consists of, a nucleotide sequence that is the same as, the nucleotides sequence of SEQ ID NO: 423, or a nucleotide sequence encoding any of the amino acid sequences of SEQ ID NOS: 435 or 451-454.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 401, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 402, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 403, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 404, 420, 421, 422, 424, and 437. In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 405, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 406, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome further comprises SEQ ID NOS: 416, 419, and 425, and, optionally, SEQ ID NO 427.

In an embodiment, the recombinant AAV genome further comprises the nucleotide sequence of SEQ ID NO: 423.

In an embodiment, the recombinant AAV genome comprises or consists of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) of the component sequences shown in Figs. 19A-19G, 20A-20F, 21A-21F, 22A-22F, 23A-23F, or 24A-24F, Tables 19 or 24-26, or any of the nucleotide sequences of SEQ ID NOS: 428-433 or 436-444.

In another aspect, disclosed herein is a recombinant adenovirus-associated virus (AAV) genome comprising the following components:

[left ITR|-|spacer 1[— | irst PHI promoter|-|first gRNA|-|spacer 4|-|second PHI promoter|- second gRNAHspacer 2ΗΡΠ promoter^-ter NLSHCas9HC-ter NLSHpoly(A)

spacer 3|-|right ITR|.

wherein the left ITR component comprises, or consists of, a nucleotide sequence that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of the left ITR nucleotide sequences disclosed in Table 24, or any of the nucleotide sequences of SEQ ID NOS: 407-415; wherein the spacer 1 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 416;

wherein the first PHI promoter component (e.g., a first U6 promoter component) comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 417;

wherein the first gRNA component comprises a targeting domain and a scaffold domain, wherein the targeting domain is 16-26 nucleotides in length, and comprises, or consists of, a targeting domain sequence disclosed herein, e.g., in any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10; and

wherein the scaffold domain (also referred to herein as a tracr domain in Figs. 19A-24F) comprises, or consists of, a nucleotide sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%,

94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 418; wherein the spacer 4 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 427.

wherein the second gRNA component comprises a targeting domain and a scaffold domain,

wherein the targeting domain of the second gRNA component is 16-26 nucleotides in length and comprises, or consists of, a targeting domain sequence disclosed herein, e.g., in any of Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10; and

wherein the scaffold domain (also referred to as a tracr domain in Figs. 19A-24F) of the second gRNA component comprises, or consists of, a nucleotide sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 418.

wherein the spacer 2 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length e.g., SEQ ID NO: 419;

wherein the PII promoter component comprises, or consists of, a polymerase II promoter sequence, e.g., a constitutive or tissue specific promoter, e.g., a promoter disclosed in Table 19;

wherein the N-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 420 or a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 434;

wherein the Cas9 component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

421 or a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 435;

wherein the C-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

422 or a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 434;

wherein the poly(A) signal component comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of the nucleotide sequences disclosed in Table 26, or any of the nucleotide sequence of SEQ ID NO: 424, 455 or 456;

wherein the spacer 3 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 425; and

wherein the right ITR component comprises, or consists of, a nucleotide sequence that is the same as, or differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of the right ITR nucleotide sequences disclosed in Table 24, or SEQ ID NOS: 436-444.

In an embodiment, the left ITR component comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences of SEQ ID NOS: 407-415.

In an embodiment, the spacer 1 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 416.

In an embodiment, the first PHI promoter component (e.g., the first U6 promoter component) comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 417.

In an embodiment, the first PHI promoter is an HI promoter component that comprises an HI promoter sequence. In another embodiment, the first PHI promoter is a tRNA promoter component that comprises a tRNA promoter sequence.

In an embodiment, the targeting domain of the first gRNA component comprises, or consists of, a nucleotide sequence that is the same as a nucleotide sequence selected from Table 10. In an embodiment, the gRNA scaffold domain of the first gRNA component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 418.

In an embodiment, the spacer 4 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 427.

In an embodiment, the second PHI promoter component (e.g., the first U6 promoter component) has the same nucleotide sequence as the first PHI promoter component (e.g., the first U6 promoter component). In another embodiment, the second PHI promoter component (e.g., the second U6 promoter component) has a nucleotide sequence that is different from the first PHI promoter component (e.g., the first U6 promoter component).

In an embodiment, the second PHI promoter is an HI promoter component that comprises an HI promoter sequence. In another embodiment, the second PHI promoter is a tRNA promoter component that comprises a tRNA promoter sequence.

In an embodiment, the targeting domain of the second gRNA component comprises, or consists of, a nucleotide sequence that is the same as a nucleotide sequence selected from Table 10.

In an embodiment, the second gRNA component has the same nucleotide sequence as the first gRNA component. In another embodiment, the second gRNA component has a nucleotide sequence that is different from the second gRNA component.

In an embodiment, the spacer 2 component comprises, or consists of, a nucleotide sequence having 0 to 150 nucleotides in length e.g., SEQ ID NO: 419;

In an embodiment, the PII promoter component is a CMV promoter component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2,

3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 401. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 401.

In an embodiment, the PII promoter component is an EFS promoter component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 402. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 402.

In an embodiment, the PII promoter component is a GRK1 promoter (e.g., a human GRK1 promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 403. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 403.

In an embodiment, the PII promoter component is a CRX promoter (e.g., a human CRX promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

404. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 404.

In an embodiment, the PII promoter component is an NRL promoter (e.g., a human NRL promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO:

405. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 405.

In an embodiment, the PII promoter component is an RCVRN promoter (e.g., a human RCVRN promoter) component, and comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 406. In an embodiment, the PII promoter comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 406.

In an embodiment, the N-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 420 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 434. In an embodiment, the Cas9 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 421 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 435.

In an embodiment, the C-ter NLS component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 422 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 434.

In an embodiment, the poly(A) signal component comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences disclosed in Table 26, or any of the nucleotide sequences of SEQ ID NOS: 424, 455 or 456. In an embodiment, the poly(A) signal component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 424.

In an embodiment, the spacer 3 component comprises, or consists of, a nucleotide sequence that is the same as the nucleotide sequence of SEQ ID NO: 425.

In an embodiment, the right ITR component comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences disclosed in Table 24, or any of the nucleotide sequences of SEQ ID NOS: 436-444.

In an embodiment, the recombinant AAV genome further comprises an affinity tag component (e.g., a 3xFLAG component). In an embodiment, the affinity tag component (e.g., the 3xFLAG component) comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 423, or a nucleotide sequence encoding any of the amino acid sequences disclosed in Table 25 or any of the amino acid sequences of SEQ ID NO: 435 or 451-454.

In an embodiment, the affinity tag component (e.g., the 3xFLAG component) is between the C-ter NLS component and the poly(A) signal component. In an embodiment, the affinity tag component (e.g., the 3xFLAG component) comprises, or consists of, a nucleotide sequence that is the same as, the nucleotide sequence of SEQ ID NO: 423 or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 435.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 401, 420, 421, 422, 424, and 437. In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 402, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 403, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of

SEQ ID NOS: 408, 417, 418, 404, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 405, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome comprises the nucleotide sequences of SEQ ID NOS: 408, 417, 418, 406, 420, 421, 422, 424, and 437.

In an embodiment, the recombinant AAV genome further comprises the nucleotide sequences of SEQ ID NO: 416, 419, 425, and 427.

In an embodiment, the recombinant AAV genome further comprises the nucleotide sequence of SEQ ID NO: 423.

In an embodiment, the recombinant AAV genome comprises any of the nucleotide sequences of SEQ ID NOS: 428-433.

In an embodiment, the recombinant AAV genome comprises, or consists of, a nucleotide sequence that is the same as, differs by no more than 100, 200, 300, 400, or 500 nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homology with any of the nucleotide sequences shown in Figs. 19A-19G, 20A-20F, 21A-21F, 22A-22F, 23A-23F, or 24A-24F, or any of the nucleotide sequences of SEQ ID NOS: 428-433 or 436-444.

In an embodiment, the recombinant AAV genome comprises, or consists of, a nucleotide sequence that is the same as any of the nucleotide sequences shown in Figs. 19A-19G, 20A-20F, 21A-21F, 22A-22F, 23A-23F, or 24A-24F, or any of the nucleotide sequences of SEQ ID NOS: 428-433 or 436-444.

In an embodiment, the recombinant AAV genome comprises or consists of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) of the component sequences shown in Figs. 19A-19G, 20A-20F, 21A-21F, 22A-22F, 23A-23F, or 24A-24F, or Tables 19 or 24-26, or any of the nucleotide sequences of SEQ ID NOS: 428-433 or 436-444.

Unless otherwise indicated, when components of a recombinant AAV genome are described herein, the order can be as provided, but other orders are included as well. In other words, in an embodiment, the order is as set out in the text, but in other embodiments, the order can be different.

It is understood that the recombinant AAV genomes disclosed herein can be single stranded or double stranded. Disclosed herein are also the reverse, complementary form of any of the recombinant AAV genomes disclosed herein, and the double stranded form thereof.

In another aspect, disclosed herein is a nucleic acid molecule (e.g., an expression vector) that comprises a recombinant AAV genome disclosed herein. In an embodiment, the nucleic acid molecule further comprises a nucleotide sequence that encodes an antibiotic resistant gene (e.g., an Amp resistant gene). In an embodiment, the nucleic acid molecule further comprises replication origin sequence (e.g., a ColEl origin, an M13 origin, or both).

In another aspect, disclosed herein is a recombinant AAV viral particle comprising a recombinant AAV genome disclosed herein.

In an embodiment, the recombinant AAV viral particle has any of the serotype disclosed herein, e.g., in Table 24, or a combination thereof. In another embodiment, the recombinant AAV viral particle has a tissue specificity of retinal pigment epithelium cells, photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells, or a combination thereof.

In another aspect, disclosed herein is a method of producing a recombinant AAV viral particle disclosed herein comprising providing a recombinant AAV genome disclosed herein and one or more capsid proteins under conditions that allow for assembly of an AAV viral particle.

In another aspect, disclosed herein is a method of altering a cell comprising contacting the cell with a recombinant AAV viral particle disclosed herein.

In another aspect, disclosed herein is a method of treating a subject having or likely to develop LCAIO comprising contacting the subject (or a cell from the subject) with a recombinant viral particle disclosed herein.

In another aspect, disclosed herein is a recombinant AAV viral particle comprising a recombinant AAV genome disclosed herein for use in treating LCAIO in a subject.

In another aspect, disclosed herein is use of a recombinant AAV viral particle comprising a recombinant AAV genome disclosed herein in the manufacture of a medicament for treating LCAIO in a subject.

The gRNA molecules and methods, as disclosed herein, can be used in combination with a governing gRNA molecule, comprising a targeting domain which is complementary to a target domain on a nucleic acid that encodes a component of the CRISPR/Cas system introduced into a cell or subject. In an embodiment, the governing gRNA molecule targets a nucleic acid that encodes a Cas9 molecule or a nucleic acid that encodes a target gene gRNA molecule. In an embodiment, the governing gRNA comprises a targeting domain that is complementary to a target domain in a sequence that encodes a Cas9 component, e.g., a Cas9 molecule or target gene gRNA molecule. In an embodiment, the target domain is designed with, or has, minimal homology to other nucleic acid sequences in the cell, e.g., to minimize off-target cleavage. For example, the targeting domain on the governing gRNA can be selected to reduce or minimize off-target effects. In an embodiment, a target domain for a governing gRNA can be disposed in the control or coding region of a Cas9 molecule or disposed between a control region and a transcribed region. In an embodiment, a target domain for a governing gRNA can be disposed in the control or coding region of a target gene gRNA molecule or disposed between a control region and a transcribed region for a target gene gRNA. While not wishing to be bound by theory, in an embodiment, it is believed that altering, e.g., inactivating, a nucleic acid that encodes a Cas9 molecule or a nucleic acid that encodes a target gene gRNA molecule can be effected by cleavage of the targeted nucleic acid sequence or by binding of a Cas9

molecule/governing gRNA molecule complex to the targeted nucleic acid sequence.

The compositions, reaction mixtures and kits, as disclosed herein, can also include a governing gRNA molecule, e.g., a governing gRNA molecule disclosed herein,

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1G are representations of several exemplary gRNAs.

Fig. 1A depicts a modular gRNA molecule derived in part (or modeled on a sequence in part) from Streptococcus pyogenes (S. pyogenes) as a duplexed structure (SEQ ID NOS: 42 and 43, respectively, in order of appearance);

Fig. IB depicts a unimolecular (or chimeric) gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 44);

Fig. 1C depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45);

Fig. ID depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 46);

Fig. IE depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 47);

Fig. IF depicts a modular gRNA molecule derived in part from Streptococcus thermophilus (S. thermophilus) as a duplexed structure (SEQ ID NOS: 48 and 49, respectively, in order of appearance);

Fig. 1G depicts an alignment of modular gRNA molecules of S. pyogenes and S.

thermophilus (SEQ ID NOS: 50-53, respectively, in order of appearance).

Figs. 2A-2G depict an alignment of Cas9 sequences from Chylinski et al. (RNA Biol. 2013; 10(5): 726-737). The N-terminal RuvC-like domain is boxed and indicated with a "Y". The other two RuvC-like domains are boxed and indicated with a "B". The HNH-like domain is boxed and indicated by a "G". Sm: S. mutans (SEQ ID NO: 1); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophilus (SEQ ID NO: 3); Li: L. innocua (SEQ ID NO: 4). Motif: this is a motif based on the four sequences: residues conserved in all four sequences are indicated by single letter amino acid abbreviation; "*" indicates any amino acid found in the corresponding position of any of the four sequences; and "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids.

Figs. 3A-3B show an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al (SEQ ID NOS: 54-103, respectively, in order of appearance). The last line of Fig. 3B identifies 4 highly conserved residues. Figs. 4A-4B show an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS: 104-177, respectively, in order of appearance). The last line of Fig. 4B identifies 3 highly conserved residues.

Figs. 5A-5C show an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al (SEQ ID NOS: 178-252, respectively, in order of appearance). The last line of Fig. 5C identifies conserved residues.

Figs. 6A-6B show an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS: 253-302, respectively, in order of appearance). The last line of Fig. 6B identifies 3 highly conserved residues.

Figs. 7A-7B depict an alignment of Cas9 sequences from S. pyogenes and Neisseria meningitidis (N. meningitidis). The N-terminal RuvC-like domain is boxed and indicated with a "Y". The other two RuvC-like domains are boxed and indicated with a "B". The HNH-like domain is boxed and indicated with a "G". Sp: S. pyogenes; Nm: N. meningitidis. Motif: this is a motif based on the two sequences: residues conserved in both sequences are indicated by a single amino acid designation; "*" indicates any amino acid found in the corresponding position of any of the two sequences; "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, and "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.

Fig. 8 shows a nucleic acid sequence encoding Cas9 of N. meningitidis (SEQ ID NO: 303). Sequence indicated by an "R" is an SV40 NLS; sequence indicated as "G" is an HA tag; and sequence indicated by an "O" is a synthetic NLS sequence; the remaining (unmarked) sequence is the open reading frame (ORF).

Figs. 9A-9B are schematic representations of the domain organization of S. pyogenes Cas

9. Fig. 9A shows the organization of the Cas9 domains, including amino acid positions, in reference to the two lobes of Cas9 (recognition (REC) and nuclease (NUC) lobes). Fig. 9B shows the percent homology of each domain across 83 Cas9 orthologs.)

Fig. 10 shows the nucleotide locations of the Alu repeats, cryptic exon and point mutation, c.2991+1655 A to G in the human CEP290 locus. "X" indicates the cryptic exon. The blue triangle indicates the LCA target position C.2991+1655A to G. Fig. 11A-11B show the rates of indels induced by various gRNAs at the CEP290 locus. Fig. 11A shows gene editing (% indels) as assessed by sequencing for S. pyogenes and S. aureus gRNAs when co-expressed with Cas9 in patient-derived IVS26 primary fibroblasts. Fig. 11B shows gene editing (% indels) as assessed by sequencing for S. aureus gRNAs when co- expressed with Cas9 in HEK293 cells.

Figs. 12A-12B show changes in expression of the wildtype and mutant (including cryptic exon) alleles of CEP290 in cells transfected with Cas9 and the indicated gRNA pairs. Total RNA was isolated from modified cells and qRT-PCR with Taqman primer-probe sets was used to quantify expression. Expression is normalized to the Beta-Actin housekeeping gene and each sample is normalized to the GFP control sample (cells transfected with only GFP). Error bars represent standard deviation of 4 technical replicates.

Fig. 13 shows changes in gene expression of the wildtype and mutant (including cryptic exon) alleles of CEP290 in cells transfected with Cas9 and pairs of gRNAs shown to have in initial qRT-PCR screening. Total RNA was isolated from modified cells and qRT-PCR with Taqman primer-probe sets was used to quantify expression. Expression is normalized to the Beta-Actin housekeeping gene and each sample is normalized to the GFP control sample (cells transfected with only GFP). Error bars represent standard error of the mean of two to six biological replicates.

Fig. 14 shows deletion rates in cells transfected with indicated gRNA pairs and Cas9 as measured by droplet digital PCR (ddPCR). % deletion was calculated by dividing the number of positive droplets in deletion assay by the number of positive droplets in a control assay. Three biological replicates are shown for two different gRNA pairs.

Fig. 15 shows deletion rates in 293T cells transfected with exemplary AAV expression plasmids. pSSlO encodes EFS-driven saCas9 without gRNA. pSS15 and pSS17 encode EFS- driven saCas9 and one U6-driven gRNA, CEP290-64 and CEP290-323 respectively. pSS 11 encodes EFS-driven saCas9 and two U6-driven gRNAs, CEP290-64 and CEP290-323 in the same vector. Deletion PCR were performed with gDNA exacted from 293T cells post transfection. The size of the PCR amplicons indicates the presence or absence of deletion events, and the deletion ratio was calculated.

Fig. 16 shows the composition of structural proteins in AAV2 viral preps expressing

Cas9. Reference AAV2 vectors (lanes 1 & 2) were obtained from Vector Core at University of North Carolina, Chapel Hill. AAV2-CMV-GFP (lane 3) and AAV2-CMV-saCas9-minpA (lane4) were packaged and purified with "Triple Transfection Protocol" followed by CsCl ultracentrifugation. Titers were obtained by quantitative PCR with primers annealing to the ITR structures on these vectors. Viral preps were denatured and probed with Bl antibody on Western Blots to demonstrate three structural proteins composing AAV2, VP1, VP2, and VP3

respectively.

Fig. 17 depicts the deletion rates in 293T cells transduced with AAV viral vectors at MOI of 1000 viral genome (vg) per cell and 10,000 vg per cell. AAV2 viral vectors were produced with "Triple Transfection Protocol" using pHelper, pRep2Cap2, pSS8 encoding gRNAs

CEP290-64 and CEP290-323, and CMV-driven saCas9. Viral preps were titered with primers annealing to ITRs on pSS8. 6 days post transduction, gDNA were extracted from 293T cells. Deletion PCR was carried out on the CEP290 locus, and deletion rates were calculated based on the predicted amplicons. Western blotting was carried out to show the AAV-mediated saCas9 expression in 293T cells (primary antibody: anti-Flag, M2; loading control: anti-alphaTubulin).

Fig. 18A-18B depicts additional exemplary structures of unimolecular gRNA molecules.

Fig. 18A shows an exemplary structure of a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure. Fig. 18B shows an exemplary structure of a unimolecular gRNA molecule derived in part from S. aureus as a duplexed structure.

Figs. 19A-19G depicts the nucleotide sequence of an exemplary recombinant AAV genome containg a CMV promoter. Various components of the recombinant AAV genome are also indicated. N = A, T, G or C. The number of N residues can vary, e.g., from 16 to 26 nucleotides. Upper stand: 5'→3' (SEQ ID NO: 428); lower stand: 3'→5' SEQ ID NO: 445).

Figs. 20A-20F depicts the nucleotide sequence of an exemplary recombinant AAV genome containg an EFS promoter. Various components of the recombinant AAV genome are also indicated. N = A, T, G or C. The number of N residues can vary, e.g., from 16 to 26 nucleotides. Upper stand: 5'→3' (SEQ ID NO: 429); lower stand: 3'→5' (SEQ ID NO: 446).

Figs. 21A-21F depicts the nucleotide sequence of an exemplary recombinant AAV genome containg a CRK1 promoter. Various components of the recombinant AAV genome are also indicated. N = A, T, G or C. The number of N residues can vary, e.g., from 16 to 26 nucleotides. Upper stand: 5'→3' (SEQ ID NO: 430); lower stand: 3'→5' (SEQ ID NO: 447). Figs. 22A-22F depicts the nucleotide sequence of an exemplary recombinant AAV genome containg a CRX promoter. Various components of the recombinant AAV genome are also indicated. N = A, T, G or C. The number of N residues can vary, e.g., from 16 to 26 nucleotides. Upper stand: 5'→3' (SEQ ID NO: 431); lower stand: 3'→5' (SEQ ID NO: 448).

Figs. 23A-23F depicts the nucleotide sequence of an exemplary recombinant AAV genome containg a NRL promoter. Various components of the recombinant AAV genome are also indicated. N = A, T, G or C. The number of N residues can vary, e.g., from 16 to 26 nucleotides. Upper stand: 5'→3' (SEQ ID NO: 432); lower stand: 3'→5' (SEQ ID NO: 449).

Figs. 24A-24F depicts the nucleotide sequence of an exemplary recombinant AAV genome containg a NRL promoter. Various components of the recombinant AAV genome are also indicated. N = A, T, G or C. The number of N residues can vary, e.g., from 16 to 26 nucleotides. Upper stand: 5'→3' (SEQ ID NO: 433); lower stand: 3'→5' (SEQ ID NO: 450).

DETAILED DESCRIPTION

Definitions

"Domain", as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.

Calculations of homology or sequence identity between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

"Governing gRNA molecule", as used herein, refers to a gRNA molecule that comprises a targeting domain that is complementary to a target domain on a nucleic acid that comprises a sequence that encodes a component of the CRISPR/Cas system that is introduced into a cell or subject. A governing gRNA does not target an endogenous cell or subject sequence. In an embodiment, a governing gRNA molecule comprises a targeting domain that is complementary with a target sequence on: (a) a nucleic acid that encodes a Cas9 molecule; (b) a nucleic acid that encodes a gRNA which comprises a targeting domain that targets the CEP290 gene (a target gene gRNA); or on more than one nucleic acid that encodes a CRISPR/Cas component, e.g., both (a) and (b). In an embodiment, a nucleic acid molecule that encodes a CRISPR/Cas component, e.g., that encodes a Cas9 molecule or a target gene gRNA, comprises more than one target domain that is complementary with a governing gRNA targeting domain. While not wishing to be bound by theory, it is believed that a governing gRNA molecule complexes with a Cas9 molecule and results in Cas9 mediated inactivation of the targeted nucleic acid, e.g., by cleavage or by binding to the nucleic acid, and results in cessation or reduction of the production of a CRISPR/Cas system component. In an embodiment, the Cas9 molecule forms two complexes: a complex comprising a Cas9 molecule with a target gene gRNA, which complex will alter the CEP290 gene; and a complex comprising a Cas9 molecule with a governing gRNA molecule, which complex will act to prevent further production of a CRISPR/Cas system component, e.g., a Cas9 molecule or a target gene gRNA molecule. In an embodiment, a governing gRNA molecule/Cas9 molecule complex binds to or promotes cleavage of a control region sequence, e.g., a promoter, operably linked to a sequence that encodes a Cas9 molecule, a sequence that encodes a transcribed region, an exon, or an intron, for the Cas9 molecule. In an embodiment, a governing gRNA molecule/Cas9 molecule complex binds to or promotes cleavage of a control region sequence, e.g., a promoter, operably linked to a gRNA molecule, or a sequence that encodes the gRNA molecule. In an embodiment, the governing gRNA, e.g., a Cas9-targeting governing gRNA molecule, or a target gene gRNA-targeting governing gRNA molecule, limits the effect of the Cas9 molecule/target gene gRNA molecule complex-mediated gene targeting. In an embodiment, a governing gRNA places temporal, level of expression, or other limits, on activity of the Cas9 molecule/target gene gRNA molecule complex. In an embodiment, a governing gRNA reduces off-target or other unwanted activity. In an

embodiment, a governing gRNA molecule inhibits, e.g., entirely or substantially entirely inhibits, the production of a component of the Cas9 system and thereby limits, or governs, its activity. "Modulator", as used herein, refers to an entity, e.g., a drug that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence. In an embodiment, modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non- covalent bond, e.g., the attachment of a moiety, to the subject molecule. In an embodiment, a modulator alters the, three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule. A modulator can increase, decrease, initiate, or eliminate a subject activity.

"Large molecule", as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologies, and carbohydrates.

"Polypeptide", as used herein, refers to a polymer of amino acids having less than 100 amino acid residues. In an embodiment, it has less than 50, 20, or 10 amino acid residues.

"Non-homologous end joining" or "NHEJ", as used herein, refers to ligation mediated repair and/or non-template mediated repair including, e.g., canonical NHEJ (cNHEJ), alternative NHEJ (altNHEJ), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

"Reference molecule", e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S.

pyogenes, S. aureus, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.

"Replacement", or "replaced", as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present. "Small molecule", as used herein, refers to a compound having a molecular weight less than about 2 kD, e.g., less than about 2 kD, less than about 1.5 kD, less than about 1 kD, or less than about 0.75 kD.

"Subject", as used herein, means either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In an embodiment, the subject is a human. In other embodiments, the subject is poultry.

"Treat", "treating" and "treatment", as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease.

"X" as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.

Methods of Altering CEP290

CEP290 encodes a centrosomal protein that plays a role in centrosome and cilia development. The CEP290 gene is involved in forming cilia around cells, particularly in the photoreceptors at the back of the retina, which are needed to detect light and color.

Disclosed herein are methods and compositions for altering the LCA10 target position in the CEP290 gene. LCA10 target position can be altered (e.g., corrected) by gene editing, e.g., using CRISPR-Cas9 mediated methods. The alteration (e.g., correction) of the mutant CEP290 gene can be mediated by any mechanism. Exemplary mechanisms that can be associated with the alteration (e.g., correction) of the mutatnt CEP290 gene include, but are not limited to, nonhomologous end joining (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template mediated), SDSA

(synthesis dependent strand annealing), single strand annealing or single strand invasion.

Methods described herein introduce one or more breaks near the site of the LCA target position (e.g., C.2991+1655A to G) in at least one allele of the CEP290 gene. In an embodiment, the one or more breaks are repaired by NHEJ. During repair of the one or more breaks, DNA sequences are inserted and/or deleted resulting in the loss or destruction of the cryptic splice site resulting from the mutation at the LCA10 target position (e.g., C.2991+1655A to G). The method can include acquiring knowledge of the mutation carried by the subject, e.g., by sequencing the appropriate portion of the CEP290 gene.

Altering the LCAIO target position refers to (1) break-induced introduction of an indel (also referred to herein as NHEJ-mediated introduction of an indel) in close proximity to or including a LCAIO target position (e.g., C.2991+1655A to G), or (2) break-induced deletion (also referred to herein as NHEJ-mediated deletion) of genomic sequence including the mutation at a LCAIO target position (e.g., C.2991+1655A to G). Both approaches give rise to the loss or destruction of the cryptic splice site.

In an embodiment, the method comprises introducing a break-induced indel in close proximity to or including the LCAIO target position (e.g., C.2991+1655A to G). As described herein, in one embodiment, the method comprises the introduction of a double strand break sufficiently close to (e.g., either 5' or 3' to) the LCAIO target position, e.g., C.2991+1655A to G, such that the break-induced indel could be reasonably expected to span the mutation. A single gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, is configured to position a double strand break sufficiently close to the LCAIO target position in the CEP290 gene. In an embodiment, the break is positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat. The double strand break may be positioned within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of the LCAIO target position, or within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) downstream of the LCAIO target position (see Fig. 9). While not wishing to be bound by theory, in an embodiment, it is believed that NHEJ- mediated repair of the double strand break allows for the NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position.

In another embodiment, the method comprises the introduction of a pair of single strand breaks sufficiently close to (either 5' or 3' to, respectively) the mutation at the LCAIO target position (e.g., C.2991+1655A to G) such that the break- induced indel could be reasonably expected to span the mutation. Two gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, are configured to position the two single strand breaks sufficiently close to the LCAIO target position in the CEP290 gene. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat. In an embodiment, the pair of single strand breaks is positioned within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of the LCAIO target position, or within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) downstream of the LCAIO target position (see Fig. 9). While not wishing to be bound by theory, in an embodiment, it is believed that NHEJ mediated repair of the pair of single strand breaks allows for the NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position. In an embodiment, the pair of single strand breaks may be accompanied by an additional double strand break, positioned by a third gRNA molecule, as is discussed below. In another embodiment, the pair of single strand breaks may be

accompanied by two additional single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule, as is discussed below.

In an embodiment, the method comprises introducing a break-induced deletion of genomic sequence including the mutation at the LCAIO target position (e.g., C.2991+1655A to G). As described herein, in one embodiment, the method comprises the introduction of two double strand breaks— one 5' and the other 3' to (i.e., flanking) the LCAIO target position. Two gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, are configured to position the two double strand breaks on opposite sides of the LCAIO target position in the CEP290 gene. In an embodiment, the first double strand break is positioned upstream of the LCAIO target position within intron 26 (e.g., within 1654 nucleotides), and the second double strand break is positioned downstream of the LCAIO target position within intron 26 (e.g., within 4183 nucleotides) (see Fig. 10). In an embodiment, the breaks (i.e., the two double strand breaks) are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat, or the endogenous CEP290 splice sites.

The first double strand break may be positioned as follows:

(1) upstream of the 5' end of the Alu repeat in intron 26,

(2) between the 3' end of the Alu repeat and the LCAIO target position in intron

26, or

(3) within the Alu repeat provided that a sufficient length of the gRNA fall outside of the repeat so as to avoid binding to other Alu repeats in the genome, and the second double strand break to be paired with the first double strand break may be positioned downstream of the LCAIO target position in intron 26.

For example, the first double strand break may be positioned: (1) within 1162 nucleotides upstream of the 5' end of the Alu repeat,

(2) within 1000 nucleotides upstream of the 5' end of the Alu repeat,

(3) within 900 nucleotides upstream of the 5' end of the Alu repeat,

(4) within 800 nucleotides upstream of the 5' end of the Alu repeat, (5) within 700 nucleotides upstream of the 5' end of the Alu repeat,

(6) within 600 nucleotides upstream of the 5' end of the Alu repeat,

(7) within 500 nucleotides upstream of the 5' end of the Alu repeat,

(8) within 400 nucleotides upstream of the 5' end of the Alu repeat,

(9) within 300 nucleotides upstream of the 5' end of the Alu repeat, (10) within 200 nucleotides upstream of the 5' end of the Alu repeat,

(11) within 100 nucleotides upstream of the 5' end of the Alu repeat,

(12) within 50 nucleotides upstream of the 5' end of the Alu repeat,

(13) within the Alu repeat provided that a sufficient length of the gRNA falls outside of the repeat so as to avoid binding to other Alu repeats in the genome,

(14) within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25,

30, 35 or 40 nucleotides) upstream of the LCA10 target position, or

(15) within 17 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16 or 17 nucleotides) upstream of the LCA10 target position,

and the second double strand breaks to be paired with the first double strand break may be positioned:

(1) within 4183 nucleotides downstream of the LCA10 target position,

(2) within 4000 nucleotides downstream of the LCA10 target position,

(3) within 3000 nucleotides downstream of the LCA10 target position,

(4) within 2000 nucleotides downstream of the LCA10 target position,

(5) within 1000 nucleotides downstream of the LCA10 target position,

(6) within 700 nucleotides downstream of the LCA10 target position,

(7) within 500 nucleotides downstream of the LCA10 target position,

(8) within 300 nucleotides downstream of the LCA10 target position,

(9) within 100 nucleotides downstream of the LCA10 target position,

(10) within 60 nucleotides downstream of the LCA10 target position, or (11) within 40 (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides) nucleotides downstream of the LCAIO target position.

While not wishing to be bound by theory, in an embodiment, it is believed that the two double strand breaks allow for break- induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene.

The method also comprises the introduction of two sets of breaks, e.g., one double strand break (either 5' or 3' to the mutation at the LCAIO target position, e.g., C.2991+1655A to G) and a pair of single strand breaks (on the other side of the LCAIO target position opposite from the double strand break) such that the two sets of breaks are positioned to flank the LCAIO target position. Three gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, are configured to position the one double strand break and the pair of single strand breaks on opposite sides of the LCAIO target position in the CEP290 gene. In an embodiment, the first set of breaks (either the double strand break or the pair of single strand breaks) is positioned upstream of the LCAIO target position within intron 26 (e.g., within 1654 nucleotides), and the second set of breaks (either the double strand break or the pair of single strand breaks) are positioned downstream of the LCAIO target position within intron 26 (e.g., within 4183 nucleotides) (see Fig. 10). In an embodiment, the two sets of breaks (i.e., the double strand break and the pair of single strand breaks) are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat, or the endogenous CEP290 splice sites.

The first set of breaks (either the double strand break or the pair of single strand breaks) may be positioned:

(1) upstream of the 5' end of the Alu repeat in intron 26,

(2) between the 3' end of the Alu repeat and the LCAIO target position in intron

26, or

(3) within the Alu repeat provided that a sufficient length of the gRNA falls outside of the repeat so as to avoid binding to other Alu repeats in the genome, and the second set of breaks to be paired with the first set of breaks (either the double strand break or the pair of single strand breaks) may be positioned downstream of the LCAIO target position in intron 26.

For example, the first set of breaks (either the double strand break or the pair of single strand breaks) may be positioned: (1) within 1162 nucleotides upstream of the 5' end of the Alu repeat,

(2) within 1000 nucleotides upstream of the 5' end of the Alu repeat,

(3) within 900 nucleotides upstream of the 5' end of the Alu repeat,

(4) within 800 nucleotides upstream of the 5' end of the Alu repeat, (5) within 700 nucleotides upstream of the 5' end of the Alu repeat,

(6) within 600 nucleotides upstream of the 5' end of the Alu repeat,

(7) within 500 nucleotides upstream of the 5' end of the Alu repeat,

(8) within 400 nucleotides upstream of the 5' end of the Alu repeat,

(9) within 300 nucleotides upstream of the 5' end of the Alu repeat, (10) within 200 nucleotides upstream of the 5' end of the Alu repeat,

(11) within 100 nucleotides upstream of the 5' end of the Alu repeat,

(12) within 50 nucleotides upstream of the 5' end of the Alu repeat,

(13) within the Alu repeat provided that a sufficient length of the gRNA falls outside of the repeat so as to avoid binding to other Alu repeats in the genome,

(14) within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25,

30, 35 or 40 nucleotides) upstream of the LCA10 target position, or

(15) within 17 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16 or 17 nucleotides) upstream of the LCA10 target position,

and the second set of breaks to be paired with the first set of breaks (either the double strand break or the pair of single strand breaks) may be positioned:

(1) within 4183 nucleotides downstream of the LCA10 target position,

(2) within 4000 nucleotides downstream of the LCA10 target position,

(3) within 3000 nucleotides downstream of the LCA10 target position,

(4) within 2000 nucleotides downstream of the LCA10 target position,

(5) within 1000 nucleotides downstream of the LCA10 target position,

(6) within 700 nucleotides downstream of the LCA10 target position,

(7) within 500 nucleotides downstream of the LCA10 target position,

(8) within 300 nucleotides downstream of the LCA10 target position,

(9) within 100 nucleotides downstream of the LCA10 target position,

(10) within 60 nucleotides downstream of the LCA10 target position, or (11) within 40 (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides) nucleotides downstream of the LCAIO target position.

While not wishing to be bound by theory, it is believed that the two sets of breaks (either the double strand break or the pair of single strand breaks) allow for break- induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene.

The method also comprises the introduction of two sets of breaks, e.g., two pairs of single strand breaks, wherein the two sets of single- stranded breaks are positioned to flank the LCAIO target position. In an embodiment, the first set of breaks (e.g., the first pair of single strand breaks) is 5' to the mutation at the LCAIO target position (e.g., C.2991+1655A to G) and the second set of breaks (e.g., the second pair of single strand breaks) is 3' to the mutation at the LCAIO target position. Four gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, are configured to position the two pairs of single strand breaks on opposite sides of the LCAIO target position in the CEP290 gene. In an embodiment, the first set of breaks (e.g., the first pair of single strand breaks) is positioned upstream of the LCAIO target position within intron 26 (e.g., within 1654 nucleotides), and the second set of breaks (e.g., the second pair of single strand breaks) is positioned downstream of the LCAIO target position within intron 26 (e.g., within 4183 nucleotides) (see Fig. 10). In an embodiment, the two sets of breaks (i.e., the two pairs of single strand breaks) are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat, or the endogenous CEP290 splice sites.

The first set of breaks (e.g., the first pair of single strand breaks) may be positioned:

(1) upstream of the 5' end of the Alu repeat in intron 26,

(2) between the 3' end of the Alu repeat and the LCAIO target position in intron

26, or

(3) within the Alu repeat provided that a sufficient length of the gRNA falls outside of the repeat so as to avoid binding to other Alu repeats in the genome, and the second set of breaks to be paired with the first set of breaks (e.g., the second pair of single strand breaks) may be positioned downstream of the LCAIO target position in intron 26.

For example, the first set of breaks (e.g., the first pair of single strand breaks) may be positioned:

(1) within 1162 nucleotides upstream of the 5' end of the Alu repeat,

(2) within 1000 nucleotides upstream of the 5' end of the Alu repeat, (3) within 900 nucleotides upstream of the 5' end of the Alu repeat,

(4) within 800 nucleotides upstream of the 5' end of the Alu repeat,

(5) within 700 nucleotides upstream of the 5' end of the Alu repeat,

(6) within 600 nucleotides upstream of the 5' end of the Alu repeat, (7) within 500 nucleotides upstream of the 5' end of the Alu repeat,

(8) within 400 nucleotides upstream of the 5' end of the Alu repeat,

(9) within 300 nucleotides upstream of the 5' end of the Alu repeat,

(10) within 200 nucleotides upstream of the 5' end of the Alu repeat,

(11) within 100 nucleotides upstream of the 5' end of the Alu repeat, (12) within 50 nucleotides upstream of the 5' end of the Alu repeat,

(13) within the Alu repeat provided that a sufficient length of the gRNA falls outside of the repeat so as to avoid binding to other Alu repeats in the genome,

(14) within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of the LCA10 target position, or

(15) within 17 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16 or 17 nucleotides) upstream of the LCA10 target position,

and the second set of breaks to be paired with the first set of breaks (e.g., the second pair of single strand breaks) may be positioned:

(I) within 4183 nucleotides downstream of the LCA10 target position,

(2) within 4000 nucleotides downstream of the LCA10 target position,

(3) within 3000 nucleotides downstream of the LCA10 target position,

(4) within 2000 nucleotides downstream of the LCA10 target position,

(5) within 1000 nucleotides downstream of the LCA10 target position,

(6) within 700 nucleotides downstream of the LCA10 target position,

(7) within 500 nucleotides downstream of the LCA10 target position,

(8) within 300 nucleotides downstream of the LCA10 target position,

(9) within 100 nucleotides downstream of the LCA10 target position,

(10) within 60 nucleotides downstream of the LCA10 target position, or

(I I) within 40 (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or 40 nucleotides) nucleotides downstream of the LCA10 target position. While not wishing to be bound by theory, it is believed that the two sets of breaks (e.g., the two pairs of single strand breaks) allow for break- induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene.

Methods to Treat or Prevent LCAIO

Described herein are methods for treating or delaying the onset or progression of Leber's Congenital Amaurosis 10 (LCAIO) caused by a c.2991+1655 A to G (adenine to guanine) mutation in the CEP290 gene. The disclosed methods for treating or delaying the onset or progression of LCAIO alter the CEP290 gene by genome editing using a gRNA targeting the LCAIO target position and a Cas9 enzyme. Details on gRNAs targeting the LCAIO target position and Cas9 enzymes are provided below.

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

glare.

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

sensitivity to glare.

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

In an embodiment, treatment is initiated

A subject's vision can evaluated, e.g., prior to treatment, or after treatment, e.g., to monitor the progress of the treatment. In an embodiment, the subject's vision is evaluated prior to treatment, e.g., to determine the need for treatment. In an embodiment, the subject's vision is evaluated after treatment has been initiated, e.g., to access the effectiveness of the treatment. Vision can be evaluated by one or more of: evaluating changes in function relative to the contralateral eye, e.g., by utilizing retinal analytical techniques; by evaluating mean, median and distribution of change in best corrected visual acuity (BCVA); evaluation by Optical Coherence Tomography; evaluation of changes in visual field using perimetry; evaluation by full-field electroretinography (ERG); evaluation by slit lamp examination; evaluation of intraocular pressure; evaluation of autofluorescence, evaluation with fundoscopy; evaluation with fundus photography; evaluation with fluorescein angiography (FA); or evaluation of visual field sensitivity (FFST).

In an embodiment, a subject's vision may be assessed by measuring the subject's mobility, e.g., the subject's ability to maneuver in space.

In an embodiment, treatment is initiated in a subject who has tested positive for a mutation in the CEP290 gene, e.g., prior to disease onset or in the earliest stages of disease.

In an embodiment, a subject has a family member that has been diagnosed with LCA10. For example, the subject has a family member that has been diagnosed with LCA10, and the subject demonstrates a symptom or sign of the disease or has been found to have a mutation in the CEP290 gene.

In an embodiment, a cell (e.g., a retinal cell, e.g., a photoreceptor cell) from a subject suffering from or likely to develop LCA10 is treated ex vivo. In an embodiment, the cell is removed from the subject, altered as described herein, and introduced into, e.g., returned to, the subject.

In an embodiment, a cell (e.g., a retinal cell, e.g., a photoreceptor cell) altered to correct a mutation in the LCA10 target position is introduced into the subject.

In an embodiment, the cell is a retinual cell (e.g., retinal pigment epithelium cell), a photoreceptor cell, a horizontal cell, a bipolar cell, an amacrine cell, or a ganglion cell. In an embodiment, it is contemplated herein that a population of cells (e.g., a population of retinal cells, e.g., a population of photoreceptor cells) from a subject may be contacted ex vivo to alter a mutation in CEP290, e.g., a 2991+1655 A to G. In an embodiment, such cells are introduced to the subject's body to prevent or treat LCA10.

In an embodiment, the population of cells are a population of retinual cells (e.g., retinal pigment epithelium cells), photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, or a combination thereof. In an embodiment, the method described herein comprises delivery of gRNA or other components described herein, e.g., a Cas9 molecule, by one or more AAV vectors, e.g., one or more AAV vectors described herein. I. gRNA Molecules

A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid. gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as "chimeric" gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below.

Several exemplary gRNA structures, with domains indicated thereon, are provided in Fig. 1. While not wishing to be bound by theory, with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in Fig. 1 and other depictions provided herein.

In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5' to

3':

a targeting domain (which is complementary to a target nucleic acid in the CEP290 gene, e.g., a targeting domain from any of Tables 1A-1D, Tables 2A- 2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A- 7D, Tables 8A-8E, Tables 9A-9B, or Table 10);

a first complementarity domain;

a linking domain;

a second complementarity domain (which is complementary to the first

complementarity domain);

a proximal domain; and

optionally, a tail domain.

In an embodiment, a modular gRNA comprises:

a first strand comprising, preferably from 5' to 3' ; a targeting domain (which is complementary to a target nucleic acid in the CEP290 gene, e.g., a targeting domain from Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10); and

a first complementarity domain; and

a second strand, comprising, preferably from 5' to 3':

optionally, a 5' extension domain;

a second complementarity domain;

a proximal domain; and

optionally, a tail domain.

The domains are discussed briefly below:

The Targeting Domain

Figs. 1A-1G provide examples of the placement of targeting domains.

The targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in an embodiment, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises in the 5' to 3' direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50 nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section VIII herein.

In an embodiment, the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain is 17 nucleotides in length. In an embodiment, the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

Targeting domains are discussed in more detail below.

The First Complementarity Domain

Figs. 1A-1G provide examples of first complementarity domains.

The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first

complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first

complementary domain is 7 to 25 nucleotides in length. In an embodiment, the first

complementary domain is 7 to 22 nucleotides in length. In an embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an embodiment, the first

complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In an embodiment, the first complentarity domain comprises 3 subdomains, which, in the

5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3' subdomain is 3 to 25, e.g., 4-22, 4-18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, nucleotides in length.

The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus, or S. thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section VIII herein.

First complementarity domains are discussed in more detail below.

The Linking Domain

Figs. 1A-1G provide examples of linking domains.

A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains, see, e.g., Figs. IB-IE. In an embodiment, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. Typically the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

In modular gRNA molecules the two molecules are associated by virtue of the

hybridization of the complementarity domains see e.g., Fig. 1A. A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length. In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5' to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section VIII herein.

Linking domains are discussed in more detail below.

The 5' Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5' to the second complementarity domain, referred to herein as the 5' extension domain, see, e.g., Fig. 1A. In an embodiment, the 5' extension domain is, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length. In an embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. The Second Complementarity Domain

Figs. 1A-1G provide examples of second complementarity domains.

The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, e.g., as shown in Figs. 1A-1B, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.

In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region. In an embodiment the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the second complentarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 tol8, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.

In an embodiment, the 5' subdomain and the 3' subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.

The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, S.

aureus, or S. thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section VIII herein.

A Proximal domain

Figs. 1A-1G provide examples of proximal domains.

In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, S. aureus, or S. thermophilus, proximal domain.

Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section VIII herein. A Tail Domain

Figs. 1A-1G provide examples of tail domains. As can be seen by inspection of the tail domains in Figs. 1A and 1B-1F, a broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiment, the tail domain nucleotides are from or share homology with sequence from the 5' end of a naturally occurring tail domain, see e.g., Fig. ID or IE. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.

In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, S. aureus, or S. thermophilus, tail domain.

In an embodiment, the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3' end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When alternate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.

The domains of gRNA molecules are described in more detail below. The Targeting Domain

The "targeting domain" of the gRNA is complementary to the "target domain" on the target nucleic acid. The strand of the target nucleic acid comprising the core domain target is referred to herein as the "complementary strand" of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al., Nat Biotechnol 2014 (doi:

10.1038/nbt.2808) and Sternberg SH et al, Nature 2014 (doi: 10.1038/naturel3011).

In an embodiment, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain is 19 nucleotides in length. In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

In an embodiment, the targeting

5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.

In an embodiment, the targeting domain is 20+/-5 nucleotides in length.

In an embodiment, the targeting domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.

n an embodimen tt,, the targeting domain is 30+/- 10 nucleotides in length,

n an embodimen tt,, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other

embodiments, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

Typically the targeting domain has full complementarity with the target sequence. In some embodiments the targeting domain has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end.

In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end.

In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In some embodiments, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain ("non-complementary nucleotides"), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.

In an embodiment, there are no noncomplementary nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other

modification(s) from Section VIII. In an embodiment, a nucleotide of the targeting domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII.

In some embodiments, the targeting domain includes 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the targeting domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.

In some embodiments, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.

Modifications in the targeting domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV gRNA's having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section IV. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In some embodiments, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In other embodiments, 1, 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.

In an embodiment, the targeting domain comprises, preferably in the 5'→3' direction: a secondary domain and a core domain. These domains are discussed in more detail below. The Core Domain and Secondary Domain of the Targeting Domain

The "core domain" of the targeting domain is complementary to the "core domain target" on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain).

In an embodiment, the secondary domain is absent or optional.

In an embodiment, the core domain and targeting domain, are independently, 6 +1-2, 1+1- 2, 8+/-2, 9+/-2, 10+/-2, 11+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, 16+-2, 17+/-2, or 18+/-2, nucleotides in length.

In an embodiment, the core domain and targeting domain, are independently, 10+/-2 nucleotides in length.

In an embodiment, the core domain and targeting domain, are independently, 10+/-4 nucleotides in length.

In an embodiment, the core domain and targeting domain, are independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, nucleotides in length.

In an embodiment, the core domain and targeting domain, are independently 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20 10 to 20 or 15 to 20 nucleotides in length.

In an embodiment, the core domain and targeting domain, are independently 3 to 15, e.g., 6 to 15, 7 to 14, 7 to 13, 6 to 12, 7 to 12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10 or 8 to 9 nucleotides in length.

The core domain is complementary with the core domain target. Typically the core domain has exact complementarity with the core domain target. In some embodiments, the core domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

The "secondary domain" of the targeting domain of the gRNA is complementary to the "secondary domain target" of the target nucleic acid.

In an embodiment, the secondary domain is positioned 5' to the core domain.

In an embodiment, the secondary domain is absent or optional. In an embodiment, if the targeting domain is 26 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.

In an embodiment, if the targeting domain is 25 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.

In an embodiment, if the targeting domain is 24 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 11 to 16 nucleotides in length.

In an embodiment, if the targeting domain is 23 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 15 nucleotides in length.

In an embodiment, if the targeting domain is 22 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.

In an embodiment, if the targeting domain is 21 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.

In an embodiment, if the targeting domain is 20 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.

In an embodiment, if the targeting domain is 19 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 11 nucleotides in length.

In an embodiment, if the targeting domain is 18 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.

In an embodiment, if the targeting domain is 17 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length. In an embodiment, if the targeting domain is 16 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.

In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides in length.

The secondary domain is complementary with the secondary domain target. Typically the secondary domain has exact complementarity with the secondary domain target. In some embodiments the secondary domain can have 1, 2, 3, 4 or 5 nucleotides that are not

complementary with the corresponding nucleotide of the secondary domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the core domain comprise one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the core domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII. Typically, a core domain will contain no more than 1, 2, or 3 modifications.

Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNA's having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the secondary domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII. Typically, a secondary domain will contain no more than 1, 2, or 3 modifications.

Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNA's having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, (1) the degree of complementarity between the core domain and its target, and (2) the degree of complementarity between the secondary domain and its target, may differ. In an embodiment, (1) may be greater (2). In an embodiment, (1) may be less than (2). In an embodiment, (1) and (2) are the same, e.g., each may be completely complementary with its target.

In an embodiment, (1) the number of modification (e.g., modifications from Section VIII) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section VIII) of the nucleotides of the secondary domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be free of modifications.

The First and Second Complementarity Domains

The first complementarity domain is complementary with the second complementarity domain.

Typically the first domain does not have exact complementarity with the second complementarity domain target. In some embodiments, the first complementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain. In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1, 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5' end of the second complementarity domain.

In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.

In an embodiment, the first and second complementarity domains are:

independently, 6 +1-2, 1+1-2, 8+/-2, 9+1-2, 10+/-2, 11+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, 16+/-2, 17+/-2, 18+/-2, 19+/-2, or 20+/-2, 21+/-2, 22+/-2, 23+/-2, or 24+/-2 nucleotides in length;

independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, nucleotides in length; or

independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.

In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.

In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section VIII.

In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the domain can comprise a 2'

modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII.

In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second

complementary domains, independently, include 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the first and second complementary domains, independently, include as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.

In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.

Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNA's having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section IV. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the first complementarity domain has at least 60, 70, 80, 85%, 90% or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus, or S. thermophilus, first complementarity domain, or a first complementarity domain described herein, e.g., from Figs. 1A-1G.

In an embodiment, the second complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus, or S. thermophilus, second complementarity domain, or a second complementarity domain described herein, e.g., from Fig. 1A-1G.

The duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).

In some embodiments, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded): NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAA UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 5).

In some embodiments, the first and second complementarity domains, when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGCAUAGCA AGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG

C (SEQ ID NO: 27).

In some embodiments the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAG CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGC (SEQ ID NO: 28).

In some embodiments the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGGAAACAAA ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO: 29).

In some embodiments, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):

NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGUUAAU _. AU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:

30) ;

NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGUUUAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:

31) ; and NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGGAAACAAU ACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO: 32). The 5' Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5' to the second complementarity domain. In an embodiment, the 5' extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

In an embodiment, the 5' extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the 5' extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5' extension domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment, a nucleotide of the 5' extension domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other

modification(s) from Section VIII.

In some embodiments, the 5' extension domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.

In some embodiments, the 5' extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or more than 5 nucleotides away from one or both ends of the 5' extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5' extension domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5' extension domain.

Modifications in the 5' extension domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNA's having a candidate 5' extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate 5' extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the 5' extension domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference 5' extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus, or S. thermophilus, 5' extension domain, or a 5' extension domain described herein, e.g., from Figs. 1A-1G. The Linking Domain

In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.

In an embodiment, the linking domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.

In an embodiment, the linking domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.

In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other embodiments, the linking domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

In an embodiment, the linking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.

In an embodiment, the linking domain is a covalent bond.

In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1, 2, or 3 nucleotides of the 3' end of the first complementarity domain and/or the 5- end of the second complementarity domain. In an embodiment, the duplexed region can be 20+/-10 base pairs in length. In an embodiment, the duplexed region can be 10+/-5, 15+/-5, 20+/-5, or 30+/-5 base pairs in length. In an embodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length.

Typically the sequences forming the duplexed region have exact complementarity with one another, though in some embodiments as many as 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.

In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the linking domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other

modification(s) from Section VIII. In an embodiment a nucleotide of the linking domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII. In some embodiments, the linking domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.

Modifications in a linking domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNA's having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section IV. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the linking domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5 ,or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein, e.g., from Figs. 1A-1G.

The Proximal Domain

In an embodiment, the proximal domain is 6 +1-2, 7+/-2, 8+/-2, 9+/-2, 10+/-2, 11+/-2, 12+/-2, 13+/-2, 14+/-2, 14+/-2, 16+/-2, 17+/-2, 18+/-2, 19+/-2, or 20+/-2 nucleotides in length. In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, or 20 nucleotides in length.

In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.

In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other

modification(s) from Section VIII. In an embodiment a nucleotide of the proximal domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII.

In some embodiments, the proximal domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.

In some embodiments, the proximal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.

Modifications in the proximal domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNA's having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In an embodiment, the proximal domain has at least 60, 70, 80, 85 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus, or S. thermophilus, proximal domain, or a proximal domain described herein, e.g., from Figs. 1A-1G.

The Tail Domain

In an embodiment, the tail domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5,

70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.

In an embodiment, the tail domain is 20+/-5 nucleotides in length.

In an embodiment, the tail domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.

In an embodiment, the tail domain is 25+/- 10 nucleotides in length.

In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.

In other embodiments, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.

In an embodiment, the tail domain is 1 to 20, 1 to 1, 1 to 10, or 1 to 5 nucleotides in length.

In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the tail domain can comprise a 2' modification, e.g., a 2-acetylation, e.g., a 2' methylation, or other modification(s) from Section VIII.

In some embodiments, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.

In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In an embodiment, a further single stranded domain, exists 3' to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment it is 4 to 6 nucleotides in length.

In an embodiment, the tail domain has at least 60, 70, 80, or 90% homology with, or differs by no more than 1, 2, 3, 4, 5 ,or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, tail domain, or a tail domain described herein, e.g., from Figs. 1A-1G.

In an embodiment, the proximal and tail domain, taken together comprise the following sequences:

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO: 33),

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC

(SEQ ID NO: 34),

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAU

C (SEQ ID NO: 35),

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO: 36),

AAGGCUAGUCCGUUAUCA (SEQ ID NO: 37), or

AAGGCUAGUCCG (SEQ ID NO: 38).

In an embodiment, the tail domain comprises the 3' sequence UUUUUU, e.g., if a U6 promoter is used for transcription.

In an embodiment, the tail domain comprises the 3' sequence UUUU, e.g., if an HI promoter is used for transcription.

In an embodiment, tail domain comprises variable numbers of 3' Us depending, e.g., on the termination signal of the pol-III promoter used.

In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template if a T7 promoter is used. In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.

In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template, e., if a pol-II promoter is used to drive transcription.

Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section IV. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.

In some embodiments, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.

In an embodiment a gRNA has the following structure:

5' [targeting domain] -[first complementarity domain] -[linking domain] -[second complementarity domain] -[proximal domain] -[tail domain] -3'

wherein, the targeting domain comprises a core domain and optionally a secondary domain, and is 10 to 50 nucleotides in length;

the first complementarity domain is 5 to 25 nucleotides in length and, In an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference first complementarity domain disclosed herein;

the linking domain is 1 to 5 nucleotides in length; the proximal domain is 5 to 20 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference proximal domain disclosed herein; and the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference tail domain disclosed herein.

Exemplary Chimeric gRNAs

In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5' to

3':

a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (which is complementary to a target nucleic acid);

a first complementarity domain;

a linking domain;

a second complementarity domain (which is complementary to the first complementarity domain);

a proximal domain; and

a tail domain,

wherein,

(a) the proximal and tail domain, when taken together, comprise

at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;

(b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or

(c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is

complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides

(e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides

(e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides

(e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides

(e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides

(e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides

(e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides

(e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides

(e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides

(e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number: NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (SEQ ID NO: 480). In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. pyogenes gRNA molecule.

In some embodiments, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number: NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUAAAAC AAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU (SEQ ID NO: 481). In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. aureus gRNA molecule.

The sequences and structures of exemplary chimeric gRNAs are also shown in Figs. 18A-18B.

Exemplary Modular gRNAs

In an embodiment, a modular gRNA comprises:

a first strand comprising, preferably from 5' to 3' :

a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;

a first complementarity domain; and a second strand, comprising, preferably from 5' to 3':

optionally a 5' extension domain;

a second complementarity domain;

a proximal domain; and

a tail domain,

wherein:

(a) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;

(b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or

(c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is

complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides

(e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides

(e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length. In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides

(e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides

(e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides

(e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain. In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides

(e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain. In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides

(e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides

(e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides

(e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40,

45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41,

46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.

II. Methods for Designing gRNAs

Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein.

Targeting Domains discussed herein can be incorporated into the gRNAs described herein.

Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in Mali et al., 2013 Science 339(6121): 823-826; Hsu et al. Nat Biotechnol, 31(9): 827-32; Fu et al., 2014 Nat Biotechnol, doi: 10.1038/nbt.2808. PubMed PMID: 24463574;

Heigwer et al., 2014 Nat Methods l l(2): 122-3. doi: 10.1038/nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 Bioinformatics PubMed PMID: 24463181; Xiao A et al., 2014 Bioinformatics PubMed PMID: 24389662.

For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice using S. pyogenes Cas9, software tools can identify all potential off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA can then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for gRNA vector construction, primer design for the on-target

Surveyor assay, and primer design for high-throughput detection and quantification of off -target cleavage via next-generation sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein.

Guide RNAs (gRNAs) for use with S. pyogenes, S. aureus and N. meningitidis Cas9s were identified using a DNA sequence searching algorithm. Guide RNA design was carried out using a custom guide RNA design software based on the public tool cas-offinder (Bae et al. Bioinformatics. 2014; 30(10): 1473-1475). Said custom guide RNA design software scores guides after calculating their genomewide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the off-target sites are computationally determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. Genomic DNA sequence for each gene was obtained from the UCSC Genome browser and sequences were screened for repeat elements using the publically available RepeatMasker program.

RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.

Following identification, gRNAs were ranked into tiers based on their distance to the target site, their orthogonality and presence of a 5' G (based on identification of close matches in the human genome containing a relavant PAM, e.g., in the case of S. pyogenes, a NGG PAM, in the case of S. aureus, NNGRR (e.g, a NNGRRT or NNGRRV) PAM, and in the case of N.

meningitides, a NNNNGATT or NNNNGCTT PAM. Orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A "high level of orthogonality" or "good orthogonality" may, for example, refer to 20-mer gRNAs that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.

As an emxaple, for S. pyogenes and N. meningitides targets, 17-mer, or 20-mer gRNAs were designed. As another example, for S. aureus targets, 18-mer, 19-mer, 20-mer, 21-mer, 22- mer, 23-mer and 24-mer gRNAs were designed. Tarteting domains, disclosed herein, may comprises the 17-mer described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A- 4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 18 or more nucleotides may comprise the 17-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 18-mer described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 19 or more nucleotides may comprise the 18-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A- 5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 19-mer described in Tables 1A-1D, Tables 2A- 2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A- 8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 20 or more nucleotides may comprise the 19-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 20-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 21 or more nucleotides may comprise the 20-mer gRNAs described in Tables 1A-1D, Tables 2A-2C,

Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 21-mer described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 22 or more nucleotides may comprise the 21-mer gRNAs described in Tables 1A- ID, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A- 7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 22-mer described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A- 4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 23 or more nucleotides may comprise the 22-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 23-mer described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 24 or more nucleotides may comprise the 23-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A- 5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10. Tarteting domains, disclosed herein, may comprises the 24-mer described in Tables 1A-1D, Tables 2A- 2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A- 8E, Tables 9A-9B, or Table 10, e.g., the targeting domains of 25 or more nucleotides may comprise the 24-mer gRNAs described in Tables 1A-1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A-7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

gRNAs were identified for both single-gRNA nuclease cleavage and for a dual-gRNA paired "nickase" strategy. Criteria for selecting gRNAs and the determination for which gRNAs can be used for the dual-gRNA paired "nickase" strategy is based on two considerations:

1. gRNA pairs should be oriented on the DNA such that PAMs are facing out and

cutting with the D10A Cas9 nickase will result in 5' overhangs.

2. An assumption that cleaving with dual nickase pairs will result in deletion of the

entire intervening sequence at a reasonable frequency. However, cleaving with dual nickase pairs can also result in indel mutations at the site of only one of the gRNAs. Candidate pair members can be tested for how efficiently they remove the entire sequence versus causing indel mutations at the site of one gRNA.

The Targeting Domains discussed herein can be incorporated into the gRNAs described herein.

Three strategies were utilized to identify gRNAs for use with S. pyogenes, S. aureus and

N. meningitidis Cas9 enzymes.

In one strategy, gRNAs were designed for use with S. pyogenes and S. aureus Cas9 enzymes to induce an indel mediated by NHEJ in close proximity to or including the LCA10 target position (e.g., C.2991+1655A to G). The gRNAs were identified and ranked into 4 tiers for S. pyogenes (Tables 1A-1D). The targeting domain for tier 1 gRNA molecules to be used with S. pyogenes Cas9 molecules were selected based on (1) a short distance to the target position, e.g., within 40bp upstream and 40bp downstream of the mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a short distance and high orthogonality were required but the presence of a 5'G was not required. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G. The gRNAs were identified and ranked into 4 tiers for S. aureus, when the relavent PAM was NNGRR (Tables 2A-2C). The targeting domain for tier 1 gRNA molecules to be used with S. pyogenes Cas9 molecules were selected based on (1) a short distance to the target position, e.g., within 40 bp upstream and 40 bp downstream of the mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a short distance and high orthogonality were required but the presence of a 5'G was not required. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G. The gRNAs were identified and ranked into 5 tiers for S. aureus when the relavent PAM was NNGRRT or NNGRR V (Tables 6A-6D). The targeting domain for tier 1 gRNA molecules to be used with S. aureus Cas9 molecules were selected based on (1) a short distance to the target position, e.g., within 40 bp upstream and 40 bp downstream of the mutation, (2) a high level of orthogonality, (3) the presence of a 5' G and (4) PAM was NNGRRT. For selection of tier 2 gRNAs, a short distance and high orthogonality were required but the presence of a 5'G was not required, and PAM was NNGRRT. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality, and PAM was NNGRRT. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G, ang PAM was NNGRRT. Tier 5 required a short distance to the target position, e.g., within 40 bp upstream and 40 bp downstream of the mutation and PAM was NNGRRV. Note that tiers are non-inclusive (each gRNA is listed only once for the strategy). In certain instances, no gRNA was identified based on the criteria of the particular tier.

In a second strategy, gRNAs were designed for use with S. pyogenes, S. aureus and N. meningitidis Cas9 molecules to delete a genomic sequence including the mutation at the LCA10 target position (e.g., C.2991+1655A to G), e.g., mediated by NHEJ. The gRNAs were identified and ranked into 4 tiers for S. pyogenes (Tables 3A-3D). The targeting domain to be used with S. pyogenes Cas9 molecules for tier 1 gRNA molecules were selected based on (1) flanking the mutation without targeting unwanted chromosome elements, such as an Alu repeat, e.g., within 400bp upstream of an Alu repeat or 700 bp downstream of mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a reasonable distance and high orthogonality were required but the presence of a 5'G was not required. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G. The gRNAs were identified and ranked into 4 tiers for S.

aureus, when the relavent PAM was NNGRR (Tables 4A-4D). The targeting domain to be used with S. aureus Cas9 molecules for tier 1 gRNA molecules were selected based on (1) flanking the mutation without targeting unwanted chromosome elements, such as an Alu repeat, e.g., within 400 bp upstream of an Alu repeat or 700 bp downstream of mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a reasonable distance and high orthogonality were required but the presence of a 5'G was not required. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G. The gRNAs were identified and ranked into 2 tiers for N.

meningitides (Tables 5A-5B). The targeting domain to be used with N. meningitides Cas9 molecules for tier 1 gRNA molecules were selected based on (1) flanking the mutation without targeting unwanted chromosome elements, such as an Alu repeat, e.g., within 400bp upstream of an Alu repeat or 700 bp downstream of mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a reasonable distance and high orthogonality were required but the presence of a 5'G was not required. Note that tiers are non-inclusive (each gRNA is listed only once for the strategy). In certain instances, no gRNA was identified based on the criteria of the particular tier.In a third strategy, gRNAs were designed for use with S. pyogenes, S. aureus and N. meningitidis Cas9 molecules to delete a genomic sequence including the mutation at the LCA10 target position (e.g., C.2991+1655A to G), e.g., mediated by NHEJ. The gRNAs were identified and ranked into 4 tiers for S. pyogenes (Tables 7A-7D). The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) flanking the mutation without targeting unwanted chromosome elements, such as an Alu repeat, e.g., within 1000 bp upstream of an Alu repeat or 1000 bp downstream of mutation, (2) a high level of orthogonality, (3) the presence of a 5' G and (4) and PAM was NNGRRT. For selection of tier 2 gRNAs, a reasonable distance and high orthogonality were required but the presence of a 5'G was not required, and and PAM was NNGRRT. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality, and and PAM was NNGRRT. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G, and and PAM was NNGRRT. The gRNAs were identified and ranked into 4 tiers for S. aureus, when the relavent PAM was NNGRRT or NNGRRV (Tables 8A-8E). The targeting domain to be used with S. aureus Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) flanking the mutation without targeting unwanted chromosome elements, such as an Alu repeat, e.g., within 1000 bp upstream of an Alu repeat or lOOObp downstream of mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a reasonable distance and high orthogonality were required but the presence of a 5'G was not required. Tier 3 uses the same distance restriction and the requirement for a 5'G, but removes the requirement of good orthogonality. Tier 4 uses the same distance restriction but removes the requirement of good orthogonality and the 5'G. Tier 5 used the same distance restriction and PAM was NNGRRV. The gRNAs were identified and ranked into 2 tiers for N. meningitides (Tables 9A-9B). The targeting domain to be used with N. meningitides Cas9 molecules for tier 1 gRNA molecules were selected based on (1) flanking the mutation without targeting unwanted chromosome elements, such as an Alu repeat, e.g., within lOOObp upstream of an Alu repeat or 1000 bp downstream of mutation, (2) a high level of orthogonality, and (3) the presence of a 5' G. For selection of tier 2 gRNAs, a reasonable distance and high orthogonality were required but the presence of a 5'G was not required. Note that tiers are non-inclusive (each gRNA is listed only once for the strategy). In certain instances, no gRNA was identified based on the criteria of the particular tier.

In an embodiment, when a single gRNA molecule is used to target a Cas9 nickase to create a single strand break to introduce a break-induced indel in close proximity to or including the LCA10 target position, the gRNA is used to target either upstream of (e.g., within 40 bp upstream of the LCA10 target position), or downstream of (e.g., within 40 bp downstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, when a single gRNA molecule is used to target a Cas9 nuclease to create a double strand break to introduce a break-induced indel in close proximity to or including the LCA10 target position, the gRNA is used to target either upstream of (e.g., within 40 bp upstream of the LCA10 target position), or downstream of (e.g., within 40 bp downstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting is used to create two double strand breaks to delete a genomic sequence including the mutation at the LCA10 target position, e.g., mediated by NHEJ. In an embodiment, the first and second gRNAs are used target two Cas9 nucleases to flank, e.g., the first of gRNA is used to target upstream of (e.g., within 400 bp upstream of the Alu repeat, or within 40 bp upstream of the LCA10 target position), and the second gRNA is used to target downstream of (e.g., within 700 bp downstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting is used to create a double strand break and a pair of single strand breaks to delete a genomic sequence including the mutation at the LCA10 target position, e.g., mediated by NHEJ. In an embodiment, the first, second and third gRNAs are used to target one Cas9 nuclease and two Cas9 nickases to flank, e.g., the first gRNA that will be used with the Cas9 nuclease is used to target upstream of (e.g., within 400 bp upstream of the Alu repeat, or within 40 bp upstream of the LCA10 target position) or downstream of (e.g., within 700 bp downstream) of the LCA10 target position, and the second and third gRNAs that will be used with the Cas9 nickase pair are used to target the opposite side of the LCA10 target position (e.g., within 400 bp upstream of the Alu repeat, within 40 bp upstream of the LCA10 target position, or within 700 bp downstream of the LCA10 target position) in the CEP290 gene. In an embodiment, when four gRNAs (e.g., two pairs) are used to target four Cas9 nickases to create four single strand breaks to delete genomic sequence including the mutation at the LCA10 target position, e.g., mediated by NHEJ, the first pair and second pair of gRNAs are used to target four Cas9 nickases to flank, e.g., the first pair of gRNAs are used to target upstream of (e.g., within 400 bp upstream of the Alu repeat, or within 40 bp upstream of the LCA10 target position), and the second pair of gRNAs are used to target downstream of (e.g., within 700 bp downstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting is utilized to delete genomic sequence including the mutation at the LCA10 target position mediated by NHEJ. It is contemplated herein that in an embodiment any upstream gRNA (e.g., within 400 bp upstream of an Alu repeat, or within 40bp upstream of the LCA10 target position) in Tables 1A-1C and Tables 3A-3D can be paired with any downstream gRNA (e.g., within 700 downstream of LCA10 target position) in Tables 3A- 3D to be used with a S. pyogenes Cas9 molecule to generate dual targeting. Exemplary pairs including selecting a targeting domain that is labeled as upstream from Tables 1A-1C or Tables 3A-3D and a second targeting domain that is labeled as downstream from Tables 3A-3D. In an embodiment, a targeting domain that is labeled as upstream in Tables 1A-1C or Tables 3A-3D can be combined with any of the targeting domains that is labeled as downstream in Tables 3A- 3D.

In an embodiment, dual targeting is utilized to delete genomic sequence including the mutation at the LCA10 target position mediated by NHEJ. It is contemplated herein that in an embodiment any upstream gRNA (e.g., within 400 bp upstream of an Alu repeat, or within 40bp upstream of the LCA10 target position) in Tables 2A-2C and Tables 4A-4D can be paired with any downstream gRNA (e.g., within 700 downstream of LCA10 target position) in Tables 4A- 4D to be used with a S. aureus Cas9 molecule to generate dual targeting. Exemplary pairs include selecting a targeting domain that is labeled as upstream from Tables 2A-2C or Tables 4A-4D and a second targeting domain that is labeled as downstream from Tables 4A-4D. In an embodiment, a targeting domain that is labeled as upstream in Tables 2A-2C or Tables 4A-4D can be combined with any of the targeting domains that is labeled as downstream in Tables 4A- 4D.

In an embodiment, dual targeting is utilized to delete genomic sequence including the mutation at the LCA10 target position mediated by NHEJ. It is contemplated herein that in an embodiment any upstream gRNA (e.g., within 400 bp upstream of an Alu repeat, or within 40 bp upstream of the LCAIO target position) in Tables 5A-5B can be paired with any downstream gRNA (e.g., within 700 downstream of LCAIO target position) in Tables 5A-5B to be used with a N. meningitidis Cas9 molecule to generate dual targeting. Exemplary pairs include selecting a targeting domain that is labeled as upstream from Tables 5A-5B and a second targeting domain that is labeled as downstream from Tables 5A-5B. In an embodiment, a targeting domain that is labeled as upstream in Tables 5A-5B can be combined with any of the targeting domains that is labeled as downstream in Tables 5A-5B.

In an embodiment, dual targeting (e.g., dual double strand cleavage) is used to create two double strand breaks to delete a genomic sequence including the mutation at the LCAIO target position, e.g., mediated by NHEJ. In an embodiment, the first and second gRNAs are used target two Cas9 nucleases to flank, e.g., the first of gRNA is used to target upstream of (e.g., within 1000 bp upstream of the Alu repeat, or within 40 bp upstream of the LCAIO target position), and the second gRNA is used to target downstream of (e.g., within 1000 bp downstream of the LCA 10 target position) in the CEP290 gene.

In an embodiment, dual targeting (e.g., dual double strand cleavage) is used to create a double strand break and a pair of single strand breaks to delete a genomic sequence including the mutation at the LCAIO target position, e.g., mediated by NHEJ. In an embodiment, the first, second and third gRNAs are used to target one Cas9 nuclease and two Cas9 nickases to flank, e.g., the first gRNA that will be used with the Cas9 nuclease is used to target upstream of (e.g., within 1000 bp upstream of the Alu repeat, or within 40bp upstream of the LCAIO target position) or downstream of (e.g., within 1000 bp downstream) of the LCAIO target position, and the second and third gRNAs that will be used with the Cas9 nickase pair are used to target the opposite side of the LCAIO target position (e.g., within 1000 bp upstream of the Alu repeat, or within 40bp upstream of the LCAIO target position or within 1000 bp downstream of the LCAIO target position) in the CEP290 gene.

In an embodiment, when four gRNAs (e.g., two pairs) are used to target four Cas9 nickases to create four single strand breaks to delete genomic sequence including the mutation at the LCAIO target position, e.g., mediated by NHEJ, the first pair and second pair of gRNAs are used to target four Cas9 nickases to flank, e.g., the first pair of gRNAs are used to target upstream of (e.g., within 1000 bp upstream of the Alu repeat, or within 40bp upstream of the LCAIO target position), and the second pair of gRNAs are used to target downstream of (e.g., within 1000 bp downstream of the LCAIO target position) in the CEP290 gene.

In an embodiment, dual targeting is utilized to delete genomic sequence including the mutation at the LCAIO target position, e.g., mediated by NHEJ. It is contemplated herein that in an embodiment any upstream gRNA (e.g., within 1000 bp upstream of an Alu repeat, or within 40bp upstream of the LCAIO target position) in Tables 1A-1C, Tables 3A-3D, or Tables 7A-7D can be paired with any downstream gRNA (e.g., within 1000 downstream of LCAIO target position) in Tables 1A-1C, Tables 3A-3D, or Tables 7A-7D to be used with a S. pyogenes Cas9 molecule to generate dual targeting. Exemplary pairs including selecting a targeting domain that is labeled as upstream from Tables 1A-1C, Tables 3A-3D, or Tables 7A-7D and a second targeting domain that is labeled as downstream from Tables 1A-1C, Tables 3A-3D, or Tables 7A-7D. In an embodiment, a targeting domain that is labeled as upstream in Tables 1A-1C, Tables 3A-3D, or Tables 7A-7D can be combined with any of the targeting domains that is labeled as downstream in Tables 1A-1C, Tables 3A-3D, or Tables 7A-7D.

In an embodiment, dual targeting is utilized to delete genomic sequence including the mutation at the LCAIO target position mediated by NHEJ. It is contemplated herein that in an embodiment any upstream gRNA (e.g., within 1000 bp upstream of an Alu repeat, or within 40bp upstream of the LCAIO target position) in Tables 2A-2C, Tables 4A-4D, Tables 6A-6D, or Tables 8A-8E can be paired with any downstream gRNA (e.g., within 1000 downstream of LCAIO target position) in Tables 2A-2C, Tables 4A-4D, Tables 6A-6D, or Tables 8A-8E to be used with a S. aureus Cas9 molecule to generate dual targeting. Exemplary pairs include selecting a targeting domain that is labeled as upstream from Tables 2A-2C, Tables 4A-4D, Tables 6A-6D, or Tables 8A-8E and a second targeting domain that is labeled as downstream from Tables 2A-2C, Tables 4A-4D, Tables 6A-6D, or Tables 8A-8E. In an embodiment, a targeting domain that is labeled as upstream in Tables 2A-2C, Tables 4A-4D, Tables 6A-6D, or Tables 8A-8E can be combined with any of the targeting domains that is labeled as downstream in Tables 2A-2C, Tables 4A-4D, Tables 6A-6D, or Tables 8A-8E.

In an embodiment, dual targeting is utilized to delete genomic sequence including the mutation at the LCAIO target position, e.g., mediated by NHEJ. It is contemplated herein that in an embodiment any upstream gRNA (e.g., within 1000 bp upstream of an Alu repeat, or within 40bp upstream of the LCAIO target position) in Tables 5A-5B or Tables 9A-9B can be paired with any downstream gRNA (e.g., within 1000 downstream of LCAIO target position) in Tables 5A-5D to be used with a N. meningitidis Cas9 molecule to generate dual targeting. Exemplary pairs include selecting a targeting domain that is labeled as upstream from Tables 5A-5B or Tables 9A-9B and a second targeting domain that is labeled as downstream from Tables 5A-5B or Tables 9A-9B. In an embodiment, a targeting domain that is labeled as upstream in Tables 5A-5B or Tables 9A-9B and can be combined with any of the targeting domains that is labeled as downstream in Tables 5A-5B or Tables 9A-9B.

Any of the targeting domains in the tables described herein can be used with a Cas9 nickase molecule to generate a single strand break.

Any of the targeting domains in the tables described herein can be used with a Cas9 nuclease molecule to generate a double strand break.

In an embodiment, dual targeting (e.g., dual nicking) is used to create two nicks on opposite DNA strands by using S. pyogenes, S. aureus and N. meningitidis Cas9 nickases with two targeting domains that are complementary to opposite DNA strands, e.g., a gRNA comprising any minus strand targeting domain may be paired any gRNA comprising a plus strand targeting domain provided that the two gRNAs are oriented on the DNA such that PAMs face outward and the distance between the 5' ends of the gRNAs is 0-50bp. Exemplary nickase pairs including selecting a targeting domain from Group A and a second targeting domain from Group B in Table ID (for S. pyogenes), or selecting a targeting domain from Group A and a second targeting domain from Group B in Table 6D (for S. aureus). It is contemplated herein that in an embodiment a targeting domain of Group A can be combined with any of the targeting domains of Group B in Table ID (for S. pyogenes). For example, CEP290-B5 or CEP290-B10 can be combined with CEP290-B1 or CEP290-B6. It is contemplated herein that in an embodiment a targeting domain of Group A can be combined with any of the targeting domains of Group B in Table 6D (for S. aureus). For example, CEP290-12 or CEP290-17 can be combined with CEP290-11 or CEP290-16.

In an embodiment, dual targeting (e.g., dual double strand cleavage) is used to create two double strand breaks by using S. pyogenes, S. aureus and N. meningitidis Cas9 nucleases with two targeting domains. It is contemplated herein that in an embodiment any upstream gRNA of any of Tables 1A-1C, 2A-2C, 3A-3D, 4A-4D, 5A-5B, 6A-6C, 7A-7D, 8A-8E, or 9A-9B can be paired with any downstream gRNA of any of Tables 1A-1C, 2A-2C, 3A-3D, 4A-4D, 5A-5B, 6A-6C, 7A-7D, 8A-8E, or 9A-9B. Exemplary nucleases pairs are shown in Table 10, e.g., CEP290-323 can be combined with CEP290-11, CEP290-323 can be combined with CEP290- 64, CEP290-490 can be combined with CEP290-496, CEP290-490 can be combined with CEP290-502, CEP290-490 can be combined with CEP290-504, CEP290-492 can be combined with CEP290-502, or CEP290-492 can be combined with CEP290-504.

It is contemplated herein that any upstream gRNA described herein may be paired with any downstream gRNA described herein. When an upstream gRNA designed for use with one species of Cas9 is paired with a downstream gRNA designed for use from a different species of Cas9, both Cas9 species are used to generate a single or double-strand break, as desired.

Exemplary Targeting Domains

Table 1A provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 40 bases of the LCAIO target position, have good orthogonality, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 1A

Table IB provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 40 bases of the LCAIO target position, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table IB DNA Target Site Position relative to gRNA Name Targeting Domain

Strand Length mutation

CEP290-B6 - CUCAUACCUAUCCCUAU 17 downstream

CEP290-B20 + ACACUGCCAAUAGGGAU 17 downstream

CEP290-B10 + CAAUUACAACUGGGGCC 17 upstream

CEP290-B21 + CUAAGACACUGCCAAUA 17 downstream

CEP290-B9 + AUACUCACAAUUACAAC 17 upstream

CEP290-B1 - UAUCUCAUACCUAUCCCUAU 20 downstream

CEP290-B29 + AAGACACUGCCAAUAGGGAU 20 downstream

CEP290-B5 + UCA CAAUUACAACUGGGGCC 20 upstream

CEP290-B30 + AGAUACUCACAAUUACAACU 20 upstream

Table 1C provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the fourth tier parameters. The targeting domains are within 40 bases of the LCAIO target position and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 1C

Table ID provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene that can be used for dual targeting. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 (nickase) molecule to generate a single stranded break. Exemplary nickase pairs including selecting a targeting domain from Group A and a second targeting domain from Group B. It is contemplated herein that a targeting domain of Group A can be combined with any of the targeting domains of Group B. For example, the CEP290-B5 or CEP290-B10 can be combined with CEP290-B1 or CEP290-B6.

Table ID

Table 2A provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 40 bases of the LCAIO target position, have good orthogonality, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 2A

Table 2B provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 40 bases of the LCAIO target position, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 2B CEP290-B1003 + UGCCAAUAGGGAUAGGU 17 downstream

CEP290-B1004 + UGAGAUACUCACAAUUACAA 20 upstream

CEP290-B1005 + AUACUCACAAUUACAAC 17 upstream

Table 2C provides targeting domains for NHEJ-mediated introduction of an indel in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the fourth tier parameters. The targeting domains are within 40 bases of the LCAIO target position, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

Table 2C

Table 3A provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, have good orthogonality, and start with G. It is

contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 3A

Table 3B provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCA10 target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 3B

Table 3C provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCA10 target position in the CEP290 gene selected according to the third tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 3C

Table 3D provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCA10 target position in the CEP290 gene selected according to the fourth tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 3D DNA Target Site gRNA Name Targeting Domain

Strand Length

CEP290-B233 + AAGGCGGGGAGUCACAU 17 downstream

CEP290-B175 + UAAGGCGGGGAGUCACA 17 downstream

CEP290-B280 + UGAACUCUGUGCCAAAC 17 downstream

CEP290-B92 + CUAAGACACUGCCAAUA 17 downstream

CEP290-B268 + UUUACCCUUCAGGUAAC 17 downstream

CEP290-B154 + UGACACCACAUGCACUG 17 downstream

CEP290-B44 + ACUAAGACACUGCCAAU 17 downstream

CEP290-B231 + UUGCUCUAGAUGACAUG 17 downstream

CEP290-B242 + UGACAG U U U U UAAGGCG 17 downstream

CEP290-B226 - UGUCAAAUAUGGUGCU U 17 downstream

CEP290-B159 + AGUCACAUGGGAGUCAC 17 downstream

CEP290-B222 - AUGAGAGUGAUUAGUGG 17 downstream

CEP290-B274 + UGACAUGAGGUAAGUAG 17 downstream

CEP290-B68 - UACAUGAGAGUGAUUAG 17 downstream

CEP290-B212 + UAAGGAGGAUGUAAGAC 17 downstream

CEP290-B270 + CU UGACUUUUACCCUUC 17 downstream

CEP290-B96 + UCACUGAGCAAAACAAC 17 downstream

CEP290-B104 + AGACUUAUAUUCCAU UA 17 downstream

CEP290-B122 + CAUGGGAGUCACAGGGU 17 downstream

CEP290-B229 + UAGAAUGAUCAUUCUUG 17 downstream

CEP290-B99 + UUGACAGUUUUUAAGGC 17 downstream

CEP290-B7 - AAACU G U CAAAAG C U AC 17 downstream

CEP290-B41 + UCAUUCUUGUGGCAGUA 17 downstream

CEP290-B37 + AUGACAUGAGGUAAGUA 17 downstream

CEP290-B97 - UGUUUCAAAACUUGU UC 17 downstream

CEP290-B173 - AUAUCUGUCUUCCUUAA 17 downstream

CEP290-B136 + UGAACAAG U U U UGAAAC 17 downstream

CEP290-B71 - UUCUGCAUCUUAUACAU 17 downstream

CEP290-B172 - AUAAGUCUUUUGAUAUA 17 downstream

CEP290-B238 + UUUGACAGUUUUUAAGG 17 downstream

CEP290-B148 - UGCUCUUUUCUAUAUAU 17 downstream

CEP290-B208 + AGACUGGAGAUAGAGAC 17 downstream

CEP290-B53 + CAUAAGAAAGAACACUG 17 downstream

CEP290-B166 + UUCUUGUGGCAGUAAGG 17 downstream

CEP290-B247 - AAGCAUACUUUUUUUAA 17 downstream

CEP290-B245 + CAACUGGAAGAGAGAAA 17 downstream

CEP290-B167 + UAUGCUUAAG A A A A A A A 17 downstream

CEP290-B171 - UUUUAUUAUCUUUAUUG 17 downstream

CEP290-B140 + CUAGAUGACAUGAGGUAAGU 20 downstream

CEP290-B147 + UUUUAAGGCGGGGAGUCACA 20 downstream

CEP290-B253 + AAGACACUGCCAAUAGGGAU 20 downstream CEP290-B73 - UCCUGUUUCAAAACUUGUUC 20 downstream

CEP290-B206 - UGUGUUGAGUAUCUCCUGUU 20 downstream

CEP290-B57 + CUCUUGCUCUAGAUGACAUG 20 downstream

CEP290-B82 + CAGUAAGGAGGAUGUAAGAC 20 downstream

CEP290-B265 + AGAUGACAUGAGGUAAGUAG 20 downstream

CEP290-B105 + AAUUCACUGAGCAAAACAAC 20 downstream

CEP290-B239 + UCACAUGGGAGUCACAGGGU 20 downstream

CEP290-B180 + UAGAUGACAUGAGGUAAGUA 20 downstream

CEP290-B103 + UUUUGACAGUUUUUAAGGCG 20 downstream

CEP290-B254 - UAAUACAUGAGAGUGAUUAG 20 downstream

CEP290-B134 - UAGUUCUGCAUCUUAUACAU 20 downstream

CEP290-B151 + AAACUAAGACACUGCCAAUA 20 downstream

CEP290-B196 + AAAACUAAGACACUGCCAAU 20 downstream

CEP290-B2 - UAAAAACUGUCAAAAGCUAC 20 downstream

CEP290-B240 + CU UUUGACAGU UUUUAAGGC 20 downstream

CEP290-B116 + AAAAGACUUAUAUUCCAUUA 20 downstream

CEP290-B39 + AUACAUAAGAAAGAACACUG 20 downstream

CEP290-B91 - AAUAUAAGUCUUUUGAUAUA 20 downstream

CEP290-B126 + UGAUCAUUCUUGUGGCAGUA 20 downstream

CEP290-B202 - UACAUAUCUGUCUUCCU UAA 20 downstream

CEP290-B152 - CUUAAGCAUACUU UUUUUAA 20 downstream

CEP290-B77 + AAACAACUGGAAGAGAGAAA 20 downstream

CEP290-B145 + UCAUUCUUGUGGCAGUAAGG 20 downstream

CEP290-B72 + AAGUAUGCUU A AG A A A A A A A 20 downstream

CEP290-B221 - AUUUUUUAUUAUCUUUAUUG 20 downstream

CEP290-B424 + CUAGGACUUUCUAAUGC 17 upstream

CEP290-B425 - AUCUAAGAUCCUU UCAC 17 upstream

CEP290-B426 + UUAUCACCACACUAAAU 17 upstream

CEP290-B427 - AGCUCAAAAGCUUUUGC 17 upstream

CEP290-B428 - UGUUCUGAGUAGCUUUC 17 upstream

CEP290-B429 + ACUUUCUAAUGCUGGAG 17 upstream

CEP290-B430 - CUCUAUACCUUUUACUG 17 upstream

CEP290-B431 + CAAGAUGUCUCUUGCCU 17 upstream

CEP290-B432 - AUUAUGCCUAUUUAGUG 17 upstream

CEP290-B433 + AUGACUCAUAAUUUAGU 17 upstream

CEP290-B434 - UAGAGGCUUAUGGAUU U 17 upstream

CEP290-B435 + UAUUCUACUCCUGUGAA 17 upstream

CEP290-B437 + CUAAUGCUGGAGAGGAU 17 upstream

CEP290-B438 - AGGCAAGAGACAUCUUG 17 upstream

CEP290-B439 + AGCCUCUAUU UCUGAUG 17 upstream

CEP290-B440 - CAG CA U U AG AAAG U CC U 17 upstream

CEP290-B441 - CUGCU UUUGCCAAAGAG 17 upstream CEP290-B442 + ACAUAAUCACCUCUCUU 17 upstream

CEP290-B443 - UCAGAAAUAGAGGCUUA 17 upstream

CEP290-B446 - UUCCUCAUCAGAAAUAG 17 upstream

CEP290-B447 + ACAGAGGACAUGGAGAA 17 upstream

CEP290-B448 + UGGAGAGGAUAGGACAG 17 upstream

CEP290-B449 + AGGAAGAUGAACAAAUC 17 upstream

CEP290-B450 + AGAUGAAAAAUACUCUU 17 upstream

CEP290-B455 + AGGACUUUCUAAUGCUGGAG 20 upstream

CEP290-B456 - AUUAGCUCAAAAGCUUUUGC 20 upstream

CEP290-B457 - CUCCAGCAUUAGAAAGUCCU 20 upstream

CEP290-B458 + AACAUGACUCAUAAUUUAGU 20 upstream

CEP290-B460 - AUCUUCCUCAUCAGAAAUAG 20 upstream

CEP290-B461 + AUAAGCCUCUAUU UCUGAUG 20 upstream

CEP290-B462 + UCUUAUUCUACUCCUGUGAA 20 upstream

CEP290-B463 - CUGCUGCUUUUGCCAAAGAG 20 upstream

CEP290-B464 + UUUCUAAUGCUGGAGAGGAU 20 upstream

CEP290-B466 + AAAUUAUCACCACACUAAAU 20 upstream

CEP290-B467 + CU UGUUCUGUCCUCAGUAAA 20 upstream

CEP290-B468 - AAAAUUAUGCCUAUUUAGUG 20 upstream

CEP290-B469 - UCAUCAGAAAUAGAGGCUUA 20 upstream

CEP290-B470 - AAAUAGAGGCUUAUGGAUUU 20 upstream

CEP290-B471 + UGCUGGAGAGGAUAGGACAG 20 upstream

CEP290-B472 + AUGAGGAAGAUGAACAAAUC 20 upstream

CEP290-B474 - CUUAUCUAAGAUCCUUUCAC 20 upstream

CEP290-B475 + AGAGGAUAGGACAGAGGACA 20 upstream

CEP290-B476 + AGGACAGAGGACAUGGAGAA 20 upstream

CEP290-B477 + AAAGAUGAAAAAUACUCUUU 20 upstream

CEP290-B495 - AGCUCAAAAGCUUUUGC 17 upstream

CEP290-B529 - UGUUCUGAGUAGCUUUC 17 upstream

CEP290-B513 + AUGACUCAUAAUUUAGU 17 upstream

CEP290-B490 + UAUUCUACUCCUGUGAA 17 upstream

CEP290-B485 - CUGCU UUUGCCAAAGAG 17 upstream

CEP290-B492 + ACAUAAUCACCUCUCUU 17 upstream

CEP290-B506 + AACAUGACUCAUAAUUUAGU 20 upstream

CEP290-B500 + UCUUAUUCUACUCCUGUGAA 20 upstream

CEP290-B521 - CUGCUGCUUUUGCCAAAGAG 20 upstream

Table 4A provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, have good orthogonality, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 4A

Table 4B provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 4B

CEP290-B1024 - CAAAAGCUACCGGUUAC 17 downstream

CEP290-B1025 + UAGGAAUCCUGAAAGCUACU 20 upstream

CEP290-B1026 + CAG AACAACG UUUUCAUUUA 20 upstream

CEP290-B1027 - CAAAAGCUUUUGCUGGCUCA 20 upstream

CEP290-B1028 + AGCAAAAGCUUUUGAGCUAA 20 upstream

CEP290-B1029 + AUCUUAUUCUACUCCUGUGA 20 upstream

CEP290-B1030 + AACAACG UUUUCAUU UA 17 upstream

CEP290-B1031 - AAGCUUUUGCUGGCUCA 17 upstream

CEP290-B1032 + AAAAGCUUUUGAGCUAA 17 upstream

CEP290-B1033 + UUAUUCUACUCCUGUGA 17 upstream

Table 4C provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the third tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 4C

CEP290-B1051 + GGGGAGUCACAUGGGAGUCA 20 downstream

CEP290-B1052 - GGUGAUUAUGUUACUUUUUA 20 upstream

CEP290-B1053 - GGUGAUUAUGUUACUUUUUA 20 upstream

CEP290-B1054 + GUAAGACUGGAGAUAGAGAC 20 downstream

CEP290-B1055 + GUCACAUGGGAGUCACAGGG 20 downstream

CEP290-B1056 - GUGGUGUCAAAUAUGGUGCU 20 downstream

CEP290-B1057 + G A A A A A A A AG G U A A U G C 17 downstream

CEP290-B1058 + GAAAAGAGCACGUACAA 17 downstream

CEP290-B1059 + GAAUCCUGAAAGCUACU 17 upstream

CEP290-B1060 - GAAUGAUCAUUCUAAAC 17 downstream

CEP290-B1061 + GACAGAGGACAUGGAGA 17 upstream

CEP290-B1062 + GACUUUCUAAUGCUGGA 17 upstream

CEP290-B1063 - GAGAGUGAUUAGUGGUG 17 downstream

CEP290-B1064 + GAGCAAAACAACUGGAA 17 downstream

CEP290-B1065 + GAGGAAGAUGAACAAAU 17 upstream

CEP290-B1066 + GAGUCACAUGGGAGUCA 17 downstream

CEP290-B1067 + GAUCUUAUUCUACUCCU 17 upstream

CEP290-B1068 + GAUCUUAUUCUACUCCU 17 upstream

CEP290-B1069 + GAUGAAAAAUACUCUUU 17 upstream

CEP290-B1070 + GAUGACAUGAGGUAAGU 17 downstream

CEP290-B1071 - GAUUAUGUUACUUUUUA 17 upstream

CEP290-B1072 - GAUUAUGUUACUUUUUA 17 upstream

CEP290-B1073 + GCAAAACAACUGGAAGA 17 downstream

CEP290-B1074 + GCAGAACUAGUGUAGAC 17 downstream

CEP290-B1075 - GCUCUUUUCUAUAUAUA 17 downstream

CEP290-B1076 + GGAUAGGACAGAGGACA 17 upstream

CEP290-B1077 + GGAUGUAAGACUGGAGA 17 downstream

CEP290-B1078 + GUAAGGAGGAUGUAAGA 17 downstream

CEP290-B1079 - GUAUCUCCUGUUUGGCA 17 downstream

CEP290-B1080 - GUCAUCUAGAGCAAGAG 17 downstream

CEP290-B1081 + GUCCUCAGUAAAAGGUA 17 upstream

CEP290-B1082 + GUGAAAGGAUCUUAGAU 17 upstream

CEP290-B1083 - GUGCUCUUUUCUAUAUA 17 downstream

CEP290-B1084 - GUGUCAAAUAUGGUGCU 17 downstream

CEP290-B1085 + GUUCCCUAUAUAUAGAA 17 downstream

Table 4D provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the fourth tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 4D

CEP290-B1120 + AUGAGGAAGAUGAACAAAUC 20 upstream

CEP290-B1121 + AUGAUCAUUCUUGUGGCAGU 20 downstream

CEP290-B1122 + AUGCUGGAGAGGAUAGGACA 20 upstream

CEP290-B1123 + AUGGUUCCCUAUAUAUAGAA 20 downstream

CEP290-B1124 - AUUUAAUUUGUUUCUGUGUG 20 downstream

CEP290-B1125 + CAAAACCUAUGUAUAAGAUG 20 downstream

CEP290-B1126 + CAAAAG ACU U AU AU U CCAU U 20 downstream

CEP290-B1127 - CAAAAGCUUUUGCUGGCUCA 20 upstream

CEP290-B1128 - CAAGAAUGAUCAUUCUAAAC 20 downstream

CEP290-B1129 - CACAGAGUUCAAGCUAAUAC 20 downstream

CEP290-B1130 + CACAGGGUAGGAUUCAUGUU 20 downstream

CEP290-B1131 + CACUGCCAAUAGGGAUAGGU 20 downstream

CEP290-B1132 + CAG AACAACG UUUUCAUUUA 20 upstream

CEP290-B1133 - CAGAGUUCAAGCUAAUACAU 20 downstream

CEP290-B1134 - CAGUAAAUGAAAACGUUGUU 20 upstream

CEP290-B1135 - CAGUAAAUGAAAACGUUGUU 20 upstream

CEP290-B1136 + CAGUAAGGAGGAUGUAAGAC 20 downstream

CEP290-B1137 + CAUAAGCCUCUAUUUCUGAU 20 upstream

CEP290-B1138 - CAU AG AG ACACAU U CAG U AA 20 upstream

CEP290-B1139 + CAUCUCUUGCUCUAGAUGAC 20 downstream

CEP290-B1140 - CAUGAGAGUGAUUAGUGGUG 20 downstream

CEP290-B1141 - CAUGUCAUCUAGAGCAAGAG 20 downstream

CEP290-B1142 + CAUUUACUGAAUGUGUCUCU 20 upstream

CEP290-B1143 + CAUUUACUGAAUGUGUCUCU 20 upstream

CEP290-B1144 + CCAUUAAAAAAAGUAUGCUU 20 downstream

CEP290-B1145 + CCUAGGACUUUCUAAUGCUG 20 upstream

CEP290-B1146 + CCUCUCUUUGGCAAAAGCAG 20 upstream

CEP290-B1147 + CCUCUCUUUGGCAAAAGCAG 20 upstream

CEP290-B1148 + CCUGUGAAAGGAUCUUAGAU 20 upstream

CEP290-B1149 - CGUGCUCUUUUCUAUAUAUA 20 downstream

CEP290-B1150 - CUAAGAUCCUUUCACAGGAG 20 upstream

CEP290-B1151 + CUAGAUGACAUGAGGUAAGU 20 downstream

CEP290-B1152 + CUAUGAGCCAGCAAAAGCUU 20 upstream

CEP290-B1153 + CUCAUAAUUUAGUAGGAAUC 20 upstream

CEP290-B1154 + CUCAUAAUUUAGUAGGAAUC 20 upstream

CEP290-B1155 - CUCAUCAGAAAUAGAGGCUU 20 upstream

CEP290-B1156 + CUCUAUUUCUGAUGAGGAAG 20 upstream

CEP290-B1157 - CUUAAGCAUACUUUUUUUAA 20 downstream

CEP290-B1158 - CUUAUCUAAGAUCCUUUCAC 20 upstream

CEP290-B1159 + CUUUCUAAUGCUGGAGAGGA 20 upstream

CEP290-B1160 + CUUUUGACAGUUUUUAAGGC 20 downstream

CEP290-B1161 + UAAAACUAAGACACUGCCAA 20 downstream CEP290-B1162 + U A AG A A A A A A A AG G U A AU G C 20 downstream

CEP290-B1163 + UAAUGCUGGAGAGGAUAGGA 20 upstream

CEP290-B1164 - UACAUAUCUGUCUUCCUUAA 20 downstream

CEP290-B1165 - UACAUCCUCCUUACUGCCAC 20 downstream

CEP290-B1166 - UACAUGAGAGUGAUUAGUGG 20 downstream

CEP290-B1167 - UACCUCAUGUCAUCUAGAGC 20 downstream

CEP290-B1168 - UACGUGCUCUUUUCUAUAUA 20 downstream

CEP290-B1169 - UAGAGCAAGAGAUGAACUAG 20 downstream

CEP290-B1170 + UAGAUGACAUGAGGUAAGUA 20 downstream

CEP290-B1171 + UAGGAAUCCUGAAAGCUACU 20 upstream

CEP290-B1172 + UAGGACAGAGGACAUGGAGA 20 upstream

CEP290-B1173 + UAGGACUUUCUAAUGCUGGA 20 upstream

CEP290-B1174 + UCACUGAGCAAAACAACUGG 20 downstream

CEP290-B1175 - UCAUGUUUAUCAAUAUUAUU 20 upstream

CEP290-B1176 - UCAUGUUUAUCAAUAUUAUU 20 upstream

CEP290-B1177 + UCCACAAGAUGUCUCUUGCC 20 upstream

CEP290-B1178 + UCCAUAAGCCUCUAUUUCUG 20 upstream

CEP290-B1179 - UCCUAGGCAAGAGACAUCUU 20 upstream

CEP290-B1180 + UCUAGAUGACAUGAGGUAAG 20 downstream

CEP290-B1181 - UCUAUACCUUUUACUGAGGA 20 upstream

CEP290-B1182 + UCUGUCCUCAGUAAAAGGUA 20 upstream

CEP290-B1183 - UCUUAAGCAUACUUUUUUUA 20 downstream

CEP290-B1184 - UCUUAUCUAAGAUCCUUUCA 20 upstream

CEP290-B1185 - UCUUCCAGUUGUUUUGCUCA 20 downstream

CEP290-B1186 + UGAGCAAAACAACUGGAAGA 20 downstream

CEP290-B1187 - UGAGUAUCUCCUGUUUGGCA 20 downstream

CEP290-B1188 + UGAUCAUUCUUGUGGCAGUA 20 downstream

CEP290-B1189 + UGCCUAGGACUUUCUAAUGC 20 upstream

CEP290-B1190 + U G CC U G AAC AAG U U U U G AAA 20 downstream

CEP290-B1191 - UGGUGUCAAAUAUGGUGCUU 20 downstream

CEP290-B1192 + UGUAAGACUGGAGAUAGAGA 20 downstream

CEP290-B1193 - UGUCCUAUCCUCUCCAGCAU 20 upstream

CEP290-B1194 - UUAACGUUAUCAUUUUCCCA 20 upstream

CEP290-B1195 - UUACAUAUCUGUCUUCCUUA 20 downstream

CEP290-B1196 + UUAGAUCUUAUUCUACUCCU 20 upstream

CEP290-B1197 + UUAGAUCUUAUUCUACUCCU 20 upstream

CEP290-B1198 - UUCAGGAUUCCUACUAAAUU 20 upstream

CEP290-B1199 - UUCAGGAUUCCUACUAAAUU 20 upstream

CEP290-B1200 - UUCAUCUUCCUCAUCAGAAA 20 upstream

CEP290-B1201 + UUGCCUAGGACUUUCUAAUG 20 upstream

CEP290-B1202 - UUUCUGCUGCUUUUGCCAAA 20 upstream

CEP290-B1203 - UUUCUGCUGCUUUUGCCAAA 20 upstream CEP290-B1204 + UUUUGACAGUUUUUAAGGCG 20 downstream

CEP290-B1205 + UUUUUAAGGCGGGGAGUCAC 20 downstream

CEP290-B1206 + AAAAGCUUUUGAGCUAA 17 upstream

CEP290-B1207 + AAAGAACAUACAUAAGA 17 downstream

CEP290-B1208 + AAAUGGUUCCCUAUAUA 17 downstream

CEP290-B1209 + AACAACG UUUUCAUUUA 17 upstream

CEP290-B1210 + AACCUAUGUAUAAGAUG 17 downstream

CEP290-B1211 + AACUAAGACACUGCCAA 17 downstream

CEP290-B1212 + AAGACUGGAGAUAGAGA 17 downstream

CEP290-B1213 + AAGACUUAUAUUCCAUU 17 downstream

CEP290-B1214 + AAGAUGAAAAAUACUCU 17 upstream

CEP290-B1215 - AAGCAUACUUUUUUUAA 17 downstream

CEP290-B1216 + AAGCCUCUAUUUCUGAU 17 upstream

CEP290-B1217 - AAGCUUUUGCUGGCUCA 17 upstream

CEP290-B1218 + AAG U U U UG AAACAGG AA 17 downstream

CEP290-B1219 + ACAAGAUGUCUCUUGCC 17 upstream

CEP290-B1220 + ACAGAGGACAUGGAGAA 17 upstream

CEP290-B1221 + ACAGGAAUAGAAAUUCA 17 downstream

CEP290-B1222 + ACAUGGGAGUCACAGGG 17 downstream

CEP290-B1223 - ACGUUAUCAUUUUCCCA 17 upstream

CEP290-B1224 + ACUAAGACACUGCCAAU 17 downstream

CEP290-B1225 + AGAAAGAACACUGUGGU 17 downstream

CEP290-B1226 + AGACUGGAGAUAGAGAC 17 downstream

CEP290-B1227 + AGACUUAUAUUCCAUUA 17 downstream

CEP290-B1228 - AGAGACACAUUCAGUAA 17 upstream

CEP290-B1229 - AGAGUUCAAGCUAAUAC 17 downstream

CEP290-B1230 - AGAUCCUUUCACAGGAG 17 upstream

CEP290-B1231 + AGAUGAAAAAUACUCUU 17 upstream

CEP290-B1232 + AGAUGACAUGAGGUAAG 17 downstream

CEP290-B1233 - AGCAAGAGAUGAACUAG 17 downstream

CEP290-B1234 + AGCCUCUAUUUCUGAUG 17 upstream

CEP290-B1235 + AGGAAGAUGAACAAAUC 17 upstream

CEP290-B1236 + AGGACUUUCUAAUGCUG 17 upstream

CEP290-B1237 + AGGAGAUACUCAACACA 17 downstream

CEP290-B1238 + AGGAUAGGACAGAGGAC 17 upstream

CEP290-B1239 - AGGAUUCCUACUAAAUU 17 upstream

CEP290-B1240 - AGGAUUCCUACUAAAUU 17 upstream

CEP290-B1241 + AGGGUAGGAUUCAUGUU 17 downstream

CEP290-B1242 - AGUUCAAGCUAAUACAU 17 downstream

CEP290-B1243 + AUAAGCCUCUAUUUCUG 17 upstream

CEP290-B1244 - AUAAGUCUUUUGAUAUA 17 downstream

CEP290-B1245 + AUAAUUUAGUAGGAAUC 17 upstream CEP290-B1246 + AUAAUUUAGUAGGAAUC 17 upstream

CEP290-B1247 - AUACCUUUUACUGAGGA 17 upstream

CEP290-B1248 - AUAGAGGCUUAUGGAUU 17 upstream

CEP290-B1249 + AUAGGACAGAGGACAUG 17 upstream

CEP290-B1250 - AUAUCUGUCUUCCUUAA 17 downstream

CEP290-B1251 - AUCAGAAAUAGAGGCUU 17 upstream

CEP290-B1252 + AUCAUUCUUGUGGCAGU 17 downstream

CEP290-B1253 - AUCCUCCUUACUGCCAC 17 downstream

CEP290-B1254 - AUCUAAGAUCCUUUCAC 17 upstream

CEP290-B1255 - AUCUUCCUCAUCAGAAA 17 upstream

CEP290-B1256 + AUGACAUGAGGUAAGUA 17 downstream

CEP290-B1257 + AUGACUCAUAAUUUAGU 17 upstream

CEP290-B1258 + AUGACUCAUAAUUUAGU 17 upstream

CEP290-B1259 - AUGAGAGUGAUUAGUGG 17 downstream

CEP290-B1260 - AUUAGAAAGUCCUAGGC 17 upstream

CEP290-B1261 + AUUCUUGUGGCAGUAAG 17 downstream

CEP290-B1262 + CACUCUCAUGUAUUAGC 17 downstream

CEP290-B1263 - CAUAUCUGUCUUCCUUA 17 downstream

CEP290-B1264 + CAUGACUCAUAAUUUAG 17 upstream

CEP290-B1265 + CAUGACUCAUAAUUUAG 17 upstream

CEP290-B1266 - CAUGAGAGUGAUUAGUG 17 downstream

CEP290-B1267 + CCUAGGACUUUCUAAUG 17 upstream

CEP290-B1268 - CCUAUCCUCUCCAGCAU 17 upstream

CEP290-B1269 + CUAGGACUUUCUAAUGC 17 upstream

CEP290-B1270 - CUCAUGUCAUCUAGAGC 17 downstream

CEP290-B1271 + CUCUUGCUCUAGAUGAC 17 downstream

CEP290-B1272 + CUCUUUGG C A A A AG C AG 17 upstream

CEP290-B1273 + CUCUUUGG C A A A AG C AG 17 upstream

CEP290-B1274 + CUGAACAAGUUUUGAAA 17 downstream

CEP290-B1275 + CUGAGCAAAACAACUGG 17 downstream

CEP290-B1276 - CUGCUGCUUUUGCCAAA 17 upstream

CEP290-B1277 - CUGCUGCUUUUGCCAAA 17 upstream

CEP290-B1278 + CUGGAGAGGAUAGGACA 17 upstream

CEP290-B1279 - UAAAUGAAAACGUUGUU 17 upstream

CEP290-B1280 - UAAAUGAAAACGUUGUU 17 upstream

CEP290-B1281 - UAAGCAUACUUUUUUUA 17 downstream

CEP290-B1282 + UAAGGAGGAUGUAAGAC 17 downstream

CEP290-B1283 + UAAUGCCUGAACAAGUU 17 downstream

CEP290-B1284 - UAAUUUGUUUCUGUGUG 17 downstream

CEP290-B1285 + UACAAAAGAACAUACAU 17 downstream

CEP290-B1286 - UAGGCAAGAGACAUCUU 17 upstream

CEP290-B1287 - UAUAAGUCUUUUGAUAU 17 downstream CEP290-B1288 - UAUCUAAGAUCCUUUCA 17 upstream

CEP290-B1289 + UAUUUCUGAUGAGGAAG 17 upstream

CEP290-B1290 + UCACUGAGCAAAACAAC 17 downstream

CEP290-B1291 + UCAUUCUUGUGGCAGUA 17 downstream

CEP290-B1292 - UCCAGUUGUUUUGCUCA 17 downstream

CEP290-B1293 + UCUAAUGCUGGAGAGGA 17 upstream

CEP290-B1294 + UGAACAAG U U U UGAAAC 17 downstream

CEP290-B1295 + UGAACUCUGUGCCAAAC 17 downstream

CEP290-B1296 + UGACAG U U U U UAAGGCG 17 downstream

CEP290-B1297 + U G AG C C AG C A A A AG C U U 17 upstream

CEP290-B1298 + UGCAGAACUAGUGUAGA 17 downstream

CEP290-B1299 + UGCCAAUAGGGAUAGGU 17 downstream

CEP290-B1300 - UGCUCUUUUCUAUAUAU 17 downstream

CEP290-B1301 + UGCUGGAGAGGAUAGGA 17 upstream

CEP290-B1302 - UGUCAAAUAUGGUGCUU 17 downstream

CEP290-B1303 - UGUUUAUCAAUAUUAUU 17 upstream

CEP290-B1304 - UGUUUAUCAAUAUUAUU 17 upstream

CEP290-B1305 + UUAAAAAAAGUAUGCUU 17 downstream

CEP290-B1306 + UUAAGGCGGGGAGUCAC 17 downstream

CEP290-B1307 + UUACUGAAUGUGUCUCU 17 upstream

CEP290-B1308 + UUACUGAAUGUGUCUCU 17 upstream

CEP290-B1309 + UUAUUCUACUCCUGUGA 17 upstream

CEP290-B1310 + UUCACUGAGCAAAACAA 17 downstream

CEP290-B1311 - UUCUGCUGCUUUUGCCA 17 upstream

CEP290-B1312 - UUCUGCUGCUUUUGCCA 17 upstream

CEP290-B1313 + UUGAACUCUGUGCCAAA 17 downstream

CEP290-B1314 + UUGACAGUUUUUAAGGC 17 downstream

CEP290-B1315 - UUGUGGAUAAUGUAUCA 17 upstream

CEP290-B1316 - UUGUUCAUCUUCCUCAU 17 upstream

CEP290-B1317 - UUGUUCUGAGUAGCUUU 17 upstream

CEP290-B1318 + UUUGACAGUUUUUAAGG 17 downstream

CEP290-B1319 + UUUUGACAGUUUUUAAG 17 downstream

Table 5A provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 400bp upstream of an Alu repeat or 700bp downstream of the mutation, have good orthogonality, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 5A

Table 5B provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 400 bp upstream of an Alu repeat or 700 bp downstream of the mutation, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 5B

CEP290-B307 + AG C AG C AG A A AG C A A AC U G A 20 upstream

CEP290-B531 - CUGCU UUUGCCAAAGAG 17 upstream

CEP290-B522 + AG C AG A A AG C A A AC U G A 17 upstream

CEP290-B537 + AAAAG CAG C AG A A AG C A 17 upstream

CEP290-B504 - AACGUUGUUCUGAGUAGCUU 20 upstream

CEP290-B478 - CUGCUGCUUUUGCCAAAGAG 20 upstream

CEP290-B526 + AG CAG CAG A A AG C A A AC U G A 20 upstream

Table 6A provides targeting domains for introduction of an indel (e.g., mediated by NHEJ) in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 40 bases of the LCAIO target position, have good orthogonality, start with G and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 6A

Table 6B provides targeting domains for introduction of an indel (e.g., mediated by NHEJ) in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 40 bases of the LCAIO target position, have good orthogonality, and PAM is NNGRRT. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 6B

CEP290-40 + AACUAAGACACUGCCAAU 18

CEP290-512 - ACCUGGCCCCAGUUGUAAUU 20

CEP290-17 - CCGCACCUGGCCCCAGU UGUAAUU 24

CEP290-41 - CGCACCUGGCCCCAGUUGUAAUU 23

CEP290-42 - CACCUGGCCCCAGUUGUAAUU 21

CEP290-513 - CCUGGCCCCAGUUGUAAUU 19

CEP290-514 - CUGGCCCCAGUUGUAAUU 18

CEP290-43 + UAAAACUAAGACACUGCCAAU 21

Table 6C provides targeting domains for introduction of an indel (e.g., mediated by NHEJ) in close proximity to or including the LCAIO target position in the CEP290 gene selected according to the fifth tier parameters. The targeting domains are within 40 bases of the LCAIO target position, and PAM is NNGRRV. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

Table 6C

CEP290-57 + UGAGAUACUCACAAUUACAAC 21

CEP290-58 + AUGAGAUAUUCACAAUUACAA 21

CEP290-59 + AG AU AU U CACAAU UACAA 18

CEP290-19 + GGUAUGAGAUAUUCACAAUUACAA 24

CEP290-61 + GU AUGAGAUAUUCACAAUUACAA 23

CEP290-63 + G AG AU AU U CACAAU UACAA 19

CEP290-65 + UAUGAGAUAUUCACAAU UACAA 22

CEP290-66 + UG AG AU AU U CACAAU UACAA 20

Table 6D provides targeting domains for introduction of an indel (e.g., mediated by NHEJ) in close proximity to or including the LCAIO target position in the CEP290 gene that can be used for dual targeting. Any of the targeting domains in the table can be used with a S. aureus Cas9 (nickase) molecule to generate a single stranded break. Exemplary nickase pairs including selecting a targeting domain from Group A and a second targeting domain from Group B. It is contemplated herein that a targeting domain of Group A can be combined with any of the targeting domains of Group B. For example, the CEP290-12 or CEP290A1 can be combined with CEP290-W or CEP290-16.

Table 6D

Table 7A provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, have good orthogonality, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 7A CEP290-68 - GAAAUAGAUGUAGAUUG 17 downstream

CEP290-70 - GAAAUAUUAAGGGCUCUUCC 20 upstream

CEP290-71 + GAACAAAAGCCAGGGACCAU 20 upstream

CEP290-72 - GAACUCUAUACCUUUUACUG 20 upstream

CEP290-73 - GAAGAAUGGAAUAGAUAAUA 20 downstream

CEP290-74 + GAAUAGUUUGUUCUGGGUAC 20 upstream

CEP290-75 - GAAUGGAAUAGAUAAUA 17 downstream

CEP290-76 + GAAUUUACAGAGUGCAUCCA 20 upstream

CEP290-77 - GAGAAAAAGGAGCAUGAAAC 20 upstream

CEP290-78 - GAGAGCCACAGUGCAUG 17 downstream

CEP290-79 - GAGGUAGAAUCAAGAAG 17 downstream

CEP290-80 + GAGUGCAUCCAUGGUCC 17 upstream

CEP290-81 + GAUAACUACAAAGGGUC 17 upstream

CEP290-82 + GAUAGAGACAGGAAUAA 17 downstream

CEP290-83 + GAUGAAAAAUACUCUUU 17 upstream

CEP290-84 + GAUGACAUGAGGUAAGU 17 downstream

CEP290-85 + GAUGCAGAACUAGUGUAGAC 20 downstream

CEP290-86 + GCAGAACUAGUGUAGAC 17 downstream

CEP290-87 - GCAUGUGGUGUCAAAUA 17 downstream

CEP290-88 + GCCUGAACAAGUUU UGAAAC 20 downstream

CEP290-89 - GCUACCGGU UACCUGAA 17 downstream

CEP290-90 - GCUCUU UUCUAUAUAUA 17 downstream

CEP290-91 + GCUUGAACUCUGUGCCAAAC 20 downstream

CEP290-92 + GCUUUUGACAGUUUUUAAGG 20 downstream

CEP290-93 - GCUUUUGUUCCUUGGAA 17 upstream

CEP290-94 + GGAACAAAAGCCAGGGACCA 20 upstream

CEP290-95 + GGACUUGACUUUUACCCUUC 20 downstream

CEP290-96 + GGAGAAUAGUUUGUUCU 17 upstream

CEP290-97 + GGAGUCACAUGGGAGUCACA 20 downstream

CEP290-98 + GGAUAGGACAGAGGACA 17 upstream

CEP290-99 + GGCUGUAAGAUAACUACAAA 20 upstream

CEP290-100 + GGGAGAAUAGUUUGUUC 17 upstream

CEP290-101 + GGGAGUCACAUGGGAGUCAC 20 downstream

CEP290-102 - GGGCUCU UCCUGGACCA 17 upstream

CEP290-103 + GGGUACAGGGGUAAGAGAAA 20 upstream

CEP290-104 - GGUCCCUGGCU UUUGUUCCU 20 upstream

CEP290-105 - GUAAAGGUUCAUGAGACUAG 20 downstream

CEP290-106 + GUAACAUAAUCACCUCUCUU 20 upstream

CEP290-107 + GUAAGACUGGAGAUAGAGAC 20 downstream

CEP290-108 + GUACAGGGGUAAGAGAA 17 upstream

CEP290-109 + GUAGCUUUUGACAGUUUUUA 20 downstream

CEP290-110 + GUCACAUGGGAGUCACA 17 downstream CEP290-111 - GUGGAGAGCCACAGUGCAUG 20 downstream

CEP290-112 - GUUACAAUCUGUGAAUA 17 upstream

CEP290-113 + GUUCUGUCCUCAGUAAA 17 upstream

CEP290-114 - GUUGAGUAUCUCCUGUU 17 downstream

CEP290-115 + GUUUAGAAUGAUCAUUCUUG 20 downstream

CEP290-116 + GUUUGUUCUGGGUACAG 17 upstream

CEP290-117 - UAAAAACUGUCAAAAGCUAC 20 downstream

CEP290-118 + UAAAAGGUAUAGAGUUCAAG 20 upstream

CEP290-119 + UAAAUCAUGCAAGUGACCUA 20 upstream

CEP290-120 + UAAGAUAACUACAAAGGGUC 20 upstream

Table 7B provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 7B

CEP290-138 - AACUAUUCUCCCAUGGUCCC 20 upstream

CEP290-140 + AAGACACUGCCAAUAGGGAU 20 downstream

CEP290-141 - AAGGAAAUACAAAAACUGGA 20 downstream

CEP290-142 + AAGGUAUAGAGUUCAAG 17 upstream

CEP290-143 - AAGGUUCAUGAGACUAG 17 downstream

CEP290-144 + AAUAGUUUGUUCUGGGUACA 20 upstream

CEP290-145 - AAUAUAAGUCUUUUGAUAUA 20 downstream

CEP290-146 - AAUAUAUUAUCUAUUUAUAG 20 upstream

CEP290-147 - AAUAUUGUAAUCAAAGG 17 upstream

CEP290-148 + AAUAUUUCAGCUACUGU 17 upstream

CEP290-149 - AAUUAUUGUUGCUUUUUGAG 20 downstream

CEP290-150 + AAUUCACUGAGCAAAACAAC 20 downstream

CEP290-151 + ACAAAAGCCAGGGACCA 17 upstream

CEP290-152 + ACACUGCCAAUAGGGAU 17 downstream

CEP290-153 + ACAGAGUGCAUCCAUGGUCC 20 upstream

CEP290-154 + ACAUAAUCACCUCUCUU 17 upstream

CEP290-155 - ACCAGACAUCUAAGAGAAAA 20 upstream

CEP290-156 - ACGUGCUCUUUUCUAUAUAU 20 downstream

CEP290-157 + ACUUUCUAAUGCUGGAG 17 upstream

CEP290-158 + ACUUUUACCCUUCAGGUAAC 20 downstream

CEP290-159 - AGAAUAUUGUAAUCAAAGGA 20 upstream

CEP290-160 - AGACAUCUAAGAGAAAA 17 upstream

CEP290-161 + AGACUUAUAUUCCAUUA 17 downstream

CEP290-162 + AGAGGAUAGGACAGAGGACA 20 upstream

CEP290-163 + AGAUGACAUGAGGUAAGUAG 20 downstream

CEP290-164 + AGAUGUCUGGUUAAAAG 17 upstream

CEP290-165 + AGCCUCUAUUUCUGAUG 17 upstream

CEP290-166 - AGCUACCGGUUACCUGA 17 downstream

CEP290-167 - AGCUCAAAAGCUUUUGC 17 upstream

CEP290-168 - AGGAAAUACAAAAACUGGAU 20 downstream

CEP290-169 + AGGAAGAUGAACAAAUC 17 upstream

CEP290-170 + AGGACAGAGGACAUGGAGAA 20 upstream

CEP290-171 + AGGACUUUCUAAUGCUGGAG 20 upstream

CEP290-172 - AGGCAAGAGACAUCUUG 17 upstream

CEP290-173 - AGGUAGAAUAUUGUAAUCAA 20 upstream

CEP290-174 - AGUAGCUGAAAUAUUAA 17 upstream

CEP290-175 + AGUCACAUGGGAGUCAC 17 downstream

CEP290-176 - AGUGCAUGUGGUGUCAAAUA 20 downstream

CEP290-177 + AGUUUGUUCUGGGUACA 17 upstream

CEP290-178 + AUAAGCCUCUAUUUCUGAUG 20 upstream

CEP290-179 - AUAAGUCUUUUGAUAUA 17 downstream

CEP290-180 + AUACAUAAGAAAGAACACUG 20 downstream CEP290-181 + AUAGUUUGUUCUGGGUACAG 20 upstream

CEP290-182 - AUAUCUGUCUUCCUUAA 17 downstream

CEP290-183 - AUAUUAAGGGCUCUUCC 17 upstream

CEP290-184 - AUAUUGUAAUCAAAGGA 17 upstream

CEP290-185 + AUCAUGCAAGUGACCUA 17 upstream

CEP290-186 - AUCUAAGAUCCUU UCAC 17 upstream

CEP290-187 - AUCUUCCUCAUCAGAAAUAG 20 upstream

CEP290-188 + AUGACAUGAGGUAAGUA 17 downstream

CEP290-189 + AUGACUCAUAAUUUAGU 17 upstream

CEP290-190 - AUGAGAGUGAUUAGUGG 17 downstream

CEP290-191 + AUGAGGAAGAUGAACAAAUC 20 upstream

CEP290-192 + AUGGGAGAAUAGUUUGUUCU 20 upstream

CEP290-193 - AUUAGCUCAAAAGCUUUUGC 20 upstream

CEP290-194 - AUUAUGCCUAUUUAGUG 17 upstream

CEP290-195 + AUUCCAAGGAACAAAAGCCA 20 upstream

CEP290-196 - AU UG AGG U AG AAU CAAG AAG 20 downstream

CEP290-197 + AUUUGACACCACAUGCACUG 20 downstream

CEP290-198 + CAAAAGCCAGGGACCAU 17 upstream

CEP290-199 - CAACAGUAGCUGAAAUAUUA 20 upstream

CEP290-200 + CAAGAUGUCUCUUGCCU 17 upstream

CEP290-201 - CAGAACAAACUAUUCUCCCA 20 upstream

CEP290-202 - CAGAUUUCAUGUGUGAAGAA 20 downstream

CEP290-204 - CAG CA U U AG AAAG U CC U 17 upstream

CEP290-205 + CAGGGGUAAGAGAAAGGGAU 20 upstream

CEP290-206 + CAGUAAGGAGGAUGUAAGAC 20 downstream

CEP290-207 - CAGUAGCUGAAAUAUUA 17 upstream

CEP290-208 + CAUAAGAAAGAACACUG 17 downstream

CEP290-209 + CAUGGGAGAAUAGUUUGUUC 20 upstream

CEP290-210 + CAUGGGAGUCACAGGGU 17 downstream

CEP290-211 + CAUUCCAAGGAACAAAAGCC 20 upstream

CEP290-212 + CCACAAGAUGUCUCUUGCCU 20 upstream

CEP290-213 - CCUAGGCAAGAGACAUCUUG 20 upstream

CEP290-214 - CGUGCUCUUUUCUAUAUAUA 20 downstream

CEP290-215 - CGUUGUUCUGAGUAGCUUUC 20 upstream

CEP290-216 + CUAAGACACUGCCAAUA 17 downstream

CEP290-217 + CUAAUGCUGGAGAGGAU 17 upstream

CEP290-218 + CUAGAUGACAUGAGGUAAGU 20 downstream

CEP290-219 + CUAGGACUUUCUAAUGC 17 upstream

CEP290-220 - CUCAUACCUAUCCCUAU 17 downstream

CEP290-221 - CUCCAGCAUUAGAAAGUCCU 20 upstream

CEP290-222 - CUCUAUACCUUUUACUG 17 upstream

CEP290-223 + CUCUUGCUCUAGAUGACAUG 20 downstream CEP290-224 - CUGCUGCUUUUGCCAAAGAG 20 upstream

CEP290-225 - CUGCU UUUGCCAAAGAG 17 upstream

CEP290-226 - CUGGCUUU UGUUCCUUGGAA 20 upstream

CEP290-227 + CUGUAAGAUAACUACAA 17 upstream

CEP290-228 - CUUAAGCAUACUU UUUUUAA 20 downstream

CEP290-229 + CU UAAUAUUUCAGCUACUGU 20 upstream

CEP290-231 + CU UAGAUGUCUGGUUAAAAG 20 upstream

CEP290-232 - CUUAUCUAAGAUCCUUUCAC 20 upstream

CEP290-233 + CU UGACUUUUACCCUUC 17 downstream

CEP290-234 + CU UGUUCUGUCCUCAGUAAA 20 upstream

CEP290-235 + CU UUUGACAGU UUUUAAGGC 20 downstream

Table 7C provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the third tier parameters. The targeting domains are within lOOObp upstream of an Alu repeat, within 40bp upstream of mutation, or lOOObp downstream of the mutation, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 7C

Table 7D provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the fourth tier parameters. The targeting domains are within lOOObp upstream of an Alu repeat, within 40bp upstream of mutation, or lOOObp downstream of the mutation, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 7D

CEP290-279 + UGUGUGUGUGUGUUAUG 17 upstream

CEP290-280 + UUCUUGUGGCAGUAAGG 17 downstream

CEP290-281 + UUGAACCUGGGAGGCAG 17 downstream

CEP290-282 - UUGCCCAGGCUGGAGUGCAG 20 downstream

CEP290-283 - UUUUAUUAUCUUUAUUG 17 downstream

Table 8A provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, have good orthogonality, start with G and PAM is NNGRRT. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 8A

CEP290-323 + GUUCUGUCCUCAGUAAAAGGUA 22 upstream

CEP290-480 + GAACAACGUUUUCAUUUA 18 upstream

CEP290-296 - GUAGAAUAUCAUAAGUUACAAUCU 24 upstream

CEP290-297 - GAAUAUCAUAAGUUACAAUCU 21 upstream

CEP290-298 + GUGGCUGUAAGAUAACUACA 20 upstream

CEP290-299 + GGCUGUAAGAUAACUACA 18 upstream

CEP290-300 - GU UUAACGUUAUCAU UUUCCCA 22 upstream

CEP290-301 + GUAAGAGAAAGGGAUGGGCACUUA 24 upstream

CEP290-492 + GAGAAAGGGAUGGGCACUUA 20 upstream

CEP290-491 + GAAAGGGAUGGGCACUUA 18 upstream

CEP290-483 - G U AAAU G AAAACG U U G U U 18 upstream

CEP290-302 + GAUAAACAUGACUCAUAAUUUAGU 24 upstream

CEP290-303 + GGAACAAAAGCCAGGGACCAUGG 23 upstream

CEP290-304 + GAACAAAAGCCAGGGACCAUGG 22 upstream

CEP290-305 + GGGAGAAUAGUUUGUUCUGGGUAC 24 upstream

CEP290-306 + GGAGAAUAGUUUGUUCUGGGUAC 23 upstream

CEP290-307 + GAGAAUAGUUUGUUCUGGGUAC 22 upstream

CEP290-490 + GAAUAGUUUGUUCUGGGUAC 20 upstream

CEP290-482 - GAAAUAGAGGCUUAUGGAUU 20 upstream

CEP290-308 + GUUCUGGGUACAGGGGUAAGAGAA 24 upstream

CEP290-494 + GGGUACAGGGGUAAGAGAA 19 upstream

CEP290-493 + GGUACAGGGGUAAGAGAA 18 upstream

CEP290-309 - GUAAAUUCUCAUCAU UUUUUAUUG 24 upstream

CEP290-310 + GGAGAGGAUAGGACAGAGGACAUG 24 upstream

CEP290-311 + GAGAGGAUAGGACAGAGGACAUG 23 upstream

CEP290-313 + GAGGAUAGGACAGAGGACAUG 21 upstream

CEP290-485 + GGAUAGGACAGAGGACAUG 19 upstream

CEP290-484 + GAUAGGACAGAGGACAUG 18 upstream

CEP290-314 - GAAUAAAUGUAGAAUUUUAAUG 22 upstream

CEP290-64 - GUCAAAAGCUACCGGU UACCUG 22 downstream

CEP290-315 + GUUUUUAAGGCGGGGAGUCACAU 23 downstream

CEP290-203 - GUCUUACAUCCUCCUUACUGCCAC 24 downstream

CEP290-316 + GAGUCACAGGGUAGGAUUCAUGUU 24 downstream

CEP290-317 + GUCACAGGGUAGGAUUCAUGUU 22 downstream

CEP290-318 - GGCACAGAGUUCAAGCUAAUACAU 24 downstream

CEP290-319 - GCACAGAGUUCAAGCUAAUACAU 23 downstream

CEP290-505 - GAGUUCAAGCUAAUACAU 18 downstream

CEP290-496 + GAUGCAGAACUAGUGUAGAC 20 downstream

CEP290-320 - GUGUUGAGUAUCUCCUGUUUGGCA 24 downstream

CEP290-321 - GU UGAGUAUCUCCUGU UUGGCA 22 downstream

CEP290-504 - GAGUAUCUCCUGUUUGGCA 19 downstream

CEP290-322 - GAAAAUCAGAUUUCAUGUGUG 21 downstream CEP290-324 - GCCACAAGAAUGAUCAUUCUAAAC 24 downstream

CEP290-325 + GGCGGGGAGUCACAUGGGAGUCA 23 downstream

CEP290-326 + GCGGGGAGUCACAUGGGAGUCA 22 downstream

CEP290-499 + GGGGAGUCACAUGGGAGUCA 20 downstream

CEP290-498 + GGGAGUCACAUGGGAGUCA 19 downstream

CEP290-497 + GGAGUCACAUGGGAGUCA 18 downstream

CEP290-327 + GCUUUUGACAGUUUUUAAGGCG 22 downstream

CEP290-328 + GAUCAUUCUUGUGGCAGUAAG 21 downstream

CEP290-329 - GAGCAAGAGAUGAACUAG 18 downstream

CEP290-500 + GCCUGAACAAGUUU UGAAAC 20 downstream

CEP290-330 - GUAGAUUGAGGUAGAAUCAAGAA 23 downstream

CEP290-506 - GAUUGAGGUAGAAUCAAGAA 20 downstream

CEP290-331 + GGAUGUAAGACUGGAGAUAGAGAC 24 downstream

CEP290-332 + GAUGUAAGACUGGAGAUAGAGAC 23 downstream

CEP290-503 + GUAAGACUGGAGAUAGAGAC 20 downstream

CEP290-333 + GGGAGUCACAUGGGAGUCACAGGG 24 downstream

CEP290-334 + GGAGUCACAUGGGAGUCACAGGG 23 downstream

CEP290-335 + GAGUCACAUGGGAGUCACAGGG 22 downstream

CEP290-502 + GUCACAUGGGAGUCACAGGG 20 downstream

CEP290-336 - GU UUACAUAUCUGUCU UCCUUAA 23 downstream

CEP290-507 - GAUUUCAUGUGUGAAGAA 18 downstream

Table 8B provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, and have good orthogonality. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 8B

CEP290-342 - AUAUUAAGGGCUCUUCCUGGACC 23 upstream

CEP290-343 - AUUAAGGGCUCUUCCUGGACC 21 upstream

CEP290-344 - AAGGGCUCUUCCUGGACC 18 upstream

CEP290-345 + AGGACUUUCUAAUGCUGGA 19 upstream

CEP290-346 + ACCAUGGGAGAAUAGUUUGUU 21 upstream

CEP290-347 + AUGGGAGAAUAGUUUGUU 18 upstream

CEP290-348 + ACUCCUGUGAAAGGAUCUUAGAU 23 upstream

CEP290-349 - AAAACGUUGUUCUGAGUAGCUUU 23 upstream

CEP290-350 - AAACGUUGUUCUGAGUAGCUUU 22 upstream

CEP290-351 - AACGUUGUUCUGAGUAGCUUU 21 upstream

CEP290-352 - ACGUUGUUCUGAGUAGCUUU 20 upstream

CEP290-353 - AUUUAUAGUGGCUGAAUGACUU 22 upstream

CEP290-354 - AUAGUGGCUGAAUGACUU 18 upstream

CEP290-355 - AUGGUCCCUGGCUUUUGUUCCU 22 upstream

CEP290-356 - AGACAUCUUGUGGAUAAUGUAUCA 24 upstream

CEP290-357 - ACAUCUUGUGGAUAAUGUAUCA 22 upstream

CEP290-358 - AUCUUGUGGAUAAUGUAUCA 20 upstream

CEP290-359 - A A AG U CC U AG G C A AG AGACAUCUU 24 upstream

CEP290-360 - AAGUCCUAGG C A AG AGACAUCUU 23 upstream

CEP290-361 - AGUCCUAGGCAAGAGACAUCUU 22 upstream

CEP290-362 + AG CC AG C A A A AG CUUUUGAGCUAA 24 upstream

CEP290-363 + AGCAAAAGCUUUUGAGCUAA 20 upstream

CEP290-364 + AGAUCUUAUUCUACUCCUGUGA 22 upstream

CEP290-365 + AUCUUAUUCUACUCCUGUGA 20 upstream

CEP290-366 - AUCUAAGAUCCUU UCACAGGAG 22 upstream

CEP290-369 - AAGAUCCUUUCACAGGAG 18 upstream

CEP290-370 - AGCUUUCAGGAUUCCUACUAAAUU 24 upstream

CEP290-371 + ACUCAGAACAACGUUU UCAUUUA 23 upstream

CEP290-372 + AGAACAACGUUUUCAUUUA 19 upstream

CEP290-373 - AGAAUAUCAUAAGUUACAAUCU 22 upstream

CEP290-375 - AAUAUCAUAAGUUACAAUCU 20 upstream

CEP290-376 - AUAUCAUAAGUUACAAUCU 19 upstream

CEP290-377 + AAGUGGCUGUAAGAUAACUACA 22 upstream

CEP290-378 + AGUGGCUGUAAGAUAACUACA 21 upstream

CEP290-379 - AUGUUUAACGUUAUCAUUUUCCCA 24 upstream

CEP290-380 - AACGUUAUCAUUUUCCCA 18 upstream

CEP290-381 + AAGAGAAAGGGAUGGGCACUUA 22 upstream

CEP290-382 + AGAGAAAGGGAUGGGCACUUA 21 upstream

CEP290-383 + AGAAAGGGAUGGGCACUUA 19 upstream

CEP290-384 - AUUCAGUAAAUGAAAACGUUGUU 23 upstream

CEP290-385 - AGUAAAUGAAAACGUUGUU 19 upstream

CEP290-386 + AUAAACAUGACUCAUAAUUUAGU 23 upstream CEP290-387 + AAACAUGACUCAUAAUU UAGU 21 upstream

CEP290-388 + AACAUGACUCAUAAUUUAGU 20 upstream

CEP290-389 + ACAUGACUCAUAAUUUAGU 19 upstream

CEP290-390 - AUUCUUAUCUAAGAUCCUUUCAC 23 upstream

CEP290-391 + AGGAACAAAAGCCAGGGACCAUGG 24 upstream

CEP290-392 + AACAAAAGCCAGGGACCAUGG 21 upstream

CEP290-393 + ACAAAAGCCAGGGACCAUGG 20 upstream

CEP290-394 + AAAAGCCAGGGACCAUGG 18 upstream

CEP290-395 + AGAAUAGUUUGUUCUGGGUAC 21 upstream

CEP290-396 + AAUAGUUUGUUCUGGGUAC 19 upstream

CEP290-397 + AUAGUUUGUUCUGGGUAC 18 upstream

CEP290-398 - AUCAGAAAUAGAGGCUUAUGGAUU 24 upstream

CEP290-399 - AGAAAUAGAGGCUUAUGGAUU 21 upstream

CEP290-400 - AAAUAGAGGCUUAUGGAUU 19 upstream

CEP290-401 - AAUAGAGGCUUAUGGAUU 18 upstream

CEP290-402 - AAUAUAUUAUCUAUUUAUAGUGG 23 upstream

CEP290-403 - AUAUAUUAUCUAUUUAUAGUGG 22 upstream

CEP290-404 - AUAUUAUCUAUUUAUAGUGG 20 upstream

CEP290-405 - AUUAUCUAUUUAUAGUGG 18 upstream

CEP290-406 - AAAUUCUCAUCAUUUU UUAUUG 22 upstream

CEP290-407 - AAUUCUCAUCAUUUUU UAUUG 21 upstream

CEP290-408 - AUUCUCAUCAUUUUUUAUUG 20 upstream

CEP290-409 + AGAGGAUAGGACAGAGGACAUG 22 upstream

CEP290-410 + AGGAUAGGACAGAGGACAUG 20 upstream

CEP290-411 - AGAAUAAAUGUAGAAUUUUAAUG 23 upstream

CEP290-412 - AAUAAAUGUAGAAUUUUAAUG 21 upstream

CEP290-413 - AUAAAUGUAGAAUUUUAAUG 20 upstream

CEP290-414 - AAAUGUAGAAUUUUAAUG 18 upstream

CEP290-415 - AUUUUUUAUUGUAGAAUAAAUG 22 upstream

CEP290-416 + CUAAAUCAUGCAAGUGACCUAAG 23 upstream

CEP290-417 - CCUUAGGUCACUUGCAUGAUUUAG 24 upstream

CEP290-418 - CUUAGGUCACUUGCAUGAUUUAG 23 upstream

CEP290-419 + CCUAGGACUUUCUAAUGCUGGA 22 upstream

CEP290-420 + CUAGGACUUUCUAAUGCUGGA 21 upstream

CEP290-421 + CCAUGGGAGAAUAGUUUGUU 20 upstream

CEP290-422 + CAUGGGAGAAUAGUUUGUU 19 upstream

CEP290-423 + CUCCUG UGAAAGGAUCUUAGAU 22 upstream

CEP290-424 + CCUGUGAAAGGAUCUUAGAU 20 upstream

CEP290-426 + CUGUGAAAGGAUCUUAGAU 19 upstream

CEP290-427 - CCCUGGCUU UUGUUCCU UGGA 21 upstream

CEP290-428 - CCUGGCU UUUGUUCCU UGGA 20 upstream

CEP290-429 - CUGGCUUU UGUUCCUUGGA 19 upstream CEP290-430 - CGUUGUUCUGAGUAGCUUU 19 upstream

CEP290-431 - CUAUUUAUAGUGGCUGAAUGACUU 24 upstream

CEP290-432 - CCAUGGUCCCUGGCUUU UGUUCCU 24 upstream

CEP290-433 - CAUGGUCCCUGGCUUUUGUUCCU 23 upstream

CEP290-434 - CAUCUUGUGGAUAAUGUAUCA 21 upstream

CEP290-435 - CUUGUGGAUAAUGUAUCA 18 upstream

CEP290-437 - CCUAGGCAAGAGACAUCUU 19 upstream

CEP290-438 - CUAGGCAAGAGACAUCU U 18 upstream

CEP290-439 + CCAGCAAAAGCUUUUGAGCUAA 22 upstream

CEP290-440 + CAG CA AAAG CUUUUGAGCUAA 21 upstream

CEP290-441 + CAAAAGCUUUUGAGCUAA 18 upstream

CEP290-442 + CU UAUUCUACUCCUGUGA 18 upstream

CEP290-443 - CU AAG AU CCU U U CACAGG AG 20 upstream

CEP290-444 - CUUCCUCAUCAGAAAUAGAGGCUU 24 upstream

CEP290-445 - CCUCAUCAGAAAUAGAGGCUU 21 upstream

CEP290-446 - CUCAUCAGAAAUAGAGGCUU 20 upstream

CEP290-447 - CAUCAGAAAUAGAGGCUU 18 upstream

CEP290-448 - CUU UCAGGAUUCCUACUAAAUU 22 upstream

CEP290-449 - CAGGAUUCCUACUAAAUU 18 upstream

CEP290-450 + CUGUCCUCAGUAAAAGGUA 19 upstream

CEP290-451 + CUCAGAACAACGUUU UCAUUUA 22 upstream

CEP290-452 + CAG AACAACG UUUUCAUUUA 20 upstream

CEP290-453 + CAAGUGGCUGUAAGAUAACUACA 23 upstream

CEP290-454 - CAU U CAG U AAAUG AAAACG U UG U U 24 upstream

CEP290-457 - CAG U AAAUG AAAACG UUGUU 20 upstream

CEP290-458 + CAUGACUCAUAAUUUAGU 18 upstream

CEP290-459 - CUUAUCUAAGAUCCUUUCAC 20 upstream

CEP290-460 + CAAAAGCCAGGGACCAUGG 19 upstream

CEP290-461 - CAGAAAUAGAGGCUUAUGGAUU 22 upstream

CEP290-462 + CUGGGUACAGGGGUAAGAGAA 21 upstream

CEP290-463 - CAAUAUAUUAUCUAUU UAUAGUGG 24 upstream

CEP290-464 - CAU UUUUUAUUGUAGAAUAAAUG 23 upstream

CEP290-465 + UAAAUCAUGCAAGUGACCUAAG 22 upstream

CEP290-466 + UCAUGCAAGUGACCUAAG 18 upstream

CEP290-467 - UUAGGUCACUUGCAUGAUUUAG 22 upstream

CEP290-468 - UAGGUCACUUGCAUGAUUUAG 21 upstream

CEP290-469 - UAUUAAGGGCUCUUCCUGGACC 22 upstream

CEP290-470 - UUAAGGGCUCUUCCUGGACC 20 upstream

CEP290-471 - UAAGGGCUCUUCCUGGACC 19 upstream

CEP290-472 + UGCCUAGGACU UUCUAAUGCUGGA 24 upstream

CEP290-473 + UAGGACUUUCUAAUGCUGGA 20 upstream

CEP290-474 + UACUCCUGUGAAAGGAUCUUAGAU 24 upstream CEP290-475 + UCCUGUGAAAGGAUCU UAGAU 21 upstream

CEP290-476 + UGUGAAAGGAUCUUAGAU 18 upstream

CEP290-477 - UCCCUGGCU UUUGUUCCUUGGA 22 upstream

CEP290-515 - UGGCUUUUGUUCCUUGGA 18 upstream

CEP290-516 - UAUUUAUAGUGGCUGAAUGACUU 23 upstream

CEP290-517 - UUUAUAGUGGCUGAAUGACUU 21 upstream

CEP290-518 - UUAUAGUGGCUGAAUGACUU 20 upstream

CEP290-519 - UAUAGUGGCUGAAUGACUU 19 upstream

CEP290-520 - UGGUCCCUGGCUUUUGUUCCU 21 upstream

CEP290-521 - UCCCUGGCU UUUGUUCCU 18 upstream

CEP290-522 - UCU UGUGGAUAAUGUAUCA 19 upstream

CEP290-523 - UCCUAGGCAAGAGACAUCUU 20 upstream

CEP290-524 + UUAGAUCUUAUUCUACUCCUGUGA 24 upstream

CEP290-525 + UAGAUCUUAUUCUACUCCUGUGA 23 upstream

CEP290-526 + UCUUAUUCUACUCCUGUGA 19 upstream

CEP290-527 - UUAUCUAAGAUCCUUUCACAGGAG 24 upstream

CEP290-528 - UAUCUAAGAUCCU UUCACAGGAG 23 upstream

CEP290-529 - UCUAAGAUCCUU UCACAGGAG 21 upstream

CEP290-530 - UAAGAUCCUUUCACAGGAG 19 upstream

CEP290-531 - UUCCUCAUCAGAAAUAGAGGCUU 23 upstream

CEP290-532 - UCCUCAUCAGAAAUAGAGGCUU 22 upstream

CEP290-533 - UCAUCAGAAAUAGAGGCUU 19 upstream

CEP290-534 - UUUCAGGAUUCCUACUAAAUU 21 upstream

CEP290-535 - UUCAGGAUUCCUACUAAAUU 20 upstream

CEP290-536 - UCAGGAUUCCUACUAAAUU 19 upstream

CEP290-537 + UUGUUCUGUCCUCAGUAAAAGGUA 24 upstream

CEP290-538 + UGUUCUGUCCUCAGUAAAAGGUA 23 upstream

CEP290-539 + UUCUGUCCUCAGUAAAAGGUA 21 upstream

CEP290-540 + UCUGUCCUCAGUAAAAGGUA 20 upstream

CEP290-541 + UGUCCUCAGUAAAAGGUA 18 upstream

CEP290-542 + UACUCAGAACAACGUU UUCAUUUA 24 upstream

CEP290-543 + U CAG AACAACG UUUUCAUUUA 21 upstream

CEP290-544 - UAGAAUAUCAUAAGUUACAAUCU 23 upstream

CEP290-545 - UAUCAUAAGUUACAAUCU 18 upstream

CEP290-546 + UCAAGUGGCUGUAAGAUAACUACA 24 upstream

CEP290-547 + UGGCUGUAAGAUAACUACA 19 upstream

CEP290-548 - UGUUUAACGUUAUCAUUUUCCCA 23 upstream

CEP290-549 - UUUAACGUUAUCAUUU UCCCA 21 upstream

CEP290-550 - UUAACGUUAUCAUUUUCCCA 20 upstream

CEP290-551 - UAACGUUAUCAUUUUCCCA 19 upstream

CEP290-552 + UAAGAGAAAGGGAUGGGCACUUA 23 upstream

CEP290-553 - UUCAGUAAAUGAAAACGUUGUU 22 upstream CEP290-554 - UCAGUAAAUGAAAACGUUGUU 21 upstream

CEP290-555 + UAAACAUGACUCAUAAU UUAGU 22 upstream

CEP290-556 - UAUUCUUAUCUAAGAUCCUUUCAC 24 upstream

CEP290-557 - UUCUUAUCUAAGAUCCU UUCAC 22 upstream

CEP290-558 - UCU UAUCUAAGAUCCUU UCAC 21 upstream

CEP290-559 - UUAUCUAAGAUCCUUUCAC 19 upstream

CEP290-560 - UAUCUAAGAUCCU UUCAC 18 upstream

CEP290-561 - UCAGAAAUAGAGGCUUAUGGAUU 23 upstream

CEP290-562 + UUCUGGGUACAGGGGUAAGAGAA 23 upstream

CEP290-563 + UCUGGGUACAGGGGUAAGAGAA 22 upstream

CEP290-564 + UGGGUACAGGGGUAAGAGAA 20 upstream

CEP290-565 - UAUAUUAUCUAUUUAUAGUGG 21 upstream

CEP290-566 - UAUUAUCUAUUUAUAGUGG 19 upstream

CEP290-567 - UAAAUUCUCAUCAUUU UUUAUUG 23 upstream

CEP290-568 - UUCUCAUCAUUUUUUAUUG 19 upstream

CEP290-569 - UCUCAUCAUUUUUUAU UG 18 upstream

CEP290-570 - UAGAAUAAAUGUAGAAUUUUAAUG 24 upstream

CEP290-571 - UAAAUGUAGAAUUUUAAUG 19 upstream

CEP290-572 - UCAUUUUUUAUUGUAGAAUAAAUG 24 upstream

CEP290-573 - UUUUUUAUUGUAGAAUAAAUG 21 upstream

CEP290-574 - UUUUUAUUGUAGAAUAAAUG 20 upstream

CEP290-575 - UUUUAUUGUAGAAUAAAUG 19 upstream

CEP290-576 - UUUAUUGUAGAAUAAAUG 18 upstream

CEP290-577 - AAAAGCUACCGGUUACCUG 19 downstream

CEP290-578 - AAAGCUACCGGUUACCUG 18 downstream

CEP290-579 + AGUUUUUAAGGCGGGGAGUCACAU 24 downstream

CEP290-580 - ACAUCCUCCUUACUGCCAC 19 downstream

CEP290-581 + AGUCACAGGGUAGGAU UCAUGUU 23 downstream

CEP290-582 + ACAGGGUAGGAUUCAUGUU 19 downstream

CEP290-583 - ACAGAGUUCAAGCUAAUACAU 21 downstream

CEP290-584 - AGAGUUCAAGCUAAUACAU 19 downstream

CEP290-585 + AUAAGAUGCAGAACUAGUGUAGAC 24 downstream

CEP290-586 + AAGAUGCAGAACUAGUGUAGAC 22 downstream

CEP290-587 + AGAUGCAGAACUAGUGUAGAC 21 downstream

CEP290-588 + AUGCAGAACUAGUGUAGAC 19 downstream

CEP290-589 - AGUAUCUCCUGUUUGGCA 18 downstream

CEP290-590 - ACGAAAAUCAGAUUUCAUGUGUG 23 downstream

CEP290-591 - AAAAUCAGAUUUCAUGUGUG 20 downstream

CEP290-592 - AAAUCAGAUUUCAUGUGUG 19 downstream

CEP290-593 - AAUCAGAUUUCAUGUGUG 18 downstream

CEP290-594 - ACAAGAAUGAUCAUUCUAAAC 21 downstream

CEP290-595 - AAGAAUGAUCAUUCUAAAC 19 downstream CEP290-596 - AGAAUGAUCAUUCUAAAC 18 downstream

CEP290-597 + AGGCGGGGAGUCACAUGGGAGUCA 24 downstream

CEP290-598 + AGCUUUUGACAGUUUU UAAGGCG 23 downstream

CEP290-599 + AAUGAUCAUUCUUGUGGCAGUAAG 24 downstream

CEP290-600 + AUGAUCAUUCUUGUGGCAGUAAG 23 downstream

CEP290-601 + AUCAUUCUUGUGGCAGUAAG 20 downstream

CEP290-602 - AUCUAGAGCAAGAGAUGAACUAG 23 downstream

CEP290-603 - AGAGCAAGAGAUGAACUAG 19 downstream

CEP290-604 + AAUG CC U G AAC AAG U U U UGAAAC 23 downstream

CEP290-605 + AUGCCUGAACAAGUUU UGAAAC 22 downstream

CEP290-606 - AGAUUGAGGUAGAAUCAAGAA 21 downstream

CEP290-607 - AU UG AGG U AG AAU CAAG AA 19 downstream

CEP290-608 + AUGUAAGACUGGAGAUAGAGAC 22 downstream

CEP290-609 + AAGACUGGAGAUAGAGAC 18 downstream

CEP290-610 + AGUCACAUGGGAGUCACAGGG 21 downstream

CEP290-611 - ACAUAUCUGUCU UCCUUAA 19 downstream

CEP290-612 - AAAUCAGAUUUCAUGUGUGAAGAA 24 downstream

CEP290-613 - AAUCAGAUUUCAUGUGUGAAGAA 23 downstream

CEP290-614 - AUCAGAUUUCAUGUGUGAAGAA 22 downstream

CEP290-615 - AGAUUUCAUGUGUGAAGAA 19 downstream

CEP290-616 + AAAUAAAACUAAGACACUGCCAAU 24 downstream

CEP290-617 + AAUAAAACUAAGACACUGCCAAU 23 downstream

CEP290-618 + AUAAAACUAAGACACUGCCAAU 22 downstream

CEP290-619 + AAAACUAAGACACUGCCAAU 20 downstream

CEP290-620 + AAACUAAGACACUGCCAAU 19 downstream

CEP290-621 + AACUAAGACACUGCCAAU 18 downstream

CEP290-622 - AACUAUUUAAUUUGUU UCUGUGUG 24 downstream

CEP290-623 - ACUAUUUAAUUUGUU UCUGUGUG 23 downstream

CEP290-624 - AUUUAAUUUGUUUCUGUGUG 20 downstream

CEP290-625 - CUGUCAAAAGCUACCGGUUACCUG 24 downstream

CEP290-626 - CAAAAGCUACCGGUUACCUG 20 downstream

CEP290-627 - CUUACAUCCUCCUUACUGCCAC 22 downstream

CEP290-628 - CAUCCUCCUUACUGCCAC 18 downstream

CEP290-629 + CACAGGGUAGGAUUCAUGUU 20 downstream

CEP290-630 + CAGGGUAGGAUUCAUGUU 18 downstream

CEP290-631 - CACAGAGUUCAAGCUAAUACAU 22 downstream

CEP290-632 - CAGAGUUCAAGCUAAUACAU 20 downstream

CEP290-633 - CACGAAAAUCAGAUUUCAUGUGUG 24 downstream

CEP290-634 - CGAAAAUCAGAUUUCAUGUGUG 22 downstream

CEP290-635 - CCACAAG AAUG AU CAU U CUAAAC 23 downstream

CEP290-636 - CACAAGAAUGAUCAUUCUAAAC 22 downstream

CEP290-637 - CAAGAAUGAUCAUUCUAAAC 20 downstream CEP290-638 + CGGGGAGUCACAUGGGAGUCA 21 downstream

CEP290-639 + CU UUUGACAGU UUUUAAGGCG 21 downstream

CEP290-640 + CAUUCUUGUGGCAGUAAG 18 downstream

CEP290-641 - CAUCUAGAGCAAGAGAUGAACUAG 24 downstream

CEP290-642 - CUAGAGCAAGAGAUGAACUAG 21 downstream

CEP290-643 + CC U G A ACAAG U U U UGAAAC 19 downstream

CEP290-644 + CUGAACAAGUUUUGAAAC 18 downstream

CEP290-645 - CUCUCU UCCAGUUGUUUUGCUCA 23 downstream

CEP290-646 - CUCUUCCAGUUGUUUUGCUCA 21 downstream

CEP290-647 - CUUCCAGU UGUUUUGCUCA 19 downstream

CEP290-648 + CACAUGGGAGUCACAGGG 18 downstream

CEP290-649 - CAUAUCUGUCUUCCUUAA 18 downstream

CEP290-650 - CAGAUUUCAUGUGUGAAGAA 20 downstream

CEP290-651 - CUAUUUAAUUUGUU UCUGUGUG 22 downstream

CEP290-652 - UGUCAAAAGCUACCGGUUACCUG 23 downstream

CEP290-653 - UCAAAAGCUACCGGUUACCUG 21 downstream

CEP290-654 + UUUUUAAGGCGGGGAGUCACAU 22 downstream

CEP290-655 + UUUUAAGGCGGGGAGUCACAU 21 downstream

CEP290-656 + UUUAAGGCGGGGAGUCACAU 20 downstream

CEP290-657 + UUAAGGCGGGGAGUCACAU 19 downstream

CEP290-658 + UAAGGCGGGGAGUCACAU 18 downstream

CEP290-659 - UCU UACAUCCUCCUUACUGCCAC 23 downstream

CEP290-660 - UUACAUCCUCCUUACUGCCAC 21 downstream

CEP290-661 - UACAUCCUCCUUACUGCCAC 20 downstream

CEP290-662 + UCACAGGGUAGGAUUCAUGUU 21 downstream

CEP290-663 + UAAGAUGCAGAACUAGUGUAGAC 23 downstream

CEP290-664 + UGCAGAACUAGUGUAGAC 18 downstream

CEP290-665 - UGUUGAGUAUCUCCUGUUUGGCA 23 downstream

CEP290-666 - UUGAGUAUCUCCUGUUUGGCA 21 downstream

CEP290-667 - UGAGUAUCUCCUGUUUGGCA 20 downstream

CEP290-668 + UAGCUUUUGACAGUUU UUAAGGCG 24 downstream

CEP290-669 + UUUUGACAGUUUUUAAGGCG 20 downstream

CEP290-670 + UUUGACAGUUUUUAAGGCG 19 downstream

CEP290-671 + U UG ACAG U U U U UAAGGCG 18 downstream

CEP290-672 + UGAUCAUUCUUGUGGCAGUAAG 22 downstream

CEP290-673 + UCAUUCUUGUGGCAGUAAG 19 downstream

CEP290-674 - UCUAGAGCAAGAGAUGAACUAG 22 downstream

CEP290-675 - UAGAGCAAGAGAUGAACUAG 20 downstream

CEP290-676 + UAAUGCCUGAACAAGUU UUGAAAC 24 downstream

CEP290-677 + UGCCUGAACAAGUU UUGAAAC 21 downstream

CEP290-678 - UG U AG AU UG AGG U AG AAU CAAG AA 24 downstream

CEP290-679 - UAGAUUGAGGUAGAAUCAAGAA 22 downstream CEP290-680 - U UG AGG U AG AAU CAAG AA 18 downstream

CEP290-681 + UGUAAGACUGGAGAUAGAGAC 21 downstream

CEP290-682 + UAAGACUGGAGAUAGAGAC 19 downstream

CEP290-683 - UCUCUCUUCCAGUUGUUUUGCUCA 24 downstream

CEP290-684 - UCUCUUCCAGUUGUUU UGCUCA 22 downstream

CEP290-685 - UCU UCCAGUUGUUU UGCUCA 20 downstream

CEP290-686 - UUCCAGUUGUUUUGCUCA 18 downstream

CEP290-687 + UCACAUGGGAGUCACAGGG 19 downstream

CEP290-688 - UGUUUACAUAUCUGUCUUCCUUAA 24 downstream

CEP290-689 - UUUACAUAUCUGUCUUCCUUAA 22 downstream

CEP290-690 - UUACAUAUCUGUCUUCCUUAA 21 downstream

CEP290-691 - UACAUAUCUGUCUUCCU UAA 20 downstream

CEP290-692 - UCAGAUUUCAUGUGUGAAGAA 21 downstream

CEP290-693 + UAAAACUAAGACACUGCCAAU 21 downstream

CEP290-694 - UAUUUAAUUUGUUUCUGUGUG 21 downstream

CEP290-695 - UUUAAUUUGUUUCUGUGUG 19 downstream

CEP290-696 - UUAAUUUGUUUCUGUGUG 18 downstream

Table 8C provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the third tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, start with G and PAM is NNGRRT. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 8C

CEP290-704 - GUGUUGCCCAGGCUGGAGUGCAG 23 downstream

CEP290-705 - GUUGCCCAGGCUGGAGUGCAG 21 downstream

CEP290-706 - GCCCAGGCUGGAGUGCAG 18 downstream

CEP290-707 - GUUGUUUUUUUUUUUGAAA 19 downstream

CEP290-708 - GAGUCUCACUGUGUUGCCCAGGC 23 downstream

CEP290-709 - GUCUCACUGUGUUGCCCAGGC 21 downstream

Table 8D provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCA10 target position in the CEP290 gene selected according to the fourth tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation and PAM is

NNGRRT. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 8D

CEP290-730 - AAAAAAUAAAAAAACUAGAG 20 upstream

CEP290-731 - AAAAAUAAAAAAACUAGAG 19 upstream

CEP290-732 - A A A A U A A A A A A AC U AG AG 18 upstream

CEP290-733 + CAAUAAAAAAUGAUGAGAAUUUA 23 upstream

CEP290-734 - UGUAGAAUAAAUUUAUUUAAUG 22 upstream

CEP290-735 - UAGAAUAAAUUUAUUUAAUG 20 upstream

CEP290-736 + U U U AU U CU ACAAU AAAAAAUG AU 23 upstream

CEP290-737 + U UAUUCUACAAUAAAAAAUGAU 22 upstream

CEP290-738 + UAUUCUACAAUAAAAAAUGAU 21 upstream

CEP290-739 + UUCUACAAUAAAAAAUGAU 19 upstream

CEP290-740 + UCUACAAUAAAAAAUGAU 18 upstream

CEP290-741 + UAAAAAAUGAUGAGAAUUUA 20 upstream

CEP290-742 - UGUAGAAUAAAAAAUAAAAAAAC 23 upstream

CEP290-743 - UAGAAUAAAAAAUAAAAAAAC 21 upstream

CEP290-744 - UAAAAAAUAAAAAAACUAGAG 21 upstream

CEP290-745 - AAAAGAAAUAGAUGUAGAUUGAGG 24 downstream

CEP290-746 - AAAGAAAUAGAUGUAGAUUGAGG 23 downstream

CEP290-747 - AAGAAAUAGAUGUAGAUUGAGG 22 downstream

CEP290-748 - AGAAAUAGAUGUAGAUUGAGG 21 downstream

CEP290-749 - AAAUAGAUGUAGAUUGAGG 19 downstream

CEP290-750 - AAUAGAUGUAGAUUGAGG 18 downstream

CEP290-751 - AUAAUAAGGAAAUACAAAAACUGG 24 downstream

CEP290-752 - AAUAAGGAAAUACAAAAACUGG 22 downstream

CEP290-753 - A U A AG G A A A U AC A A A A AC U G G 21 downstream

CEP290-754 - AAGGAAAUACAAAAACUGG 19 downstream

CEP290-755 - AGGAAAUACAAAAACUGG 18 downstream

CEP290-756 - AUAGAUAAUAAGGAAAUACAAAAA 24 downstream

CEP290-757 - AGAUAAUAAGGAAAUACAAAAA 22 downstream

CEP290-758 - AUAAUAAGGAAAUACAAAAA 20 downstream

CEP290-759 - A A U A AG G AAA U AC A A A A A 18 downstream

CEP290-760 + AAAAAAAAAAACAACAAAAA 20 downstream

CEP290-761 + AAAAAAAAAACAACAAAAA 19 downstream

CEP290-762 + AAAAAAAAACAACAAAAA 18 downstream

CEP290-763 - AGAGUCUCACUGUGUUGCCCAGGC 24 downstream

CEP290-764 - AGUCUCACUGUGUUGCCCAGGC 22 downstream

CEP290-765 + CAAAAAAAAAAACAACAAAAA 21 downstream

CEP290-766 - CUCACUGUGUUGCCCAGGC 19 downstream

CEP290-767 - UAAUAAGGAAAUACAAAAACUGG 23 downstream

CEP290-768 - U AAGGAAAUACAAAAACUGG 20 downstream

CEP290-769 - U AGAUAAUAAGGAAAUACAAAAA 23 downstream

CEP290-770 - UAAUAAGGAAAUACAAAAA 19 downstream

CEP290-771 - UGUGUUGCCCAGGCUGGAGUGCAG 24 downstream CEP290-772 - UGUUGCCCAGGCUGGAGUGCAG 22 downstream

CEP290-773 - UUGCCCAGGCUGGAGUGCAG 20 downstream

CEP290-774 - UGCCCAGGCUGGAGUGCAG 19 downstream

CEP290-775 + U U U CAAAAAAAAAAAC AACAAAAA 24 downstream

CEP290-776 + U U C AAAAAAAAAAACAACAAAAA 23 downstream

CEP290-777 + U C AAAAAAAAAAACAACAAAAA 22 downstream

CEP290-778 - UUUUUGUUGUUUUUUUUUUUGAAA 24 downstream

CEP290-779 - UUUUGUUGU UUUUUUUUUUGAAA 23 downstream

CEP290-780 - UUUGUUGUU UUUUUUUUUGAAA 22 downstream

CEP290-781 - UUGUUGUUU UUUUUUUUGAAA 21 downstream

CEP290-782 - UGUUGUUUU UUUUUUUGAAA 20 downstream

CEP290-783 - UUGUUUUUUUUUUUGAAA 18 downstream

CEP290-784 - UCUCACUGUGUUGCCCAGGC 20 downstream

CEP290-785 - UCACUGUGUUGCCCAGGC 18 downstream

Table 8E provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCA10 target position in the CEP290 gene selected according to the fifth tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation and PAM is

NNGRRV. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 8E

CEP290-799 + AGCUAAAUCAUGCAAGUGACCUA 23 upstream

CEP290-800 + AAAUCAUGCAAGUGACCUA 19 upstream

CEP290-801 + AAUCAUGCAAGUGACCUA 18 upstream

CEP290-802 - AAACCUCUUUUAACCAGACAUCU 23 upstream

CEP290-803 - AACCUCUUUUAACCAGACAUCU 22 upstream

CEP290-804 - ACCUCU UUUAACCAGACAUCU 21 upstream

CEP290-805 + AGUUUGUUCUGGGUACAGGGGUAA 24 upstream

CEP290-806 + AUGACUCAUAAUUUAGUAGGAAUC 24 upstream

CEP290-807 + ACUCAUAAUUUAGUAGGAAUC 21 upstream

CEP290-808 - AAUGGAUGUAGCCAACAGUAG 21 upstream

CEP290-809 - AUGGAUGUAGCCAACAGUAG 20 upstream

CEP290-810 + AUCACCUCUCUUUGGCAAAAGCAG 24 upstream

CEP290-811 + ACCUCUCUUUGGCAAAAGCAG 21 upstream

CEP290-812 - AGGUAGAAUAUUGUAAUCAAAGG 23 upstream

CEP290-813 - AGAAUAUUGUAAUCAAAGG 19 upstream

CEP290-814 + AAGGAACAAAAGCCAGGGACC 21 upstream

CEP290-815 + AGGAACAAAAGCCAGGGACC 20 upstream

CEP290-816 + ACAUCCAUUCCAAGGAACAAAAGC 24 upstream

CEP290-817 + AUCCAUUCCAAGG A AC A A A AG C 22 upstream

CEP290-818 + AUUCCAAGGAACAAAAGC 18 upstream

CEP290-819 + AGAAUUAGAUCUUAUUCUACUCCU 24 upstream

CEP290-820 + AAUUAGAUCUUAUUCUACUCCU 22 upstream

CEP290-821 + AUUAGAUCUUAUUCUACUCCU 21 upstream

CEP290-822 + AGAUCUUAUUCUACUCCU 18 upstream

CEP290-823 - AUUUGUUCAUCUUCCUCAU 19 upstream

CEP290-824 - AGAGGUGAUUAUGUUACUUUUUA 23 upstream

CEP290-825 - AGGUGAUUAUGUUACUUUUUA 21 upstream

CEP290-826 - AACCUCUUUUAACCAGACAUCUAA 24 upstream

CEP290-827 - ACCUCU UUUAACCAGACAUCUAA 23 upstream

CEP290-828 + AUAAACAUGACUCAUAAUUUAG 22 upstream

CEP290-829 + AAACAUGACUCAUAAUU UAG 20 upstream

CEP290-830 + AACAUGACUCAUAAUUUAG 19 upstream

CEP290-831 + ACAUGACUCAUAAUUUAG 18 upstream

CEP290-832 - ACAGG U AG AAU AU UG U AAU CAAAG 24 upstream

CEP290-833 - AGGUAGAAUAUUGUAAUCAAAG 22 upstream

CEP290-834 - AGAAUAUUGUAAUCAAAG 18 upstream

CEP290-835 + AUAGUUUGUUCUGGGUACAGGGGU 24 upstream

CEP290-836 + AGUUUGUUCUGGGUACAGGGGU 22 upstream

CEP290-837 - AGACAUCUAAGAG A A A A AG G AG C 23 upstream

CEP290-838 - ACAUCUAAGAGAAAAAGGAGC 21 upstream

CEP290-839 - AUCUAAGAG A A A A AG G AG C 19 upstream

CEP290-840 + AGAGGAUAGGACAGAGGACA 20 upstream CEP290-841 + AGGAUAGGACAGAGGACA 18 upstream

CEP290-842 + AGGAAAGAUGAAAAAUACUCUU 22 upstream

CEP290-843 + AAAGAUGAAAAAUACUCUU 19 upstream

CEP290-844 + AAGAUGAAAAAUACUCUU 18 upstream

CEP290-845 + AGGAAAGAUGAAAAAUACUCUUU 23 upstream

CEP290-846 + AAAGAUGAAAAAUACUCUUU 20 upstream

CEP290-847 + AAGAUGAAAAAUACUCUUU 19 upstream

CEP290-848 + AGAUGAAAAAUACUCUU U 18 upstream

CEP290-849 + AGGAAAGAUGAAAAAUACUCU 21 upstream

CEP290-850 + AAAGAUGAAAAAUACUCU 18 upstream

CEP290-851 + AUAGGACAGAGGACAUGGAGAA 22 upstream

CEP290-852 + AGGACAGAGGACAUGGAGAA 20 upstream

CEP290-853 + AGGAUAGGACAGAGGACAUGGAGA 24 upstream

CEP290-854 + AUAGGACAGAGGACAUGGAGA 21 upstream

CEP290-855 + AGGACAGAGGACAUGGAGA 19 upstream

CEP290-856 + AAGGAACAAAAGCCAGGGACCAU 23 upstream

CEP290-857 + AGGAACAAAAGCCAGGGACCAU 22 upstream

CEP290-858 + AACAAAAGCCAGGGACCAU 19 upstream

CEP290-859 + ACAAAAGCCAGGGACCAU 18 upstream

CEP290-860 + ACAU U U AU U CU ACAAU AAAAAAUG 24 upstream

CEP290-861 + AUUUAUUCUACAAUAAAAAAUG 22 upstream

CEP290-862 + AUUCUACAAUAAAAAAUG 18 upstream

CEP290-863 + AUUGUGUGUGUGUGUGUGUGUUAU 24 upstream

CEP290-864 + CUACUGUUGGCUACAUCCAUUCC 23 upstream

CEP290-865 + CUGUUGGCUACAUCCAUUCC 20 upstream

CEP290-866 + CAGAGUGCAUCCAUGGUC 18 upstream

CEP290-867 - CUCCAGCAUUAGAAAGUCCUAGGC 24 upstream

CEP290-868 - CCAGCAUUAGAAAGUCCUAGGC 22 upstream

CEP290-869 - CAGCAUUAGAAAGUCCUAGGC 21 upstream

CEP290-870 - CAU UAGAAAGUCCUAGGC 18 upstream

CEP290-871 - CCCAUGGUCCCUGGCU UUUGUUCC 24 upstream

CEP290-872 - CCAUGGUCCCUGGCUUU UGUUCC 23 upstream

CEP290-873 - CAUGGUCCCUGGCUUUUGUUCC 22 upstream

CEP290-874 - CUCAUAGAGACACAUUCAGUAA 22 upstream

CEP290-875 - CAU AG AG ACACAU U CAG U AA 20 upstream

CEP290-876 - CUCAAAAGCUUUUGCUGGCUCA 22 upstream

CEP290-877 - CAAAAGCUUUUGCUGGCUCA 20 upstream

CEP290-878 + CCAUAAGCCUCUAUUUCUGAU 21 upstream

CEP290-879 + CAUAAGCCUCUAUUUCUGAU 20 upstream

CEP290-880 + CAGCUAAAUCAUGCAAGUGACCUA 24 upstream

CEP290-881 + CUAAAUCAUGCAAGUGACCUA 21 upstream

CEP290-882 - CAAACCUCUU UUAACCAGACAUCU 24 upstream CEP290-883 - CCUCUU UUAACCAGACAUCU 20 upstream

CEP290-884 - CUCUUUUAACCAGACAUCU 19 upstream

CEP290-885 + CUCAUAAUUUAGUAGGAAUC 20 upstream

CEP290-886 + CAUAAUUUAGUAGGAAUC 18 upstream

CEP290-887 + CACCUCUCUUUGGCAAAAGCAG 22 upstream

CEP290-888 + CCUCUCU UUGGCAAAAGCAG 20 upstream

CEP290-889 + CUCUCU UUGGCAAAAGCAG 19 upstream

CEP290-890 - CAGG U AG AAU AU UG U AAU CAAAGG 24 upstream

CEP290-891 + CCAAGGAACAAAAGCCAGGGACC 23 upstream

CEP290-892 + CAAGGAACAAAAGCCAGGGACC 22 upstream

CEP290-893 + CA U CCA U U CCAAG G AAC AAAAG C 23 upstream

CEP290-894 + CCAU U CCAAGG AACAAAAG C 20 upstream

CEP290-895 + CAUUCCAAGGAACAAAAGC 19 upstream

CEP290-896 + CUCUUGCCUAGGACUUUCUAAUGC 24 upstream

CEP290-897 + CU UGCCUAGGACUUUCUAAUGC 22 upstream

CEP290-898 + CCUAGGACUUUCUAAUGC 18 upstream

CEP290-899 - CCUGAUUUGUUCAUCU UCCUCAU 23 upstream

CEP290-900 - CUGAUUUGUUCAUCUUCCUCAU 22 upstream

CEP290-901 - CCUCUU UUAACCAGACAUCUAA 22 upstream

CEP290-902 - CUCUUUUAACCAGACAUCUAA 21 upstream

CEP290-903 - CU U U U AACCAG ACAU CU AA 19 upstream

CEP290-904 - CCUCUGUCCUAUCCUCUCCAGCAU 24 upstream

CEP290-905 - CUCUGUCCUAUCCUCUCCAGCAU 23 upstream

CEP290-906 - CUGUCCUAUCCUCUCCAGCAU 21 upstream

CEP290-907 - CAGG U AG AAU AU UG U AAU CAAAG 23 upstream

CEP290-908 + CUGGGUACAGGGGUAAGAGA 20 upstream

CEP290-909 - CUU UCUGCUGCUUUUGCCA 19 upstream

CEP290-910 - CAGACAUCUAAGAGAAAAAGGAGC 24 upstream

CEP290-911 - CAUCUAAGAGAAAAAGGAGC 20 upstream

CEP290-912 + CUGGAGAGGAUAGGACAGAGGACA 24 upstream

CEP290-913 + CAAGGAACAAAAGCCAGGGACCAU 24 upstream

CEP290-914 + CAU U U AU U CU ACAAU AAAAAAUG 23 upstream

CEP290-915 + GCUACUGUUGGCUACAUCCAUUCC 24 upstream

CEP290-916 + GUUGGCUACAUCCAUUCC 18 upstream

CEP290-917 - GCAUUAGAAAGUCCUAGGC 19 upstream

CEP290-918 - GGUCCCUGGCU UUUGUUCC 19 upstream

CEP290-919 - GUCCCUGGCUUU UGUUCC 18 upstream

CEP290-920 - GGCUCAUAGAGACACAU UCAGUAA 24 upstream

CEP290-921 - GCUCAUAGAGACACAUUCAGUAA 23 upstream

CEP290-922 - GCUCAAAAGCUUUUGCUGGCUCA 23 upstream

CEP290-923 + GCUAAAUCAUGCAAGUGACCUA 22 upstream

CEP290-924 + GUUUGUUCUGGGUACAGGGGUAA 23 upstream CEP290-925 + GUUCUGGGUACAGGGGUAA 19 upstream

CEP290-926 + GACUCAUAAUUUAGUAGGAAUC 22 upstream

CEP290-927 - GGAAUGGAUGUAGCCAACAGUAG 23 upstream

CEP290-928 - GAAUGGAUGUAGCCAACAGUAG 22 upstream

CEP290-929 - GGAUGUAGCCAACAGUAG 18 upstream

CEP290-930 - GGUAGAAUAUUGUAAU CAAAGG 22 upstream

CEP290-931 - GUAGAAUAUUGUAAUCAAAGG 21 upstream

CEP290-932 - GAAUAUUGUAAUCAAAGG 18 upstream

CEP290-933 + GGAACAAAAGCCAGGGACC 19 upstream

CEP290-934 + GAACAAAAGCCAGGGACC 18 upstream

CEP290-935 + GAAUUAGAUCUUAUUCUACUCCU 23 upstream

CEP290-936 + GCCUAGGACUU UCUAAUGC 19 upstream

CEP290-937 - GAUUUGUUCAUCUUCCUCAU 20 upstream

CEP290-938 - GAGAGGUGAUUAUGUUACUUUUUA 24 upstream

CEP290-939 - GAGGUGAUUAUGUUACUUUUUA 22 upstream

CEP290-940 - GGUGAUUAUGUUACUU UUUA 20 upstream

CEP290-941 - GUGAUUAUGUUACU UUUUA 19 upstream

CEP290-942 - GUCCUAUCCUCUCCAGCAU 19 upstream

CEP290-943 + GAUAAACAUGACUCAUAAUUUAG 23 upstream

CEP290-944 - GGUAGAAUAUUGUAAU CAAAG 21 upstream

CEP290-945 - GUAGAAUAUUGUAAUCAAAG 20 upstream

CEP290-946 + GUUCUGGGUACAGGGGUAAGAGA 23 upstream

CEP290-947 + GGGUACAGGGGUAAGAGA 18 upstream

CEP290-948 + GUUUGUUCUGGGUACAGGGGU 21 upstream

CEP290-949 - GU UUGCUUUCUGCUGCUUUUGCCA 24 upstream

CEP290-950 - GCUUUCUGCUGCUUUUGCCA 20 upstream

CEP290-951 - GACAUCUAAGAG A A A A AG G AG C 22 upstream

CEP290-952 + GGAGAGGAUAGGACAGAGGACA 22 upstream

CEP290-953 + GAGAGGAUAGGACAGAGGACA 21 upstream

CEP290-954 + GAGGAUAGGACAGAGGACA 19 upstream

CEP290-955 + GGAAAGAUGAAAAAUACUCUU 21 upstream

CEP290-956 + GAAAGAUGAAAAAUACUCUU 20 upstream

CEP290-957 + GGAAAGAUGAAAAAUACUCUUU 22 upstream

CEP290-958 + GAAAGAUGAAAAAUACUCUUU 21 upstream

CEP290-959 + GGAAAGAUGAAAAAUACUCU 20 upstream

CEP290-960 + GAAAGAUGAAAAAUACUCU 19 upstream

CEP290-961 + GGAUAGGACAGAGGACAUGGAGAA 24 upstream

CEP290-962 + GAUAGGACAGAGGACAUGGAGAA 23 upstream

CEP290-963 + GGACAGAGGACAUGGAGAA 19 upstream

CEP290-964 + GACAGAGGACAUGGAGAA 18 upstream

CEP290-965 + GGAUAGGACAGAGGACAUGGAGA 23 upstream

CEP290-966 + GAUAGGACAGAGGACAUGGAGA 22 upstream CEP290-967 + GGACAGAGGACAUGGAGA 18 upstream

CEP290-968 + GGAACAAAAGCCAGGGACCAU 21 upstream

CEP290-969 + GAACAAAAGCCAGGGACCAU 20 upstream

CEP290-970 + GUGUGUGUGUGUGUGUGUUAU 21 upstream

CEP290-971 + GUGUGUGUGUGUGUGUUAU 19 upstream

CEP290-972 + GUGUGUGUGUGUGUGUGUUAUG 22 upstream

CEP290-973 + GUGUGUGUGUGUGUGUUAUG 20 upstream

CEP290-974 + GUGUGUGUGUGUGUUAUG 18 upstream

CEP290-975 + UACUGUUGGCUACAUCCAUUCC 22 upstream

CEP290-976 + UGUUGGCUACAUCCAU UCC 19 upstream

CEP290-977 + UUUACAGAGUGCAUCCAUGGUC 22 upstream

CEP290-978 + UUACAGAGUGCAUCCAUGGUC 21 upstream

CEP290-979 + UACAGAGUGCAUCCAUGGUC 20 upstream

CEP290-980 - UCCAGCAUUAGAAAGUCCUAGGC 23 upstream

CEP290-981 - UGGUCCCUGGCUUUUGUUCC 20 upstream

CEP290-982 - UCAUAGAGACACAUUCAGUAA 21 upstream

CEP290-983 - UAGAGACACAUUCAGUAA 18 upstream

CEP290-984 - UCAAAAGCUUUUGCUGGCUCA 21 upstream

CEP290-985 + UCCAUAAGCCUCUAUUUCUGAU 22 upstream

CEP290-986 + UAAGCCUCUAUU UCUGAU 18 upstream

CEP290-987 + UAAAUCAUGCAAGUGACCUA 20 upstream

CEP290-988 - UCU UUUAACCAGACAUCU 18 upstream

CEP290-989 + UUUGUUCUGGGUACAGGGGUAA 22 upstream

CEP290-990 + UUGUUCUGGGUACAGGGGUAA 21 upstream

CEP290-991 + UGUUCUGGGUACAGGGGUAA 20 upstream

CEP290-992 + UUCUGGGUACAGGGGUAA 18 upstream

CEP290-993 + UGACUCAUAAUUUAGUAGGAAUC 23 upstream

CEP290-994 + UCAUAAUUUAGUAGGAAUC 19 upstream

CEP290-995 - UGGAAUGGAUGUAGCCAACAGUAG 24 upstream

CEP290-996 - UGGAUGUAGCCAACAGUAG 19 upstream

CEP290-997 + U CACCU CU CU U U GG CAAAAGCAG 23 upstream

CEP290-998 + UCUCUUUGGCAAAAGCAG 18 upstream

CEP290-999 - UAGAAUAUUGUAAUCAAAGG 20 upstream

CEP290-1000 + UCCAAGGAACAAAAGCCAGGGACC 24 upstream

CEP290-1001 + UCCAUUCCAAGG A AC A A A AG C 21 upstream

CEP290-1002 + UUAGAUCUUAUUCUACUCCU 20 upstream

CEP290-1003 + UAGAUCUUAUUCUACUCCU 19 upstream

CEP290-1004 + UCUUGCCUAGGACUUUCUAAUGC 23 upstream

CEP290-1005 + UUGCCUAGGACUUUCUAAUGC 21 upstream

CEP290-1006 + UGCCUAGGACU UUCUAAUGC 20 upstream

CEP290-1007 - UCCUGAUUUGUUCAUCUUCCUCAU 24 upstream

CEP290-1008 - UGAUUUGUUCAUCUUCCUCAU 21 upstream CEP290-1009 - UUUGUUCAUCUUCCUCAU 18 upstream

CEP290-1010 - UGAUUAUGUUACUUUUUA 18 upstream

CEP290-1011 - UCUUUUAACCAGACAUCUAA 20 upstream

CEP290-1012 - U U U U AACCAG ACAU CU AA 18 upstream

CEP290-1013 - UCUGUCCUAUCCUCUCCAGCAU 22 upstream

CEP290-1014 - UGUCCUAUCCUCUCCAGCAU 20 upstream

CEP290-1015 - UCCUAUCCUCUCCAGCAU 18 upstream

CEP290-1016 + UGAUAAACAUGACUCAUAAUUUAG 24 upstream

CEP290-1017 + UAAACAUGACUCAUAAUUUAG 21 upstream

CEP290-1018 - UAGAAUAUUGUAAUCAAAG 19 upstream

CEP290-1019 + UGUUCUGGGUACAGGGGUAAGAGA 24 upstream

CEP290-1020 + UUCUGGGUACAGGGGUAAGAGA 22 upstream

CEP290-1021 + UCUGGGUACAGGGGUAAGAGA 21 upstream

CEP290-1022 + UGGGUACAGGGGUAAGAGA 19 upstream

CEP290-1023 + UAGUUUGUUCUGGGUACAGGGGU 23 upstream

CEP290-1024 + UUUGUUCUGGGUACAGGGGU 20 upstream

CEP290-1025 + UUGUUCUGGGUACAGGGGU 19 upstream

CEP290-1026 + UGUUCUGGGUACAGGGGU 18 upstream

CEP290-1027 - UUUGCUUUCUGCUGCUUUUGCCA 23 upstream

CEP290-1028 - UUGCUUUCUGCUGCUUUUGCCA 22 upstream

CEP290-1029 - UGCUUUCUGCUGCUUUUGCCA 21 upstream

CEP290-1030 - UUUCUGCUGCUUUUGCCA 18 upstream

CEP290-1031 - U C U A AG AG A A A A AG G AG C 18 upstream

CEP290-1032 + UGGAGAGGAUAGGACAGAGGACA 23 upstream

CEP290-1033 + UUAGGAAAGAUGAAAAAUACUCUU 24 upstream

CEP290-1034 + UAGGAAAGAUGAAAAAUACUCUU 23 upstream

CEP290-1035 + UAGGAAAGAUGAAAAAUACUCUUU 24 upstream

CEP290-1036 + UUUAGGAAAGAUGAAAAAUACUCU 24 upstream

CEP290-1037 + UUAGGAAAGAUGAAAAAUACUCU 23 upstream

CEP290-1038 + UAGGAAAGAUGAAAAAUACUCU 22 upstream

CEP290-1039 + UAGGACAGAGGACAUGGAGAA 21 upstream

CEP290-1040 + UAGGACAGAGGACAUGGAGA 20 upstream

CEP290-1041 + UUUAUUCUACAAUAAAAAAUG 21 upstream

CEP290-1042 + UUAUUCUACAAUAAAAAAUG 20 upstream

CEP290-1043 + UAUUCUACAAUAAAAAAUG 19 upstream

CEP290-1044 + UUGUGUGUGUGUGUGUGUGUUAU 23 upstream

CEP290-1045 + UGUGUGUGUGUGUGUGUGUUAU 22 upstream

CEP290-1046 + UGUGUGUGUGUGUGUGUUAU 20 upstream

CEP290-1047 + UGUGUGUGUGUGUGUUAU 18 upstream

CEP290-1048 + UUGUGUGUGUGUGUGUGUGUUAUG 24 upstream

CEP290-1049 + UGUGUGUGUGUGUGUGUGUUAUG 23 upstream

CEP290-1050 + UGUGUGUGUGUGUGUGUUAUG 21 upstream CEP290-1051 + UGUGUGUGUGUGUGUUAUG 19 upstream

CEP290-1052 + ACUGUUGGCUACAUCCAUUCCA 22 upstream

CEP290-1053 + AUUAUCCACAAGAUGUCUCUUGCC 24 upstream

CEP290-1054 + AUCCACAAGAUGUCUCU UGCC 21 upstream

CEP290-1055 + AUGAGCCAG C A A A AG C U U 18 upstream

CEP290-1056 + ACAGAGUGCAUCCAUGGUCCAGG 23 upstream

CEP290-1057 + AGAGUGCAUCCAUGGUCCAGG 21 upstream

CEP290-1058 + AGUGCAUCCAUGGUCCAGG 19 upstream

CEP290-1059 - AGCUGAAAUAUUAAGGGCUCUUC 23 upstream

CEP290-1060 - AAAUAUUAAGGGCUCUUC 18 upstream

CEP290-1061 - AACUCUAUACCU UUUACUGAGGA 23 upstream

CEP290-1062 - ACUCUAUACCUUUUACUGAGGA 22 upstream

CEP290-1063 - ACUUGAACUCUAUACCUUUUACU 23 upstream

CEP290-1064 - AACUCUAUACCU UUUACU 18 upstream

CEP290-1065 + AGUAGGAAUCCUGAAAGCUACU 22 upstream

CEP290-1066 + AGGAAUCCUGAAAGCUACU 19 upstream

CEP290-1067 - AGCCAACAGUAGCUGAAAUAUU 22 upstream

CEP290-1068 - AACAGUAGCUGAAAUAUU 18 upstream

CEP290-1069 + AUCCAUUCCAAGG A AC A A A AG CC 23 upstream

CEP290-1070 + AUUCCAAGGAACAAAAGCC 19 upstream

CEP290-1071 - AUCCCU UUCUCU UACCCCUGUACC 24 upstream

CEP290-1072 + AGGACUUUCUAAUGCUGGAGAGGA 24 upstream

CEP290-1073 + ACUUUCUAAUGCUGGAGAGGA 21 upstream

CEP290-1074 + AAUGCUGGAGAGGAUAGGACA 21 upstream

CEP290-1075 + AUGCUGGAGAGGAUAGGACA 20 upstream

CEP290-1076 - AUCAUAAGUUACAAUCUGUGAAU 23 upstream

CEP290-1077 - AUAAGUUACAAUCUGUGAAU 20 upstream

CEP290-1078 - AAGUUACAAUCUGUGAAU 18 upstream

CEP290-1079 - AACCAGACAUCUAAGAGAAAA 21 upstream

CEP290-1080 - ACCAGACAUCUAAGAGAAAA 20 upstream

CEP290-1081 + AAGCCUCUAUUUCUGAUGAGGAAG 24 upstream

CEP290-1082 + AGCCUCUAUU UCUGAUGAGGAAG 23 upstream

CEP290-1083 + AUGAGGAAGAUGAACAAAUC 20 upstream

CEP290-1084 + AUUUACUGAAUGUGUCUCU 19 upstream

CEP290-1085 + ACAGGGGUAAGAGAAAGGG 19 upstream

CEP290-1086 + CUACUGUUGGCUACAUCCAUUCCA 24 upstream

CEP290-1087 + CUGUUGGCUACAUCCAUUCCA 21 upstream

CEP290-1088 + CCACAAGAUGUCUCUUGCC 19 upstream

CEP290-1089 + CACAAGAUGUCUCUUGCC 18 upstream

CEP290-1090 - CCUUUGUAGUUAUCUUACAGCCAC 24 upstream

CEP290-1091 - CU UUGUAGUUAUCUUACAGCCAC 23 upstream

CEP290-1092 + CUCUAUGAGCCAGCAAAAGCUU 22 upstream CEP290-1093 + CUAUGAGCCAGCAAAAGCUU 20 upstream

CEP290-1094 + CAGAGUGCAUCCAUGGUCCAGG 22 upstream

CEP290-1095 - CUGAAAUAUUAAGGGCUCUUC 21 upstream

CEP290-1096 - CUCUAUACCUUUUACUGAGGA 21 upstream

CEP290-1097 - CUAUACCUUUUACUGAGGA 19 upstream

CEP290-1098 - CACUUGAACUCUAUACCUUUUACU 24 upstream

CEP290-1099 - CUUGAACUCUAUACCUUUUACU 22 upstream

CEP290-1100 - CCAACAGUAGCUGAAAUAUU 20 upstream

CEP290-1101 - CAACAGUAGCUGAAAUAUU 19 upstream

CEP290-1102 + CA U CC AU U CCAAG G AAC AAAAG CC 24 upstream

CEP290-1103 + CCAUUCCAAGGAACAAAAGCC 21 upstream

CEP290-1104 + CAUUCCAAGGAACAAAAGCC 20 upstream

CEP290-1105 - CCCUUUCUCUUACCCCUGUACC 22 upstream

CEP290-1106 - CCUUUCUCUUACCCCUGUACC 21 upstream

CEP290-1107 - CUUUCUCUUACCCCUGUACC 20 upstream

CEP290-1108 + CUUUCUAAUGCUGGAGAGGA 20 upstream

CEP290-1109 + CUAAUGCUGGAGAGGAUAGGACA 23 upstream

CEP290-1110 - CAUAAGUUACAAUCUGUGAAU 21 upstream

CEP290-1111 - CCAGACAUCUAAGAGAAAA 19 upstream

CEP290-1112 - CAGACAUCUAAGAGAAAA 18 upstream

CEP290-1113 + CCUCUAUUUCUGAUGAGGAAG 21 upstream

CEP290-1114 + CUCUAUUUCUGAUGAGGAAG 20 upstream

CEP290-1115 + CUAUUUCUGAUGAGGAAG 18 upstream

CEP290-1116 + CUGAUGAGGAAGAUGAACAAAUC 23 upstream

CEP290-1117 + CAUUUACUGAAUGUGUCUCU 20 upstream

CEP290-1118 + CAGGGGUAAGAGAAAGGG 18 upstream

CEP290-1119 + GUUGGCUACAUCCAUUCCA 19 upstream

CEP290-1120 - GUAGUUAUCUUACAGCCAC 19 upstream

CEP290-1121 + GUCUCUAUGAGCCAGCAAAAGCUU 24 upstream

CEP290-1122 + GAGUGCAUCCAUGGUCCAGG 20 upstream

CEP290-1123 + GUGCAUCCAUGGUCCAGG 18 upstream

CEP290-1124 - GCUGAAAUAUUAAGGGCUCUUC 22 upstream

CEP290-1125 - GAAAUAUUAAGGGCUCUUC 19 upstream

CEP290-1126 - GAACUCUAUACCUUUUACUGAGGA 24 upstream

CEP290-1127 - GAACUCUAUACCUUUUACU 19 upstream

CEP290-1128 + GUAGGAAUCCUGAAAGCUACU 21 upstream

CEP290-1129 + GGAAUCCUGAAAGCUACU 18 upstream

CEP290-1130 - GUAGCCAACAGUAGCUGAAAUAUU 24 upstream

CEP290-1131 - GCCAACAGUAGCUGAAAUAUU 21 upstream

CEP290-1132 + GGACUUUCUAAUGCUGGAGAGGA 23 upstream

CEP290-1133 + GACUUUCUAAUGCUGGAGAGGA 22 upstream

CEP290-1134 + GCUGGAGAGGAUAGGACA 18 upstream CEP290-1135 + GCCUCUAUUUCUGAUGAGGAAG 22 upstream

CEP290-1136 + GAUGAGGAAGAUGAACAAAUC 21 upstream

CEP290-1137 + GAGGAAGAUGAACAAAUC 18 upstream

CEP290-1138 + GGGUACAGGGGUAAGAGAAAGGG 23 upstream

CEP290-1139 + GGUACAGGGGUAAGAGAAAGGG 22 upstream

CEP290-1140 + GUACAGGGGUAAGAGAAAGGG 21 upstream

CEP290-1141 + GUGUGUGUGUGUGUGUGUUAUGU 23 upstream

CEP290-1142 + GUGUGUGUGUGUGUGUUAUGU 21 upstream

CEP290-1143 + GUGUGUGUGUGUGUUAUGU 19 upstream

CEP290-1144 + UACUGUUGGCUACAUCCAUUCCA 23 upstream

CEP290-1145 + UGUUGGCUACAUCCAUUCCA 20 upstream

CEP290-1146 + UUGGCUACAUCCAUUCCA 18 upstream

CEP290-1147 + UUAUCCACAAGAUGUCUCUUGCC 23 upstream

CEP290-1148 + UAUCCACAAGAUGUCUCUUGCC 22 upstream

CEP290-1149 + UCCACAAGAUGUCUCUUGCC 20 upstream

CEP290-1150 - UUUGUAGUUAUCUUACAGCCAC 22 upstream

CEP290-1151 - UUGUAGUUAUCUUACAGCCAC 21 upstream

CEP290-1152 - UGUAGUUAUCUUACAGCCAC 20 upstream

CEP290-1153 - UAGUUAUCUUACAGCCAC 18 upstream

CEP290-1154 + UCUCUAUGAGCCAGCAAAAGCUU 23 upstream

CEP290-1155 + UCUAUGAGCCAGCAAAAGCUU 21 upstream

CEP290-1156 + UAUGAGCCAG C A A A AG C U U 19 upstream

CEP290-1157 + UACAGAGUGCAUCCAUGGUCCAGG 24 upstream

CEP290-1158 - UAGCUGAAAUAUUAAGGGCUCUUC 24 upstream

CEP290-1159 - UGAAAUAUUAAGGGCUCUUC 20 upstream

CEP290-1160 - UCUAUACCUUUUACUGAGGA 20 upstream

CEP290-1161 - UAUACCUUUUACUGAGGA 18 upstream

CEP290-1162 - UUGAACUCUAUACCUUUUACU 21 upstream

CEP290-1163 - UGAACUCUAUACCUUUUACU 20 upstream

CEP290-1164 + UUAGUAGGAAUCCUGAAAGCUACU 24 upstream

CEP290-1165 + UAGUAGGAAUCCUGAAAGCUACU 23 upstream

CEP290-1166 + UAGGAAUCCUGAAAGCUACU 20 upstream

CEP290-1167 - UAGCCAACAGUAGCUGAAAUAUU 23 upstream

CEP290-1168 + UCCAUUCCAAGG A AC A A A AG CC 22 upstream

CEP290-1169 + UUCCAAGGAACAAAAGCC 18 upstream

CEP290-1170 - UCCCUUUCUCUUACCCCUGUACC 23 upstream

CEP290-1171 - UUUCUCUUACCCCUGUACC 19 upstream

CEP290-1172 - UUCUCUUACCCCUGUACC 18 upstream

CEP290-1173 + UUUCUAAUGCUGGAGAGGA 19 upstream

CEP290-1174 + UUCUAAUGCUGGAGAGGA 18 upstream

CEP290-1175 + UCUAAUGCUGGAGAGGAUAGGACA 24 upstream

CEP290-1176 + UAAUGCUGGAGAGGAUAGGACA 22 upstream CEP290-1177 + UGCUGGAGAGGAUAGGACA 19 upstream

CEP290-1178 - UAUCAUAAGUUACAAUCUGUGAAU 24 upstream

CEP290-1179 - UCAUAAGUUACAAUCUGUGAAU 22 upstream

CEP290-1180 - UAAGUUACAAUCUGUGAAU 19 upstream

CEP290-1181 - U U U AACCAG ACAU CU AAG AG AAAA 24 upstream

CEP290-1182 - UUAACCAGACAUCUAAGAGAAAA 23 upstream

CEP290-1183 - UAACCAGACAUCUAAGAGAAAA 22 upstream

CEP290-1184 + UCUAUUUCUGAUGAGGAAG 19 upstream

CEP290-1185 + UCUGAUGAGGAAGAUGAACAAAUC 24 upstream

CEP290-1186 + UGAUGAGGAAGAUGAACAAAUC 22 upstream

CEP290-1187 + UGAGGAAGAUGAACAAAUC 19 upstream

CEP290-1188 + UUUUCAUUUACUGAAUGUGUCUCU 24 upstream

CEP290-1189 + UUUCAUUUACUGAAUGUGUCUCU 23 upstream

CEP290-1190 + UUCAUUUACUGAAUGUGUCUCU 22 upstream

CEP290-1191 + UCAUUUACUGAAUGUGUCUCU 21 upstream

CEP290-1192 + UUUACUGAAUGUGUCUCU 18 upstream

CEP290-1193 + UGGGUACAGGGGUAAGAGAAAGGG 24 upstream

CEP290-1194 + UACAGGGGUAAGAGAAAGGG 20 upstream

CEP290-1195 + UGUGUGUGUGUGUGUGUGUUAUGU 24 upstream

CEP290-1196 + UGUGUGUGUGUGUGUGUUAUGU 22 upstream

CEP290-1197 + UGUGUGUGUGUGUGUUAUGU 20 upstream

CEP290-1198 + UGUGUGUGUGUGUUAUGU 18 upstream

CEP290-1199 + AUUUACAGAGUGCAUCCAUGGUCC 24 upstream

CEP290-1200 + ACAGAGUGCAUCCAUGGUCC 20 upstream

CEP290-1201 + AGAGUGCAUCCAUGGUCC 18 upstream

CEP290-1202 - ACUUGAACUCUAUACCUUUUA 21 upstream

CEP290-1203 + AGCUAAAUCAUGCAAGUGACCU 22 upstream

CEP290-1204 + AAAUCAUGCAAGUGACCU 18 upstream

CEP290-1205 + AUCCAUAAGCCUCUAUUUCUGAUG 24 upstream

CEP290-1206 + AUAAGCCUCUAUUUCUGAUG 20 upstream

CEP290-1207 + AAGCCUCUAUUUCUGAUG 18 upstream

CEP290-1208 + AGAAUAGUUUGUUCUGGGUA 20 upstream

CEP290-1209 + AAUAGUUUGUUCUGGGUA 18 upstream

CEP290-1210 + AGGAGAAUGAUCUAGAUAAUCAUU 24 upstream

CEP290-1211 + AGAAUGAUCUAGAUAAUCAUU 21 upstream

CEP290-1212 + AAUGAUCUAGAUAAUCAUU 19 upstream

CEP290-1213 + AUGAUCUAGAUAAUCAUU 18 upstream

CEP290-1214 + AAUGCUGGAGAGGAUAGGA 19 upstream

CEP290-1215 + AUGCUGGAGAGGAUAGGA 18 upstream

CEP290-1216 + AAAAUCCAUAAGCCUCUAUUUCUG 24 upstream

CEP290-1217 + AAAUCCAUAAGCCUCUAUUUCUG 23 upstream

CEP290-1218 + AAUCCAUAAGCCUCUAUUUCUG 22 upstream CEP290-1219 + AUCCAUAAGCCUCUAUUUCUG 21 upstream

CEP290-1220 - AAACAGG U AG AAU AU UG U AAU CA 23 upstream

CEP290-1221 - AACAGG U AG AAU AU UG U AAU CA 22 upstream

CEP290-1222 - ACAGGUAGAAUAUUGUAAUCA 21 upstream

CEP290-1223 - AGGUAGAAUAUUGUAAUCA 19 upstream

CEP290-1224 + AAGGAACAAAAGCCAGGGACCA 22 upstream

CEP290-1225 + AGGAACAAAAGCCAGGGACCA 21 upstream

CEP290-1226 + AACAAAAGCCAGGGACCA 18 upstream

CEP290-1227 - AGGUAGAAUAUUGUAAUCAAAGGA 24 upstream

CEP290-1228 - AGAAUAUUGUAAUCAAAGGA 20 upstream

CEP290-1229 - AAUAUUGUAAUCAAAGGA 18 upstream

CEP290-1230 - AGUCAUGUUUAUCAAUAUUAUU 22 upstream

CEP290-1231 - AUGUUUAUCAAUAUUAUU 18 upstream

CEP290-1232 - AACCAGACAUCUAAGAGAAA 20 upstream

CEP290-1233 - ACCAGACAUCUAAGAGAAA 19 upstream

CEP290-1234 - AUUCUUAUCUAAGAUCCUUUCA 22 upstream

CEP290-1235 - AAACAGG U AG AAU AU UG U AAU CAA 24 upstream

CEP290-1236 - AACAGG U AG AAU AU UG U AAU CAA 23 upstream

CEP290-1237 - ACAGG U AG AAU AU UG U AAU CAA 22 upstream

CEP290-1238 - AGGUAGAAUAUUGUAAUCAA 20 upstream

CEP290-1239 + AUGAGGAAGAUGAACAAAU 19 upstream

CEP290-1240 + AGAGGAUAGGACAGAGGAC 19 upstream

CEP290-1241 + CAGAGUGCAUCCAUGGUCC 19 upstream

CEP290-1242 + CU UGCCUAGGACUUUCUAAUGCUG 24 upstream

CEP290-1243 + CCUAGGACUUUCUAAUGCUG 20 upstream

CEP290-1244 + CUAGGACUUUCUAAUGCUG 19 upstream

CEP290-1245 - CCACUUGAACUCUAUACCUUUUA 23 upstream

CEP290-1246 - CACUUGAACUCUAUACCUUUUA 22 upstream

CEP290-1247 - CU UGAACUCUAUACCUUUUA 20 upstream

CEP290-1248 + CAGCUAAAUCAUGCAAGUGACCU 23 upstream

CEP290-1249 + CUAAAUCAUGCAAGUGACCU 20 upstream

CEP290-1250 + CUCUUGCCUAGGACUUUCUAAUG 23 upstream

CEP290-1251 + CU UGCCUAGGACUUUCUAAUG 21 upstream

CEP290-1252 + CCAUAAGCCUCUAUUUCUGAUG 22 upstream

CEP290-1253 + CAUAAGCCUCUAUUUCUGAUG 21 upstream

CEP290-1254 + CUAAUGCUGGAGAGGAUAGGA 21 upstream

CEP290-1255 + CCAUAAGCCUCUAUUUCUG 19 upstream

CEP290-1256 + CAUAAGCCUCUAUUUCUG 18 upstream

CEP290-1257 - CAGGUAGAAUAUUGUAAUCA 20 upstream

CEP290-1258 - CU UUCUGCUGCUUUUGCCAAA 21 upstream

CEP290-1259 + CCAAGGAACAAAAGCCAGGGACCA 24 upstream

CEP290-1260 + CAAGGAACAAAAGCCAGGGACCA 23 upstream CEP290-1261 + CUCUUAGAUGUCUGGU UAA 19 upstream

CEP290-1262 - CAUGUUUAUCAAUAUUAUU 19 upstream

CEP290-1263 - CCAGACAUCUAAGAGAAA 18 upstream

CEP290-1264 - CU UAUCUAAGAUCCUUUCA 19 upstream

CEP290-1265 - CAGG U AG AAU AU UG U AAU CAA 21 upstream

CEP290-1266 + CUGAUGAGGAAGAUGAACAAAU 22 upstream

CEP290-1267 + CUGGAGAGGAUAGGACAGAGGAC 23 upstream

CEP290-1268 - CAUCUUCCUCAUCAGAAA 18 upstream

CEP290-1269 + GCCUAGGACUU UCUAAUGCUG 21 upstream

CEP290-1270 - GCCACUUGAACUCUAUACCUUUUA 24 upstream

CEP290-1271 + GCUAAAUCAUGCAAGUGACCU 21 upstream

CEP290-1272 + GCCUAGGACUU UCUAAUG 18 upstream

CEP290-1273 + GGGAGAAUAGUUUGUUCUGGGUA 23 upstream

CEP290-1274 + GGAGAAUAGUUUGUUCUGGGUA 22 upstream

CEP290-1275 + GAGAAUAGUUUGUUCUGGGUA 21 upstream

CEP290-1276 + GAAUAGUUUGUUCUGGGUA 19 upstream

CEP290-1277 + GGAGAAUGAUCUAGAUAAUCAUU 23 upstream

CEP290-1278 + GAGAAUGAUCUAGAUAAUCAUU 22 upstream

CEP290-1279 + GAAUGAUCUAGAUAAUCAUU 20 upstream

CEP290-1280 - GAAACAGGUAGAAUAUUGUAAUCA 24 upstream

CEP290-1281 - GGUAGAAUAUUGUAAUCA 18 upstream

CEP290-1282 - GCUUUCUGCUGCUUUUGCCAAA 22 upstream

CEP290-1283 + GGAACAAAAGCCAGGGACCA 20 upstream

CEP290-1284 + GAACAAAAGCCAGGGACCA 19 upstream

CEP290-1285 - GGUAGAAUAUUGUAAUCAAAGGA 23 upstream

CEP290-1286 - G U AG AAU AU UG U AAU CAAAGG A 22 upstream

CEP290-1287 - GAAUAUUGUAAUCAAAGGA 19 upstream

CEP290-1288 - GAGUCAUGUUUAUCAAUAUUAUU 23 upstream

CEP290-1289 - GUCAUGUUUAUCAAUAUUAUU 21 upstream

CEP290-1290 - GGUAGAAUAUUGUAAUCAA 19 upstream

CEP290-1291 - G U AG AAU AU UG U AAU CAA 18 upstream

CEP290-1292 + GAUGAGGAAGAUGAACAAAU 20 upstream

CEP290-1293 + GCUGGAGAGGAUAGGACAGAGGAC 24 upstream

CEP290-1294 + GGAGAGGAUAGGACAGAGGAC 21 upstream

CEP290-1295 + GAGAGGAUAGGACAGAGGAC 20 upstream

CEP290-1296 + GAGGAUAGGACAGAGGAC 18 upstream

CEP290-1297 - GUUCAUCUUCCUCAUCAGAAA 21 upstream

CEP290-1298 + UUUACAGAGUGCAUCCAUGGUCC 23 upstream

CEP290-1299 + UUACAGAGUGCAUCCAUGGUCC 22 upstream

CEP290-1300 + UACAGAGUGCAUCCAUGGUCC 21 upstream

CEP290-1301 + UUGCCUAGGACUUUCUAAUGCUG 23 upstream

CEP290-1302 + UGCCUAGGACU UUCUAAUGCUG 22 upstream CEP290-1303 + UAGGACUUUCUAAUGCUG 18 upstream

CEP290-1304 - UUGAACUCUAUACCUUUUA 19 upstream

CEP290-1305 - UGAACUCUAUACCUUUUA 18 upstream

CEP290-1306 + UCAGCUAAAUCAUGCAAGUGACCU 24 upstream

CEP290-1307 + UAAAUCAUGCAAGUGACCU 19 upstream

CEP290-1308 + UCUCUUGCCUAGGACUUUCUAAUG 24 upstream

CEP290-1309 + UCUUGCCUAGGACUUUCUAAUG 22 upstream

CEP290-1310 + UUGCCUAGGACUUUCUAAUG 20 upstream

CEP290-1311 + UGCCUAGGACUUUCUAAUG 19 upstream

CEP290-1312 + UCCAUAAGCCUCUAUUUCUGAUG 23 upstream

CEP290-1313 + UAAGCCUCUAUUUCUGAUG 19 upstream

CEP290-1314 + UGGGAGAAUAGUUUGUUCUGGGUA 24 upstream

CEP290-1315 + UUUCUAAUGCUGGAGAGGAUAGGA 24 upstream

CEP290-1316 + UUCUAAUGCUGGAGAGGAUAGGA 23 upstream

CEP290-1317 + UCUAAUGCUGGAGAGGAUAGGA 22 upstream

CEP290-1318 + UAAUGCUGGAGAGGAUAGGA 20 upstream

CEP290-1319 + UCCAUAAGCCUCUAUUUCUG 20 upstream

CEP290-1320 - UUGCUUUCUGCUGCUUUUGCCAAA 24 upstream

CEP290-1321 - UGCUUUCUGCUGCUUUUGCCAAA 23 upstream

CEP290-1322 - UUUCUGCUGCUUUUGCCAAA 20 upstream

CEP290-1323 - UUCUGCUGCUUUUGCCAAA 19 upstream

CEP290-1324 - UCUGCUGCUUUUGCCAAA 18 upstream

CEP290-1325 - UAGAAUAUUGUAAUCAAAGGA 21 upstream

CEP290-1326 + UUUUUCUCUUAGAUGUCUGGUUAA 24 upstream

CEP290-1327 + UUUUCUCUUAGAUGUCUGGUUAA 23 upstream

CEP290-1328 + UUUCUCUUAGAUGUCUGGUUAA 22 upstream

CEP290-1329 + UUCUCUUAGAUGUCUGGUUAA 21 upstream

CEP290-1330 + UCUCUUAGAUGUCUGGUUAA 20 upstream

CEP290-1331 + UCUUAGAUGUCUGGUUAA 18 upstream

CEP290-1332 - UGAGUCAUGUUUAUCAAUAUUAUU 24 upstream

CEP290-1333 - UCAUGUUUAUCAAUAUUAUU 20 upstream

CEP290-1334 - U U U U AACCAG ACAU CU AAG AG AAA 24 upstream

CEP290-1335 - U U U AACCAG ACAU CU AAG AG AAA 23 upstream

CEP290-1336 - UUAACCAGACAUCUAAGAGAAA 22 upstream

CEP290-1337 - UAACCAGACAUCUAAGAGAAA 21 upstream

CEP290-1338 - UUAUUCUUAUCUAAGAUCCUUUCA 24 upstream

CEP290-1339 - UAUUCUUAUCUAAGAUCCUUUCA 23 upstream

CEP290-1340 - UUCUUAUCUAAGAUCCUUUCA 21 upstream

CEP290-1341 - UCUUAUCUAAGAUCCUUUCA 20 upstream

CEP290-1342 - UUAUCUAAGAUCCUUUCA 18 upstream

CEP290-1343 + UUCUGAUGAGGAAGAUGAACAAAU 24 upstream

CEP290-1344 + UCUGAUGAGGAAGAUGAACAAAU 23 upstream CEP290-1345 + UGAUGAGGAAGAUGAACAAAU 21 upstream

CEP290-1346 + UGAGGAAGAUGAACAAAU 18 upstream

CEP290-1347 + UGGAGAGGAUAGGACAGAGGAC 22 upstream

CEP290-1348 - UUUGUUCAUCUUCCUCAUCAGAAA 24 upstream

CEP290-1349 - UUGUUCAUCUUCCUCAUCAGAAA 23 upstream

CEP290-1350 - UGUUCAUCUUCCUCAUCAGAAA 22 upstream

CEP290-1351 - UUCAUCUUCCUCAUCAGAAA 20 upstream

CEP290-1352 - UCAUCUUCCUCAUCAGAAA 19 upstream

CEP290-1353 - ACUUACCUCAUGUCAUCUAGAGC 23 downstream

CEP290-1354 - ACCUCAUGUCAUCUAGAGC 19 downstream

CEP290-1355 + ACAGUUUUUAAGGCGGGGAGUCAC 24 downstream

CEP290-1356 + AGUUUUUAAGGCGGGGAGUCAC 22 downstream

CEP290-1357 - ACAGAGUUCAAGCUAAUAC 19 downstream

CEP290-1358 + AUUAGCUUGAACUCUGUGCCAAAC 24 downstream

CEP290-1359 + AGCUUGAACUCUGUGCCAAAC 21 downstream

CEP290-1360 - AUGUGGUGUCAAAUAUGGUGCU 22 downstream

CEP290-1361 - AUGUGGUGUCAAAUAUGGUGCUU 23 downstream

CEP290-1362 + AGAUGACAUGAGGUAAGU 18 downstream

CEP290-1363 - AAUACAUGAGAGUGAUUAGUGG 22 downstream

CEP290-1364 - AUACAUGAGAGUGAUUAGUGG 21 downstream

CEP290-1365 - ACAUGAGAGUGAUUAGUGG 19 downstream

CEP290-16 + AAGACACUGCCAAUAGGGAUAGGU 24 downstream

CEP290-1366 + AGACACUGCCAAUAGGGAUAGGU 23 downstream

CEP290-1367 + ACACUGCCAAUAGGGAUAGGU 21 downstream

CEP290-510 + ACUGCCAAUAGGGAUAGGU 19 downstream

CEP290-1368 - AAAGGUUCAUGAGACUAGAGGUC 23 downstream

CEP290-1369 - AAGGUUCAUGAGACUAGAGGUC 22 downstream

CEP290-1370 - AGGUUCAUGAGACUAGAGGUC 21 downstream

CEP290-1371 + AAACAGGAGAUACUCAACACA 21 downstream

CEP290-1372 + AACAGGAGAUACUCAACACA 20 downstream

CEP290-1373 + ACAGGAGAUACUCAACACA 19 downstream

CEP290-1374 + AGCACGUACAAAAGAACAUACAU 23 downstream

CEP290-1375 + ACGUACAAAAGAACAUACAU 20 downstream

CEP290-1376 + AGUAAGGAGGAUGUAAGAC 19 downstream

CEP290-1377 + AG CU U UUGACAG U U U U UAAGG 21 downstream

CEP290-1378 - ACGUGCUCUUUUCUAUAUAU 20 downstream

CEP290-1379 + AAAUUCACUGAGCAAAACAACUGG 24 downstream

CEP290-1380 + AAUUCACUGAGCAAAACAACUGG 23 downstream

CEP290-1381 + AUUCACUGAGCAAAACAACUGG 22 downstream

CEP290-1382 + AC U GAG CAAAAC AACU G G 18 downstream

CEP290-1383 + AACAAGUUUUGAAACAGGAA 20 downstream

CEP290-1384 + ACAAGUUUUGAAACAGGAA 19 downstream CEP290-1385 + AAUG CC U G AAC AAG U U U U G AAA 22 downstream

CEP290-1386 + AUG CC U G AAC AAG U U U U G AAA 21 downstream

CEP290-1387 + AUUCACUGAGCAAAACAACUGGAA 24 downstream

CEP290-1388 + ACUGAGCAAAACAACUGGAA 20 downstream

CEP290-1389 + AAAAAG G U AAU G CC U G AAC AAG U U 24 downstream

CEP290-1390 + AAAAG G U AAU G CC U G AAC AAG U U 23 downstream

CEP290-1391 + AAAGGUAAUGCCUGAACAAGUU 22 downstream

CEP290-1392 + AAGGUAAUGCCUGAACAAGUU 21 downstream

CEP290-1393 + AGGUAAUGCCUGAACAAGUU 20 downstream

CEP290-1394 - ACGUGCUCUUUUCUAUAUA 19 downstream

CEP290-1395 + AUUAUCUAUUCCAUUCUUCACAC 23 downstream

CEP290-1396 + AUCUAUUCCAUUCUUCACAC 20 downstream

CEP290-1397 + AAGAGAGAAAUGGUUCCCUAUAUA 24 downstream

CEP290-1398 + AGAGAGAAAUGGUUCCCUAUAUA 23 downstream

CEP290-1399 + AGAGAAAUGGUUCCCUAUAUA 21 downstream

CEP290-1400 + AGAAAUGGUUCCCUAUAUA 19 downstream

CEP290-1401 - AGGAAAUUAUUGUUGCUUU 19 downstream

CEP290-1402 + AC U GAG CAAAAC AACU G G AAG A 22 downstream

CEP290-1403 + AGCAAAACAACUGGAAGA 18 downstream

CEP290-1404 + AUACAUAAGAAAGAACACUGUGGU 24 downstream

CEP290-1405 + ACAUAAGAAAGAACACUGUGGU 22 downstream

CEP290-1406 + AUAAGAAAGAACACUGUGGU 20 downstream

CEP290-1407 + AAGAAAGAACACUGUGGU 18 downstream

CEP290-1408 - AAGAAUGGAAUAGAUAAU 18 downstream

CEP290-1409 + AAGGAGGAUGUAAGACUGGAGA 22 downstream

CEP290-1410 + AGGAGGAUGUAAGACUGGAGA 21 downstream

CEP290-1411 + AGGAUGUAAGACUGGAGA 18 downstream

CEP290-1412 - AAAAACUUGAAAUUUGAUAGUAG 23 downstream

CEP290-1413 - AAAACUUGAAAUUUGAUAGUAG 22 downstream

CEP290-1414 - AAACUUGAAAUUUGAUAGUAG 21 downstream

CEP290-1415 - AACUUGAAAUUUGAUAGUAG 20 downstream

CEP290-1416 - ACUUGAAAUUUGAUAGUAG 19 downstream

CEP290-1417 - ACAUAUCUGUCUUCCUUA 18 downstream

CEP290-1418 + AUUAAAAAAAGUAUGCUU 18 downstream

CEP290-1419 + AUAUCAAAAGACUUAUAUUCCAUU 24 downstream

CEP290-1420 + AUCAAAAGACUUAUAUUCCAUU 22 downstream

CEP290-1421 + AAAAG ACU UAUAUUCCAUU 19 downstream

CEP290-1422 + AAAGACUUAUAUUCCAUU 18 downstream

CEP290-1423 - AAAAUCAGAUUUCAUGUGUGAAGA 24 downstream

CEP290-1424 - AAAUCAGAUUUCAUGUGUGAAGA 23 downstream

CEP290-1425 - AAUCAGAUUUCAUGUGUGAAGA 22 downstream

CEP290-1426 - AUCAGAUUUCAUGUGUGAAGA 21 downstream CEP290-1427 - AGAUUUCAUGUGUGAAGA 18 downstream

CEP290-1428 - AAUGGAAUAUAAGUCUUUUGAUAU 24 downstream

CEP290-1429 - AUGGAAUAUAAGUCUUUUGAUAU 23 downstream

CEP290-1430 - AAUAUAAGUCUUUUGAUAU 19 downstream

CEP290-1431 - AUAUAAGUCUUUUGAUAU 18 downstream

CEP290-1432 - AAGAAUGGAAUAGAUAAUA 19 downstream

CEP290-1433 - AGAAUGGAAUAGAUAAUA 18 downstream

CEP290-1434 - AAAACUGGAUGGGUAAUAAAGCAA 24 downstream

CEP290-1435 - AAACUGGAUGGGUAAUAAAGCAA 23 downstream

CEP290-1436 - AACUGGAUGGGUAAUAAAGCAA 22 downstream

CEP290-1437 - ACUGGAUGGGUAAUAAAGCAA 21 downstream

CEP290-1438 + AUAGAAAUUCACUGAGCAAAACAA 24 downstream

CEP290-1439 + AGAAAUUCACUGAGCAAAACAA 22 downstream

CEP290-1440 + AAAUUCACUGAGCAAAACAA 20 downstream

CEP290-1441 + AAUUCACUGAGCAAAACAA 19 downstream

CEP290-1442 + AUUCACUGAGCAAAACAA 18 downstream

CEP290-1443 + AGGAUGUAAGACUGGAGAUAGAGA 24 downstream

CEP290-1444 + AUGUAAGACUGGAGAUAGAGA 21 downstream

CEP290-1445 - AAAU U UG AU AG U AG AAG AAAA 21 downstream

CEP290-1446 - AAU U UG AU AG U AG AAG AAAA 20 downstream

CEP290-1447 - AU U UG AU AG U AG AAG AAAA 19 downstream

CEP290-1448 + AAAAUAAAACUAAGACACUGCCAA 24 downstream

CEP290-1449 + AAAUAAAACUAAGACACUGCCAA 23 downstream

CEP290-1450 + AAUAAAACUAAGACACUGCCAA 22 downstream

CEP290-1451 + AUAAAACUAAGACACUGCCAA 21 downstream

CEP290-1452 + AAAACUAAGACACUGCCAA 19 downstream

CEP290-1453 + AAACUAAGACACUGCCAA 18 downstream

CEP290-1454 - A A U A A AG C A A A AG A A A AAC 19 downstream

CEP290-1455 - A U A A AG C A A A AG A A A A AC 18 downstream

CEP290-1456 - AUUCUUUUUUUGUUGUUUUUUUUU 24 downstream

CEP290-1457 + ACUCCAGCCUGGGCAACACA 20 downstream

CEP290-1458 - CUUACCUCAUGUCAUCUAGAGC 22 downstream

CEP290-1459 - CCUCAUGUCAUCUAGAGC 18 downstream

CEP290-1460 + CAGUUUUUAAGGCGGGGAGUCAC 23 downstream

CEP290-1461 - CACAGAGUUCAAGCUAAUAC 20 downstream

CEP290-1462 - CAGAGUUCAAGCUAAUAC 18 downstream

CEP290-1463 + CUUGAACUCUGUGCCAAAC 19 downstream

CEP290-1464 - CAUGUGGUGUCAAAUAUGGUGCU 23 downstream

CEP290-1465 - CAUGUGGUGUCAAAUAUGGUGCUU 24 downstream

CEP290-1466 + CUCUAGAUGACAUGAGGUAAGU 22 downstream

CEP290-1467 + CUAGAUGACAUGAGGUAAGU 20 downstream

CEP290-1468 - CUAAUACAUGAGAGUGAUUAGUGG 24 downstream CEP290-1469 - CAUGAGAGUGAUUAGUGG 18 downstream

CEP290-509 + CACUGCCAAUAGGGAUAGGU 20 downstream

CEP290-511 + CUGCCAAUAGGGAUAGGU 18 downstream

CEP290-1470 + CCAAACAGGAGAUACUCAACACA 23 downstream

CEP290-1471 + CAAACAGGAGAUACUCAACACA 22 downstream

CEP290-1472 + CAGGAGAUACUCAACACA 18 downstream

CEP290-1473 + CACGUACAAAAGAACAUACAU 21 downstream

CEP290-1474 + CG U ACAAAAG AACAU ACAU 19 downstream

CEP290-1475 + CAGUAAGGAGGAUGUAAGAC 20 downstream

CEP290-1476 + CUUUUGACAGUUUUUAAGG 19 downstream

CEP290-1477 - CGUGCUCUUUUCUAUAUAU 19 downstream

CEP290-1478 + CACUGAGCAAAACAACUGG 19 downstream

CEP290-1479 + CC U G AACAAG U U U U G AAACAG G AA 24 downstream

CEP290-1480 + CU G AACAAG U U U UG AAACAGG AA 23 downstream

CEP290-1481 + CA AG U U U U G AAACAG G A A 18 downstream

CEP290-1482 + CCUGAACAAGUUUUGAAA 18 downstream

CEP290-1483 + CACUGAGCAAAACAACUGGAA 21 downstream

CEP290-1484 + CUGAGCAAAACAACUGGAA 19 downstream

CEP290-1485 - CGUGCUCUUUUCUAUAUA 18 downstream

CEP290-1486 + CUAUUCCAUUCUUCACAC 18 downstream

CEP290-1487 - CUUAGGAAAUUAUUGUUGCUUU 22 downstream

CEP290-1488 - CUUUUUGAGAGGUAAAGGUUC 21 downstream

CEP290-1489 + CACUGAGCAAAACAACUGGAAGA 23 downstream

CEP290-1490 + CUGAGCAAAACAACUGGAAGA 21 downstream

CEP290-1491 + CAUAAGAAAGAACACUGUGGU 21 downstream

CEP290-1492 - CUUGAAAUUUGAUAGUAG 18 downstream

CEP290-1493 + CCAUUAAAAAAAGUAUGCUU 20 downstream

CEP290-1494 + CAUUAAAAAAAGUAUGCUU 19 downstream

CEP290-1495 + CAAAAG ACU U AU AU U CCAU U 20 downstream

CEP290-1496 - CAGAUUUCAUGUGUGAAGA 19 downstream

CEP290-1497 - CUGGAUGGGUAAUAAAGCAA 20 downstream

CEP290-1498 - CUUAAGCAUACUUUUUUUA 19 downstream

CEP290-1499 - CUUUUUUUGUUGUUUUUUUUU 21 downstream

CEP290-1500 + CUGCACUCCAGCCUGGGCAACACA 24 downstream

CEP290-1501 + CACUCCAGCCUGGGCAACACA 21 downstream

CEP290-1502 + CUCCAGCCUGGGCAACACA 19 downstream

CEP290-1503 + GUUUUUAAGGCGGGGAGUCAC 21 downstream

CEP290-230 - GGCACAGAGUUCAAGCUAAUAC 22 downstream

CEP290-1504 - GCACAGAGUUCAAGCUAAUAC 21 downstream

CEP290-1505 + GCUUGAACUCUGUGCCAAAC 20 downstream

CEP290-139 - GCAUGUGGUGUCAAAUAUGGUGCU 24 downstream

CEP290-1506 - GUGGUGUCAAAUAUGGUGCU 20 downstream CEP290-1507 - GGUGUCAAAUAUGGUGCU 18 downstream

CEP290-1508 - GUGGUGUCAAAUAUGGUGCUU 21 downstream

CEP290-1509 - GGUGUCAAAUAUGGUGCUU 19 downstream

CEP290-1510 - GUGUCAAAUAUGGUGCUU 18 downstream

CEP290-1511 + GCUCUAGAUGACAUGAGGUAAGU 23 downstream

CEP290-11 + GACACUGCCAAUAGGGAUAGGU 22 downstream

CEP290-1512 - GGUUCAUGAGACUAGAGGUC 20 downstream

CEP290-1513 - GUUCAUGAGACUAGAGGUC 19 downstream

CEP290-1514 + GCCAAACAGGAGAUACUCAACACA 24 downstream

CEP290-1515 + GAGCACGUACAAAAGAACAUACAU 24 downstream

CEP290-1516 + GCACGUACAAAAGAACAUACAU 22 downstream

CEP290-1517 + G U ACAAAAG AACAU ACAU 18 downstream

CEP290-1518 + GUGGCAGUAAGGAGGAUGUAAGAC 24 downstream

CEP290-1519 + GGCAGUAAGGAGGAUGUAAGAC 22 downstream

CEP290-1520 + GCAGUAAGGAGGAUGUAAGAC 21 downstream

CEP290-1521 + GUAAGGAGGAUGUAAGAC 18 downstream

CEP290-1522 + GGUAGCUUUUGACAGUUUUUAAGG 24 downstream

CEP290-1523 + GUAGCUUUUGACAGUUUUUAAGG 23 downstream

CEP290-1524 + GCUUUUGACAGUUUUUAAGG 20 downstream

CEP290-1525 - GUACGUGCUCUUUUCUAUAUAU 22 downstream

CEP290-1526 - GUGCUCUUUUCUAUAUAU 18 downstream

CEP290-1527 + GAACAAG U U U U G AAAC AG G AA 21 downstream

CEP290-1528 + GUAAUGCCUGAACAAGUUUUGAAA 24 downstream

CEP290-1529 + G CCU GAACAAG U U U U G AAA 19 downstream

CEP290-1530 + GGUAAUGCCUGAACAAGUU 19 downstream

CEP290-1531 + GUAAUGCCUGAACAAGUU 18 downstream

CEP290-1532 - GUACGUGCUCUUUUCUAUAUA 21 downstream

CEP290-1533 + GAGAGAAAUGGUUCCCUAUAUA 22 downstream

CEP290-1534 + GAGAAAUGGUUCCCUAUAUA 20 downstream

CEP290-1535 + GAAAUGGUUCCCUAUAUA 18 downstream

CEP290-1536 - GCUUAGGAAAUUAUUGUUGCUUU 23 downstream

CEP290-1537 - GGAAAUUAUUGUUGCUUU 18 downstream

CEP290-1538 - GCUUUUUGAGAGGUAAAGGUUC 22 downstream

CEP290-1539 + GAGCAAAACAACUGGAAGA 19 downstream

CEP290-1540 - GUGUGAAGAAUGGAAUAGAUAAU 23 downstream

CEP290-1541 - GUGAAGAAUGGAAUAGAUAAU 21 downstream

CEP290-1542 - GAAGAAUGGAAUAGAUAAU 19 downstream

CEP290-1543 + GUAAGGAGGAUGUAAGACUGGAGA 24 downstream

CEP290-1544 + GGAGGAUGUAAGACUGGAGA 20 downstream

CEP290-1545 + GAGGAUGUAAGACUGGAGA 19 downstream

CEP290-1546 - G AAAAACU UG AAAU U UG AU AG U AG 24 downstream

CEP290-1547 - GUGUUUACAUAUCUGUCUUCCUUA 24 downstream CEP290-1548 - GUUUACAUAUCUGUCUUCCUUA 22 downstream

CEP290-1549 + GUUCCAUUAAAAAAAGUAUGCUU 23 downstream

CEP290-1550 - GGAAUAUAAGUCUUUUGAUAU 21 downstream

CEP290-1551 - GAAUAUAAGUCUUUUGAUAU 20 downstream

CEP290-1552 - GUGUGAAGAAUGGAAUAGAUAAUA 24 downstream

CEP290-1553 - GUGAAGAAUGGAAUAGAUAAUA 22 downstream

CEP290-1554 - GAAGAAUGGAAUAGAUAAUA 20 downstream

CEP290-1555 - GGAUGGGUAAUAAAGCAA 18 downstream

CEP290-1556 + GAAAUUCACUGAGCAAAACAA 21 downstream

CEP290-1557 + GGAUGUAAGACUGGAGAUAGAGA 23 downstream

CEP290-1558 + GAUGUAAGACUGGAGAUAGAGA 22 downstream

CEP290-1559 + GUAAGACUGGAGAUAGAGA 19 downstream

CEP290-1560 - GAAAU U UG AU AG U AG AAG AAAA 22 downstream

CEP290-1561 - G G G U A A U A A AG C A A A AG A A A A AC 23 downstream

CEP290-1562 - G G U A A U A A AG C A A A AG A A A A AC 22 downstream

CEP290-1563 - GUAAUAAAGCAAAAGAAAAAC 21 downstream

CEP290-1564 + GCACUCCAGCCUGGG CA ACAC A 22 downstream

CEP290-1565 - UACUUACCUCAUGUCAUCUAGAGC 24 downstream

CEP290-1566 - UUACCUCAUGUCAUCUAGAGC 21 downstream

CEP290-1567 - UACCUCAUGUCAUCUAGAGC 20 downstream

CEP290-1568 + UUUUUAAGGCGGGGAGUCAC 20 downstream

CEP290-1569 + UUUUAAGGCGGGGAGUCAC 19 downstream

CEP290-1570 + UUUAAGGCGGGGAGUCAC 18 downstream

CEP290-1571 - UUGGCACAGAGUUCAAGCUAAUAC 24 downstream

CEP290-1572 - UGGCACAGAGUUCAAGCUAAUAC 23 downstream

CEP290-1573 + UUAGCUUGAACUCUGUGCCAAAC 23 downstream

CEP290-1574 + UAGCUUGAACUCUGUGCCAAAC 22 downstream

CEP290-1575 + UUGAACUCUGUGCCAAAC 18 downstream

CEP290-1576 - UGUGGUGUCAAAUAUGGUGCU 21 downstream

CEP290-1577 - UGGUGUCAAAUAUGGUGCU 19 downstream

CEP290-1578 - UGUGGUGUCAAAUAUGGUGCUU 22 downstream

CEP290-1579 - UGGUGUCAAAUAUGGUGCUU 20 downstream

CEP290-1580 + UGCUCUAGAUGACAUGAGGUAAGU 24 downstream

CEP290-1581 + UCUAGAUGACAUGAGGUAAGU 21 downstream

CEP290-1582 + UAGAUGACAUGAGGUAAGU 19 downstream

CEP290-1583 - UAAUACAUGAGAGUGAUUAGUGG 23 downstream

CEP290-1584 - UACAUGAGAGUGAUUAGUGG 20 downstream

CEP290-1585 - UAAAGGUUCAUGAGACUAGAGGUC 24 downstream

CEP290-1586 - UUCAUGAGACUAGAGGUC 18 downstream

CEP290-1587 + UGGCAGUAAGGAGGAUGUAAGAC 23 downstream

CEP290-1588 + U AG CU U U UG ACAG U U U U UAAGG 22 downstream

CEP290-1589 + UUUUGACAGUUUUUAAGG 18 downstream CEP290-1590 - UUGUACGUGCUCUUUUCUAUAUAU 24 downstream

CEP290-1591 - UGUACGUGCUCUUUUCUAUAUAU 23 downstream

CEP290-1592 - UACGUGCUCUUUUCUAUAUAU 21 downstream

CEP290-1593 + UUCACUGAGCAAAACAACUGG 21 downstream

CEP290-1594 + UCACUGAGCAAAACAACUGG 20 downstream

CEP290-1595 + UGAACAAG U U U U G AAAC AG G AA 22 downstream

CEP290-1596 + UAAUGCCUGAACAAGUUUUGAAA 23 downstream

CEP290-1597 + U G CC U G AAC AAG U U U U G AAA 20 downstream

CEP290-1598 + UUCACUGAGCAAAACAACUGGAA 23 downstream

CEP290-1599 + U C ACU G AG C AAAACAAC U G G AA 22 downstream

CEP290-1600 + UGAGCAAAACAACUGGAA 18 downstream

CEP290-1601 - UUUGUACGUGCUCUUUUCUAUAUA 24 downstream

CEP290-1602 - UUGUACGUGCUCUUUUCUAUAUA 23 downstream

CEP290-1603 - UGUACGUGCUCUUUUCUAUAUA 22 downstream

CEP290-1604 - UACGUGCUCUUUUCUAUAUA 20 downstream

CEP290-1605 + UAUUAUCUAUUCCAUUCUUCACAC 24 downstream

CEP290-1606 + UUAUCUAUUCCAUUCUUCACAC 22 downstream

CEP290-1607 + UAUCUAUUCCAUUCUUCACAC 21 downstream

CEP290-1608 + UCUAUUCCAUUCUUCACAC 19 downstream

CEP290-1609 - UGCUUAGGAAAUUAUUGUUGCUUU 24 downstream

CEP290-1610 - UUAGGAAAUUAUUGUUGCUUU 21 downstream

CEP290-1611 - UAGGAAAUUAUUGUUGCUUU 20 downstream

CEP290-1612 - UUGCUUUUUGAGAGGUAAAGGUUC 24 downstream

CEP290-1613 - UGCUUUUUGAGAGGUAAAGGUUC 23 downstream

CEP290-1614 - UUUUUGAGAGGUAAAGGUUC 20 downstream

CEP290-1615 - UUUUGAGAGGUAAAGGUUC 19 downstream

CEP290-1616 - UUUGAGAGGUAAAGGUUC 18 downstream

CEP290-1617 + UCACUGAGCAAAACAACUGGAAGA 24 downstream

CEP290-1618 + UGAGCAAAACAACUGGAAGA 20 downstream

CEP290-1619 + UACAUAAGAAAGAACACUGUGGU 23 downstream

CEP290-1620 + UAAGAAAGAACACUGUGGU 19 downstream

CEP290-1621 - UGUGUGAAGAAUGGAAUAGAUAAU 24 downstream

CEP290-1622 - UGUGAAGAAUGGAAUAGAUAAU 22 downstream

CEP290-1623 - UGAAGAAUGGAAUAGAUAAU 20 downstream

CEP290-1624 + UAAGGAGGAUGUAAGACUGGAGA 23 downstream

CEP290-1625 - UGUUUACAUAUCUGUCUUCCUUA 23 downstream

CEP290-1626 - UUUACAUAUCUGUCUUCCUUA 21 downstream

CEP290-1627 - UUACAUAUCUGUCUUCCUUA 20 downstream

CEP290-1628 - UACAUAUCUGUCUUCCUUA 19 downstream

CEP290-1629 + UGUUCCAUUAAAAAAAGUAUGCUU 24 downstream

CEP290-1630 + UUCCAUUAAAAAAAGUAUGCUU 22 downstream

CEP290-1631 + UCCAUUAAAAAAAGUAUGCUU 21 downstream CEP290-1632 + UAUCAAAAGACUUAUAUUCCAUU 23 downstream

CEP290-1633 + UCAAAAGACUUAUAUUCCAUU 21 downstream

CEP290-1634 - UCAGAUUUCAUGUGUGAAGA 20 downstream

CEP290-1635 - UGGAAUAUAAGUCUUUUGAUAU 22 downstream

CEP290-1636 - UGUGAAGAAUGGAAUAGAUAAUA 23 downstream

CEP290-1637 - UGAAGAAUGGAAUAGAUAAUA 21 downstream

CEP290-1638 - UGGAUGGGUAAUAAAGCAA 19 downstream

CEP290-1639 + UAGAAAUUCACUGAGCAAAACAA 23 downstream

CEP290-1640 + UGUAAGACUGGAGAUAGAGA 20 downstream

CEP290-1641 + UAAGACUGGAGAUAGAGA 18 downstream

CEP290-1642 - U UG AAAU U UG AU AG U AG AAG AAAA 24 downstream

CEP290-1643 - UG AAAU U UG AU AG U AG AAG AAAA 23 downstream

CEP290-1644 - U U UG AU AG U AG AAG AAAA 18 downstream

CEP290-1645 + UAAAACUAAGACACUGCCAA 20 downstream

CEP290-1646 - UUUUUCUUAAGCAUACUUUUUUUA 24 downstream

CEP290-1647 - UUUUCUUAAGCAUACUUUUUUUA 23 downstream

CEP290-1648 - UUUCUUAAGCAUACUUUUUUUA 22 downstream

CEP290-1649 - UUCUUAAGCAUACUUUUUUUA 21 downstream

CEP290-1650 - UCUUAAGCAUACUUUUUUUA 20 downstream

CEP290-1651 - UUAAGCAUACUUUUUUUA 18 downstream

CEP290-1652 - U G G G U A A U A A AG C A A A AG A A A A AC 24 downstream

CEP290-1653 - U A A U A A AG C A A A AG A AAA AC 20 downstream

CEP290-1654 - UUCUUUUUUUGUUGUUUUUUUUU 23 downstream

CEP290-1655 - UCUUUUUUUGUUGUUUUUUUUU 22 downstream

CEP290-1656 - UUUUUUUGUUGUUUUUUUUU 20 downstream

CEP290-1657 - UUUUUUGUUGUUUUUUUUU 19 downstream

CEP290-1658 - UUUUUGUUGUUUUUUUUU 18 downstream

CEP290-1659 + U G CAC U CCAG CC U G GG C AACAC A 23 downstream

CEP290-1660 + UCCAGCCUGGGCAACACA 18 downstream

CEP290-1661 + AUUUUCGUGACCUCUAGUCUC 21 downstream

CEP290-1662 + ACUAAUCACUCUCAUGUAUUAGC 23 downstream

CEP290-1663 + AAUCACUCUCAUGUAUUAGC 20 downstream

CEP290-1664 + AUCACUCUCAUGUAUUAGC 19 downstream

CEP290-1665 + AGAUGACAUGAGGUAAGUA 19 downstream

CEP290-1666 - ACCUCAUGUCAUCUAGAGCAAGAG 24 downstream

CEP290-1667 - AUGUCAUCUAGAGCAAGAG 19 downstream

CEP290-1668 - AAUACAUGAGAGUGAUUAGUGGUG 24 downstream

CEP290-1669 - AUACAUGAGAGUGAUUAGUGGUG 23 downstream

CEP290-1670 - ACAUGAGAGUGAUUAGUGGUG 21 downstream

CEP290-1671 - AUGAGAGUGAUUAGUGGUG 19 downstream

CEP290-1672 - ACGUGCUCUUUUCUAUAUAUA 21 downstream

CEP290-1673 + ACAAAACCUAUGUAUAAGAUG 21 downstream CEP290-1674 + AAAACCUAUGUAUAAGAUG 19 downstream

CEP290-1675 + AAACCUAUGUAUAAGAUG 18 downstream

CEP290-1676 + AUAUAUAGAAAAGAGCACGUACAA 24 downstream

CEP290-1677 + AUAUAGAAAAGAGCACGUACAA 22 downstream

CEP290-1678 + AUAGAAAAGAGCACGUACAA 20 downstream

CEP290-1679 + AG AAAAG AG CACGUACAA 18 downstream

CEP290-1680 + AGAAAUGGUUCCCUAUAUAUAGAA 24 downstream

CEP290-1681 + AAAUGGUUCCCUAUAUAUAGAA 22 downstream

CEP290-1682 + AAUGGUUCCCUAUAUAUAGAA 21 downstream

CEP290-1683 + AUGGUUCCCUAUAUAUAGAA 20 downstream

CEP290-1684 - AUGGAAUAUAAGUCUUUUGAUAUA 24 downstream

CEP290-1685 - AAUAUAAGUCUUUUGAUAUA 20 downstream

CEP290-1686 - AUAUAAGUCUUUUGAUAUA 19 downstream

CEP290-1687 + ACGUACAAAAGAACAUACAUAAGA 24 downstream

CEP290-1688 + ACAAAAGAACAUACAUAAGA 20 downstream

CEP290-1689 + AAAAGAACAUACAUAAGA 18 downstream

CEP290-1690 + A AG A A A A A A A AG G U A A U G C 19 downstream

CEP290-1691 + AG A A A A A A A AG G U A A U G C 18 downstream

CEP290-1692 + AAACAGGAAUAGAAAUUCA 19 downstream

CEP290-1693 + AACAGGAAUAGAAAUUCA 18 downstream

CEP290-1694 + AAGAUCACUCCACUGCACUCCAGC 24 downstream

CEP290-1695 + AGAUCACUCCACUGCACUCCAGC 23 downstream

CEP290-1696 + AUCACUCCACUGCACUCCAGC 21 downstream

CEP290-1697 + ACUCCACUGCACUCCAGC 18 downstream

CEP290-1698 - CCCCUACUUACCUCAUGUCAUC 22 downstream

CEP290-1699 - CCCUACUUACCUCAUGUCAUC 21 downstream

CEP290-1700 - CCUACUUACCUCAUGUCAUC 20 downstream

CEP290-1701 - CUACUUACCUCAUGUCAUC 19 downstream

CEP290-1702 + CUGAUUUUCGUGACCUCUAGUCUC 24 downstream

CEP290-1703 + CACUAAUCACUCUCAUGUAUUAGC 24 downstream

CEP290-1704 + CUAAUCACUCUCAUGUAUUAGC 22 downstream

CEP290-1705 + CUCUAGAUGACAUGAGGUAAGUA 23 downstream

CEP290-1706 + CUAGAUGACAUGAGGUAAGUA 21 downstream

CEP290-1707 - CCU CAU G U CAU CU AG AG CAAG AG 23 downstream

CEP290-1708 - CU CAUG U CAU CU AG AGCAAG AG 22 downstream

CEP290-1709 - CAUGUCAUCUAGAGCAAGAG 20 downstream

CEP290-1710 - CAUGAGAGUGAUUAGUGGUG 20 downstream

CEP290-1711 - CGUGCUCUUUUCUAUAUAUA 20 downstream

CEP290-1712 + CAAAACCUAUGUAUAAGAUG 20 downstream

CEP290-1713 + CG U ACAAAAGAACAUACAUAAGA 23 downstream

CEP290-1714 + CAAAAGAACAUACAUAAGA 19 downstream

CEP290-1715 + C U U A AG A A A A A A A AG G U A A U G C 22 downstream CEP290-1716 - CUUAAGCAUACUUUUUUUAA 20 downstream

CEP290-1717 + CACUCCACUGCACUCCAGC 19 downstream

CEP290-132 - GUCCCCUACUUACCUCAUGUCAUC 24 downstream

CEP290-1718 + GAUUUUCGUGACCUCUAGUCUC 22 downstream

CEP290-1719 + GCUCUAGAUGACAUGAGGUAAGUA 24 downstream

CEP290-1720 + GAUGACAUGAGGUAAGUA 18 downstream

CEP290-1721 - GUACGUGCUCUUUUCUAUAUAUA 23 downstream

CEP290-1722 - GUGCUCUUUUCUAUAUAUA 19 downstream

CEP290-1723 + GUACAAAACCUAUGUAUAAGAUG 23 downstream

CEP290-1724 + GAAAUGGUUCCCUAUAUAUAGAA 23 downstream

CEP290-1725 + GGUUCCCUAUAUAUAGAA 18 downstream

CEP290-1726 - GGAAUAUAAGUCUUUUGAUAUA 22 downstream

CEP290-1727 - GAAUAUAAGUCUUUUGAUAUA 21 downstream

CEP290-1728 + G U ACAAAAG AACAU ACAU AAG A 22 downstream

CEP290-1729 + G C U U A AG A A A A A A A AG G U A A U G C 23 downstream

CEP290-1730 + GAAACAGGAAUAGAAAUUCA 20 downstream

CEP290-1731 + GAUCACUCCACUGCACUCCAGC 22 downstream

CEP290-1732 - UCCCCUACUUACCUCAUGUCAUC 23 downstream

CEP290-1733 - UACUUACCUCAUGUCAUC 18 downstream

CEP290-1734 + UGAUUUUCGUGACCUCUAGUCUC 23 downstream

CEP290-1735 + UUUUCGUGACCUCUAGUCUC 20 downstream

CEP290-1736 + UUUCGUGACCUCUAGUCUC 19 downstream

CEP290-1737 + UUCGUGACCUCUAGUCUC 18 downstream

CEP290-1738 + UAAUCACUCUCAUGUAUUAGC 21 downstream

CEP290-1739 + UCACUCUCAUGUAUUAGC 18 downstream

CEP290-1740 + UCUAGAUGACAUGAGGUAAGUA 22 downstream

CEP290-1741 + UAGAUGACAUGAGGUAAGUA 20 downstream

CEP290-1742 - UCAUGUCAUCUAGAGCAAGAG 21 downstream

CEP290-1743 - UGUCAUCUAGAGCAAGAG 18 downstream

CEP290-1744 - UACAUGAGAGUGAUUAGUGGUG 22 downstream

CEP290-1745 - UGAGAGUGAUUAGUGGUG 18 downstream

CEP290-1746 - UGUACGUGCUCUUUUCUAUAUAUA 24 downstream

CEP290-1747 - UACGUGCUCUUUUCUAUAUAUA 22 downstream

CEP290-1748 - UGCUCUUUUCUAUAUAUA 18 downstream

CEP290-1749 + UGUACAAAACCUAUGUAUAAGAUG 24 downstream

CEP290-1750 + UACAAAACCUAUGUAUAAGAUG 22 downstream

CEP290-1751 + UAUAUAGAAAAGAGCACGUACAA 23 downstream

CEP290-1752 + UAUAGAAAAGAGCACGUACAA 21 downstream

CEP290-1753 + UAGAAAAGAGCACGUACAA 19 downstream

CEP290-1754 + UGGUUCCCUAUAUAUAGAA 19 downstream

CEP290-1755 - UGGAAUAUAAGUCUUUUGAUAUA 23 downstream

CEP290-1756 - UAUAAGUCUUUUGAUAUA 18 downstream CEP290-1757 + UACAAAAGAACAUACAUAAGA 21 downstream

CEP290-1758 + U G C U U A AG A A A A A A A AG G U A A U G C 24 downstream

CEP290-1759 + U U A AG A A A A A A A AG G UAAUGC 21 downstream

CEP290-1760 + U A AG A A A A A A A AG G U A AU G C 20 downstream

CEP290-1761 + U U U UG AAACAGG AAU AG AAAU U CA 24 downstream

CEP290-1762 + U U UG AAACAGG AAU AG AAAU U CA 23 downstream

CEP290-1763 + UUGAAACAGGAAUAGAAAUUCA 22 downstream

CEP290-1764 + UGAAACAGGAAUAGAAAUUCA 21 downstream

CEP290-1765 - UUUUCUUAAGCAUACUUUUUUUAA 24 downstream

CEP290-1766 - UUUCUUAAGCAUACUUUUUUUAA 23 downstream

CEP290-1767 - UUCUUAAGCAUACUUUUUUUAA 22 downstream

CEP290-1768 - UCUUAAGCAUACUUUUUUUAA 21 downstream

CEP290-1769 - U UAAG CAU ACU U U U U U U AA 19 downstream

CEP290-1770 - UAAGCAUACUUUUUUUAA 18 downstream

CEP290-1771 + UCACUCCACUGCACUCCAGC 20 downstream

CEP290-1772 + AGUUUUUAAGGCGGGGAGUCACA 23 downstream

CEP290-1773 - AAACUGUCAAAAGCUACCGGUUAC 24 downstream

CEP290-1774 - AACUGUCAAAAGCUACCGGUUAC 23 downstream

CEP290-252 - ACUGUCAAAAGCUACCGGUUAC 22 downstream

CEP290-1775 + AGUUCAUCUCUUGCUCUAGAUGAC 24 downstream

CEP290-1776 + AUCUCUUGCUCUAGAUGAC 19 downstream

CEP290-1777 - ACGAAAAUCAGAUUUCAUGU 20 downstream

CEP290-1778 - AAUACAUGAGAGUGAUUAGUG 21 downstream

CEP290-1779 - AUACAUGAGAGUGAUUAGUG 20 downstream

CEP290-1780 - ACAUGAGAGUGAUUAGUG 18 downstream

CEP290-1781 + AUUAGCUUGAACUCUGUGCCAAA 23 downstream

CEP290-1782 + AGCUUGAACUCUGUGCCAAA 20 downstream

CEP290-1783 - AUGUAGAUUGAGGUAGAAUCAAG 23 downstream

CEP290-1784 - AGAUUGAGGUAGAAUCAAG 19 downstream

CEP290-1785 + AUAAGAUGCAGAACUAGUGUAGA 23 downstream

CEP290-1786 + AAGAUGCAGAACUAGUGUAGA 21 downstream

CEP290-1787 + AGAUGCAGAACUAGUGUAGA 20 downstream

CEP290-1788 + AUGCAGAACUAGUGUAGA 18 downstream

CEP290-1789 - AUAGAUGUAGAUUGAGGUAGAAUC 24 downstream

CEP290-1790 - AGAUGUAGAUUGAGGUAGAAUC 22 downstream

CEP290-1791 - AUGUAGAUUGAGGUAGAAUC 20 downstream

CEP290-1792 + AGAAUGAUCAUUCUUGUGGCAGUA 24 downstream

CEP290-1793 + AAUGAUCAUUCUUGUGGCAGUA 22 downstream

CEP290-1794 + AUGAUCAUUCUUGUGGCAGUA 21 downstream

CEP290-1795 + AUCAUUCUUGUGGCAGUA 18 downstream

CEP290-1796 + AGAAUGAUCAUUCUUGUGGCAGU 23 downstream

CEP290-1797 + AAUGAUCAUUCUUGUGGCAGU 21 downstream CEP290-1798 + AUGAUCAUUCUUGUGGCAGU 20 downstream

CEP290-1799 - AGAGGUAAAGGUUCAUGAGAC 21 downstream

CEP290-1800 - AGGUAAAGGUUCAUGAGAC 19 downstream

CEP290-1801 + AGCUUUUGACAGUUUUUAAG 20 downstream

CEP290-1802 + AGCUUUUGACAGUUUUUAAGGC 22 downstream

CEP290-1803 + AGAAAUUCACUGAGCAAAACAAC 23 downstream

CEP290-1804 + AAAUUCACUGAGCAAAACAAC 21 downstream

CEP290-1805 + AAUUCACUGAGCAAAACAAC 20 downstream

CEP290-1806 + AUUCACUGAGCAAAACAAC 19 downstream

CEP290-1807 + AGUAAGGAGGAUGUAAGA 18 downstream

CEP290-1808 + AUCAAAAGACUUAUAUUCCAUUA 23 downstream

CEP290-1809 + AAAAGACUUAUAUUCCAUUA 20 downstream

CEP290-1810 + AAAGACUUAUAUUCCAUUA 19 downstream

CEP290-1811 + AAGACUUAUAUUCCAUUA 18 downstream

CEP290-1812 - AGGAAAUUAUUGUUGCUUUUU 21 downstream

CEP290-1813 - AAAUUAUUGUUGCUUUUU 18 downstream

CEP290-1814 - AAAG AAAAACU UG AAAU U UG AU AG 24 downstream

CEP290-1815 - AAG AAAAACU UG AAAU U UG AU AG 23 downstream

CEP290-1816 - AG AAAAACU UG AAAU U UG AU AG 22 downstream

CEP290-1817 - AAAAACU UGAAAUUUGAU AG 20 downstream

CEP290-1818 - AAAACUUGAAAUUUGAUAG 19 downstream

CEP290-1819 - AAACUUGAAAUUUGAUAG 18 downstream

CEP290-1820 - AAGAAAAAAGAAAUAGAUGUAGA 23 downstream

CEP290-1821 - AG AAAAAAG AAAU AG AUG U AG A 22 downstream

CEP290-1822 - AAAAAAGAAAUAGAUGUAGA 20 downstream

CEP290-1823 - A A A A AG AAA UAGAUGUAGA 19 downstream

CEP290-1824 - A A A AG AAA UAGAUGUAGA 18 downstream

CEP290-1825 - AGAGUCUCACUGUGUUGCCCAGG 23 downstream

CEP290-1826 - AGUCUCACUGUGUUGCCCAGG 21 downstream

CEP290-1827 + CAGUUUUUAAGGCGGGGAGUCACA 24 downstream

CEP290-1828 - CUGUCAAAAGCUACCGGUUAC 21 downstream

CEP290-1829 + CAUCUCUUGCUCUAGAUGAC 20 downstream

CEP290-1830 - CACGAAAAUCAGAUUUCAUGU 21 downstream

CEP290-1831 - CGAAAAUCAGAUUUCAUGU 19 downstream

CEP290-1832 - CUAAUACAUGAGAGUGAUUAGUG 23 downstream

CEP290-1833 + CUUGAACUCUGUGCCAAA 18 downstream

CEP290-1834 + CUCUAGAUGACAUGAGGUAAG 21 downstream

CEP290-1835 + CUAGAUGACAUGAGGUAAG 19 downstream

CEP290-1836 + CGGUAGCUUUUGACAGUUUUUAAG 24 downstream

CEP290-1837 + CUUUUGACAGUUUUUAAG 18 downstream

CEP290-1838 + CUUUUGACAGUUUUUAAGGC 20 downstream

CEP290-1839 + CAGUAAGGAGGAUGUAAGA 19 downstream CEP290-1840 + CAAAAG ACU U AU AU UCCAUUA 21 downstream

CEP290-1841 - CUUAGGAAAUUAUUGUUGCUUUUU 24 downstream

CEP290-1842 - CUGUGUUGCCCAGGCUGGAGUGCA 24 downstream

CEP290-1843 - CAGAGUCUCACUGUGUUGCCCAGG 24 downstream

CEP290-1844 - CUCACUGUGUUGCCCAGG 18 downstream

CEP290-1845 + GUUUUUAAGGCGGGGAGUCACA 22 downstream

CEP290-1846 - GUCAAAAGCUACCGGUUAC 19 downstream

CEP290-1847 + GUUCAUCUCUUGCUCUAGAUGAC 23 downstream

CEP290-1848 - GGUCACGAAAAUCAGAUUUCAUGU 24 downstream

CEP290-1849 - GUCACGAAAAUCAGAUUUCAUGU 23 downstream

CEP290-1850 - GAAAAUCAGAUUUCAUGU 18 downstream

CEP290-1851 - GCUAAUACAUGAGAGUGAUUAGUG 24 downstream

CEP290-1852 + GCUUGAACUCUGUGCCAAA 19 downstream

CEP290-1853 + GCUCUAGAUGACAUGAGGUAAG 22 downstream

CEP290-1854 - GAUGUAGAUUGAGGUAGAAUCAAG 24 downstream

CEP290-1855 - G U AG AU UG AGG U AG AAU CAAG 21 downstream

CEP290-1856 - GAUUGAGGUAGAAUCAAG 18 downstream

CEP290-1857 + GAUGCAGAACUAGUGUAGA 19 downstream

CEP290-1858 - GAUGUAGAUUGAGGUAGAAUC 21 downstream

CEP290-1859 - GUAGAUUGAGGUAGAAUC 18 downstream

CEP290-1860 + GAAUGAUCAUUCUUGUGGCAGUA 23 downstream

CEP290-1861 + GAUCAUUCUUGUGGCAGUA 19 downstream

CEP290-1862 + GAAUGAUCAUUCUUGUGGCAGU 22 downstream

CEP290-1863 + GAUCAUUCUUGUGGCAGU 18 downstream

CEP290-1864 - GAGAGGUAAAGGUUCAUGAGAC 22 downstream

CEP290-1865 - G AGG UAAAGG U U CAUG AG AC 20 downstream

CEP290-1866 - GGUAAAGGUUCAUGAGAC 18 downstream

CEP290-1867 + GGUAGCUUUUGACAGUUUUUAAG 23 downstream

CEP290-1868 + GUAGCUUUUGACAGUUUUUAAG 22 downstream

CEP290-1869 + GCUUUUGACAGUUUUUAAG 19 downstream

CEP290-1870 + GUAGCUUUUGACAGUUUUUAAGGC 24 downstream

CEP290-1871 + GCUUUUGACAGUUUUUAAGGC 21 downstream

CEP290-1872 + GAAAUUCACUGAGCAAAACAAC 22 downstream

CEP290-1873 + GUGGCAGUAAGGAGGAUGUAAGA 23 downstream

CEP290-1874 + GGCAGUAAGGAGGAUGUAAGA 21 downstream

CEP290-1875 + GCAGUAAGGAGGAUGUAAGA 20 downstream

CEP290-1876 - GGAAAUUAUUGUUGCUUUUU 20 downstream

CEP290-1877 - GAAAUUAUUGUUGCUUUUU 19 downstream

CEP290-1878 - GAAAAACUUGAAAUUUGAUAG 21 downstream

CEP290-1879 - G A AG A A A A A AG AAA UAGAUGUAGA 24 downstream

CEP290-1880 - GAAAAAAGAAAUAGAUGUAGA 21 downstream

CEP290-1881 - GUGUUGCCCAGGCUGGAGUGCA 22 downstream CEP290-1882 - GUUGCCCAGGCUGGAGUGCA 20 downstream

CEP290-1883 - GAGUCUCACUGUGUUGCCCAGG 22 downstream

CEP290-1884 - GUCUCACUGUGUUGCCCAGG 20 downstream

CEP290-1885 + UUUUUAAGGCGGGGAGUCACA 21 downstream

CEP290-1886 + UUUUAAGGCGGGGAGUCACA 20 downstream

CEP290-1887 + UUUAAGGCGGGGAGUCACA 19 downstream

CEP290-1888 + UUAAGGCGGGGAGUCACA 18 downstream

CEP290-1889 - UGUCAAAAGCUACCGGUUAC 20 downstream

CEP290-1890 - UCAAAAGCUACCGGUUAC 18 downstream

CEP290-1891 + UUCAUCUCUUGCUCUAGAUGAC 22 downstream

CEP290-1892 + UCAUCUCUUGCUCUAGAUGAC 21 downstream

CEP290-1893 + UCUCUUGCUCUAGAUGAC 18 downstream

CEP290-1894 - UCACGAAAAUCAGAUUUCAUGU 22 downstream

CEP290-1895 - UAAUACAUGAGAGUGAUUAGUG 22 downstream

CEP290-1896 - UACAUGAGAGUGAUUAGUG 19 downstream

CEP290-1897 + UAUUAGCUUGAACUCUGUGCCAAA 24 downstream

CEP290-1898 + UUAGCUUGAACUCUGUGCCAAA 22 downstream

CEP290-1899 + UAGCUUGAACUCUGUGCCAAA 21 downstream

CEP290-1900 + UUGCUCUAGAUGACAUGAGGUAAG 24 downstream

CEP290-1901 + UGCUCUAGAUGACAUGAGGUAAG 23 downstream

CEP290-1902 + UCUAGAUGACAUGAGGUAAG 20 downstream

CEP290-1903 + UAGAUGACAUGAGGUAAG 18 downstream

CEP290-1904 - UGUAGAUUGAGGUAGAAUCAAG 22 downstream

CEP290-1905 - UAGAUUGAGGUAGAAUCAAG 20 downstream

CEP290-1906 + UAUAAGAUGCAGAACUAGUGUAGA 24 downstream

CEP290-1907 + UAAGAUGCAGAACUAGUGUAGA 22 downstream

CEP290-1908 - UAGAUGUAGAUUGAGGUAGAAUC 23 downstream

CEP290-1909 - UGUAGAUUGAGGUAGAAUC 19 downstream

CEP290-1910 + UGAUCAUUCUUGUGGCAGUA 20 downstream

CEP290-1911 + UAGAAUGAUCAUUCUUGUGGCAGU 24 downstream

CEP290-1912 + UGAUCAUUCUUGUGGCAGU 19 downstream

CEP290-1913 - UUGAGAGGUAAAGGUUCAUGAGAC 24 downstream

CEP290-1914 - UGAGAGGUAAAGGUUCAUGAGAC 23 downstream

CEP290-1915 + U AG CU U UUGACAG U U U U UAAG 21 downstream

CEP290-1916 + UAGCUUUUGACAGUUUUUAAGGC 23 downstream

CEP290-1917 + UUUUGACAGUUUUUAAGGC 19 downstream

CEP290-1918 + UUUGACAGUUUUUAAGGC 18 downstream

CEP290-1919 + UAGAAAUUCACUGAGCAAAACAAC 24 downstream

CEP290-1920 + UUCACUGAGCAAAACAAC 18 downstream

CEP290-1921 + UGUGGCAGUAAGGAGGAUGUAAGA 24 downstream

CEP290-1922 + UGGCAGUAAGGAGGAUGUAAGA 22 downstream

CEP290-1923 + UAUCAAAAGACUUAUAUUCCAUUA 24 downstream CEP290-1924 + UCAAAAGACUUAUAUUCCAUUA 22 downstream

CEP290-1925 - UUAGGAAAUUAUUGUUGCUUUUU 23 downstream

CEP290-1926 - UAGGAAAUUAUUGUUGCUUUUU 22 downstream

CEP290-1927 - UGUGUUGCCCAGGCUGGAGUGCA 23 downstream

CEP290-1928 - UGUUGCCCAGGCUGGAGUGCA 21 downstream

CEP290-1929 - UUGCCCAGGCUGGAGUGCA 19 downstream

CEP290-1930 - UGCCCAGGCUGGAGUGCA 18 downstream

CEP290-1931 - UCUCACUGUGUUGCCCAGG 19 downstream

CEP290-13 + AUGAGAUACUCACAAUUACAAC 22 upstream

CEP290-18 + GUAUGAGAUACUCACAAUUACAAC 24 upstream

CEP290-14 + UAUGAGAUACUCACAAUUACAAC 23 upstream

CEP290-19 + GGUAUGAGAUAUUCACAAUUACAA 24 upstream

Table 9A provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the first tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, have good orthogonality, and start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 9A

Table 9B provides targeting domains for break-induced deletion of genomic sequence including the mutation at the LCAIO target position in the CEP290 gene selected according to the second tier parameters. The targeting domains are within 1000 bp upstream of an Alu repeat, within 40bp upstream of mutation, or 1000 bp downstream of the mutation, have good orthogonality, and do not start with G. It is contemplated herein that the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single- stranded break (Cas9 nickase).

Table 9B

Table 10 provides targeting domains for break- induced deletion of genomic sequence including the mutation at the LCA10 target position in the CEP290 gene by dual targeting (e.g., dual double strand cleavage). Exemplary gRNA pairs to be used with S. aureus Cas9 are shown in Table 10, e.g., CEP290-323 can be combined with CEP290-11, CEP290-323 can be combined with CEP290-64, CEP290-490 can be combined with CEP290-496, CEP290-490 can be combined with CEP290-502, CEP290-490 can be combined with CEP290-504, CEP290-492 can be combined with CEP290-502, or CEP290-492 can be combined with CEP290-504.

Table 10

III. Cas9 Molecules

Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes, S. aureus, and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules Cas9 molecules from the other species can replace them. Such species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp.,

Cycliphilusdenitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus,

Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp.,

Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida,

Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris,

Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae,

Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

A Cas9 molecule, or Cas9 polypeptide, as that term is used herein, refers to a molecule or polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, homes or localizes to a site which comprises a target domain and PAM sequence. Cas9 molecule and Cas9 polypeptide, as those terms are used herein, refer to naturally occurring Cas9 molecules and to engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table 11.

Cas9 Domains

Crystal structures have been determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., Science, 343(6176): 1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935- 949, 2014; and Anders et al., Nature, 2014, doi: 10.1038/naturel3579).

A naturally occurring Cas9 molecule comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains described herein. Figs. 8A-8B provide a schematic of the organization of important Cas9 domains in the primary structure. The domain nomenclature and the numbering of the amino acid residues encompassed by each domain used throughout this disclosure is as described in Nishimasu et al. The numbering of the amino acid residues is with reference to Cas9 from S. pyogenes.

The REC lobe comprises the arginine-rich bridge helix (BH), the REC1 domain, and the REC2 domain. The REC lobe does not share structural similarity with other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long a helix and arginine rich region and comprises amino acids 60-93 of the sequence of S. pyogenes Cas9. The REC1 domain is important for recognition of the repeat: anti-repeat duplex, e.g., of a gRNA or a tracrRNA, and is therefore critical for Cas9 activity by recognizing the target sequence. The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and 308 to 717 of the sequence of S. pyogenes Cas9. These two REC1 domains, though separated by the REC2 domain in the linear primary structure, assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or parts thereof, may also play a role in the recognition of the repeat: anti-repeat duplex. The REC2 domain comprises amino acids 180-307 of the sequence of S. pyogenes Cas9.

The NUC lobe comprises the RuvC domain (also referred to herein as RuvC-like domain), the HNH domain (also referred to herein as HNH-like domain), and the PAM- interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The RuvC domain is assembled from the three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1- 59, 718-769, and 909-1098, respectively, of the sequence of S. pyogenes Cas9. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule. The HNH domain lies between the RuvC II-III motifs and comprises amino acids 775-908 of the sequence of S. pyogenes Cas9. The PI domain interacts with the PAM of the target nucleic acid molecule, and comprises amino acids 1099-1368 of the sequence of S. pyogenes Cas9.

A RuvC-like domain and an HNH-like domain

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises an HNH-like domain and a RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH-like domain. A Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more of the following domains: a RuvC- like domain and an HNH-like domain. In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide and the eaCas9 molecule or eaCas9 polypeptide comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below.

RuvC-like domains

In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non- complementary strand of the target nucleic acid molecule. The Cas9 molecule or Cas9 polypeptide can include more than one RuvC-like domain (e.g., one, two, three or more RuvC- like domains). In an embodiment, a RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 18, 17, 16 or 15 amino acids in length. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.

N-terminal RuvC-like domains

Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules or Cas9 polypeptide can comprise an N-terminal RuvC-like domain. Exemplary N- terminal RuvC-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:

D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9 (SEQ ID NO: 8),

wherein,

XI is selected from I, V, M, L and T (e.g., selected from I, V, and L);

X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);

X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X4 is selected from S, Y, N and F (e.g., S);

X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);

X6 is selected from W, F, V, Y, S and L (e.g., W);

X7 is selected from A, S, C, V and G (e.g., selected from A and S);

X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and

X9 is selected from any amino acid or is absent, designated by Δ (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID

NO:8, by as many as 1 but no more than 2, 3, 4, or 5 residues. In embodiment, the N-terminal RuvC-like domain is cleavage competent.

In embodiment, the N-terminal RuvC-like domain is cleavage incompetent.

In an embodiment, a eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:

D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9, (SEQ ID NO: 9),

wherein

XI is selected from I, V, M, L and T (e.g., selected from I, V, and L);

X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);

X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);

X6 is selected from W, F, V, Y, S and L (e.g., W);

X7 is selected from A, S, C, V and G (e.g., selected from A and S);

X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and

X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:9 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:

D-I-G-X2-X3-S-V-G-W-A-X8-X9 (SEQ ID NO: 10),

wherein

X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);

X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and

X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A,

Y, M and R or selected from e.g., T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 10 by as many as 1 but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:

D-I-G-T-N-S-V-G-W-A-V-X (SEQ ID NO: 11), wherein

X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V, I, L and T (e.g., the eaCas9 molecule can comprise an N-terminal RuvC-like domain shown in Figs. 2A-2G (is depicted as Y)).

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 11 by as many as 1 but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N- terminal RuvC like domain disclosed herein, e.g., in Figs. 3A-3B or Figs. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, or all 3 of the highly conserved residues identified in Figs. 3A-3B or Figs. 7A-7B are present.

In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N- terminal RuvC-like domain disclosed herein, e.g., in Figs. 4A-4B or Figs. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, 3 or all 4 of the highly conserved residues identified in Figs. 4A-4B or Figs. 7A-7B are present.

Additional RuvC-like domains

In addition to the N-terminal RuvC-like domain, the Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more additional RuvC-like domains. In an embodiment, the Cas9 molecule or Cas9 polypeptide can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.

An additional RuvC-like domain can comprise an amino acid sequence:

I-X1-X2-E-X3-A-R-E (SEQ ID NO: 12), wherein

XI is V or H,

X2 is I, L or V (e.g., I or V); and

X3 is M or T.

In an embodiment, the additional RuvC-like domain comprises the amino acid sequence: I-V-X2-E-M-A-R-E (SEQ ID NO: 13), wherein

X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule or eaCas9 polypeptide can comprise an additional RuvC-like domain shown in Fig. 2A-2G or Figs. 7A-7B (depicted as B)).

An additional RuvC-like domain can comprise an amino acid sequence: H-H-A-X1-D-A-X2-X3 (SEQ ID NO: 14), wherein

XI is H or L;

X2 is R or V; and

X3 is E or V.

In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:

H-H-A-H-D-A-Y-L (SEQ ID NO: 15).

In an embodiment, the additional RuvC-like domain differs from a sequence of SEQ ID NO: 13, 15, 12 or 14 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In some embodiments, the sequence flanking the N-terminal RuvC-like domain is a sequences of formula V:

K-X1'-Y-X2'-X3'-X4'-Z-T-D-X9'-Y, (SEQ ID NO: 16).

wherein

Χ is selected from K and P,

X2' is selected from V, L, I, and F (e.g., V, I and L);

X3' is selected from G, A and S (e.g., G),

X4' is selected from L, I, V and F (e.g., L);

X9' is selected from D, E, N and Q; and

Z is an N-terminal RuvC-like domain, e.g., as described above.

HNH-like domains

In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VI:

X 1 -X2-X3-H-X4-X5-P-X6-X7-X8-X9-X 10-X 11 -X 12-X 13-X 14-X 15-N-X 16-X 17-X 18- X19-X20-X21-X22-X23-N (SEQ ID NO: 17), wherein

XI is selected from D, E, Q and N (e.g., D and E);

X2 is selected from L, I, R, Q, V, M and K;

X3 is selected from D and E; X4 is selected from I, V, T, A and L (e.g., A, I and V);

X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

X6 is selected from Q, H, R, K, Y, I, L, F and W;

X7 is selected from S, A, D, T and K (e.g., S and A);

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

XI 1 is selected from D, S, N, R, L and T (e.g., D);

X12 is selected from D, N and S;

X13 is selected from S, A, T, G and R (e.g., S);

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);

X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X16 is selected from K, L, R, M, T and F (e.g., L, R and K);

X17 is selected from V, L, I, A and T;

X18 is selected from L, I, V and A (e.g., L and I);

X19 is selected from T, V, C, E, S and A (e.g., T and V);

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X21 is selected from S, P, R, K, N, A, H, Q, G and L;

X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 16 by at least one but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain is cleavage competent.

In an embodiment, the HNH-like domain is cleavage incompetent.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:

X 1 -X2-X3-H-X4-X5-P-X6-S-X8-X9-X 10-D-D-S-X 14-X 15-N-K- V-L-X 19-X20-X21 - X22-X23-N (SEQ ID NO: 18),

wherein

XI is selected from D and E;

X2 is selected from L, I, R, Q, V, M and K; X3 is selected from D and E;

X4 is selected from I, V, T, A and L (e.g., A, I and V);

X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

X6 is selected from Q, H, R, K, Y, I, L, F and W;

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);

X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X19 is selected from T, V, C, E, S and A (e.g., T and V);

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X21 is selected from S, P, R, K, N, A, H, Q, G and L;

X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 15 by 1,

2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:

X 1 - V-X3-H-I- V-P-X6-S-X8-X9-X 10-D-D-S-X 14-X 15-N-K- V-L-T-X20-X21 -X22-X23- N (SEQ ID NO: 19),

wherein

XI is selected from D and E;

X3 is selected from D and E;

X6 is selected from Q, H, R, K, Y, I, L and W;

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);

X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X21 is selected from S, P, R, K, N, A, H, Q, G and L; X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO:GG by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VIII:

D-X2-D-H-I-X5-P-Q-X7-F-X9-X 10-D-X 12-S-I-D-N-X 16- V-L-X 19-X20-S-X22-X23-N (SEQ ID NO:20),

wherein

X2 is selected from I and V;

X5 is selected from I and V;

X7 is selected from A and S;

X9 is selected from I and L;

X10 is selected from K and T;

X12 is selected from D and N;

X16 is selected from R, K and L; X19 is selected from T and V;

X20 is selected from S and R;

X22 is selected from K, D and A; and

X23 is selected from E, K, G and N (e.g., the eaCas9 molecule or eaCas9 polypeptide can comprise an HNH-like domain as described herein).

In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 19 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises the amino acid sequence of formula IX:

L- Y- Y-L-Q-N-G-X 1 ' -D-M- Y-X2' -X3 ' -X4' -X5 ' -L-D-I— X6' -X7 ' -L-S-X8 ' - Y-Z-N-R- X9'-K-X10'-D-X11'-V-P (SEQ ID NO:21),

wherein

Χ is selected from K and R;

X2' is selected from V and T;

X3' is selected from G and D;

X4' is selected from E, Q and D; Χ5' is selected from E and D;

X6' is selected from D, N and H;

XV is selected from Y, R and N;

X8' is selected from Q, D and N; X9' is selected from G and E;

X10' is selected from S and G;

XI 1 ' is selected from D and N; and

Z is an HNH-like domain, e.g., as described above.

In an embodiment, the eaCas9 molecule or eaCas9 polypeptide comprises an amino acid sequence that differs from a sequence of SEQ ID NO:21 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in Figs. 5A-5C or Figs. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 or both of the highly conserved residues identified in Figs. 5A- 5C or Figs. 7A-7B are present.

In an embodiment, the HNH -like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in Figs. 6A-6B or Figs. 7A-7B , as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, all 3 of the highly conserved residues identified in Figs. 6A-6B or Figs. 7A-7B are present. Cas9 Activities

Nuclease and Helicase Activities

In an embodiment, the Cas9 molecule or Cas9 polypeptide is capable of cleaving a target nucleic acid molecule. Typically wild type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and Cas9 polypeptides can be engineered to alter nuclease cleavage (or other properties), e.g., to provide a Cas9 molecule or Cas9 peolypeptide which is a nickase, or which lacks the ability to cleave target nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 molecule or eaCas9 polypeptide.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following activities: a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule;

a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;

an endonuclease activity;

an exonuclease activity; and

a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.

In an embodiment, an enzymatically active or eaCas9 molecule or eaCas9 polypeptide cleaves both strands and results in a double stranded break. In an embodiment, an eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an active, or cleavage competent, HNH-like domain and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an inactive, or cleavage incompetent, HNH- like domain and an active, or cleavage competent, N-terminal RuvC-like domain.

Some Cas9 molecules or Cas9 polypeptides have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule localize to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 molecule or eiCas9 polypeptide. For example, an eiCas9 molecule or eiCas9 polypeptide can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1 % of the cleavage activity of a reference Cas9 molecule or eiCas9 polypeptide, as measured by an assay described herein. Targeting and PAMs

A Cas9 molecule or Cas9 polypeptide, is a polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, localizes to a site which comprises a target domain and PAM sequence.

In an embodiment, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG, NAG, NGA and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali et al., Science 2013; 339(6121): 823-826. In an embodiment, an eaCas9 molecule of S. thermophilus recognizes the sequence motif NGGNG and NNAGAAW (W = A or T) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al., Science 2010; 327(5962): 167-170, and Deveau et al., J Bacteriol 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. mutans recognizes the sequence motif NGG and/or NAAR (R = A or G) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al., J Bacteriol 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R = A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRN (R = A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRT (R = A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRV (R = A or G, V = A, G or C) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of Neisseria meningitidis recognizes the sequence motif NNNNGATT or NNNGCTT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al., PNAS Early Edition 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al., Science 2012 337:816. In the aforementioned embodiments, N can be any nucleotide residue, e.g., any of A, G, C or T.

As is discussed herein, Cas9 molecules can be engineered to alter the PAM specificity of the Cas9 molecule.

Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA BIOLOGY 2013 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family, a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family. Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S.

gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clipl l262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Another exemplary Cas9 molecule is a Cas9 molecule of Neisseria meningitidis (Hou et al., PNAS Early Edition 2013, 1- 6).

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence:

having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with;

differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared with;

differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or

is identical to any Cas9 molecule sequence described herein, or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA BIOLOGY 2013 10:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6; SEQ ID NOS: l-4. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to home to a target nucleic acid.

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises the amino acid sequence of the consensus sequence of Figs. 2A-2G, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutans and L. innocua, and "-" indicates any amino acid. In an embodiment, a Cas9 molecule or Cas9 polypeptide differs from the sequence of the consensus sequence disclosed in Figs. 2A-2G by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises the amino acid sequence of SEQ ID NO:7 of Figs. 7A-7B, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, or N. meningitidis, "-" indicates any amino acid, and "-" indicates any amino acid or absent. In an embodiment, a Cas9 molecule or Cas9 polypeptide differs from the sequence of SEQ ID NO:6 or 7 disclosed in Figs. 7A-7B by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

A comparison of the sequence of a number of Cas9 molecules indicate that certain regions are conserved. These are identified below as:

region 1 (residues 1 to 180, or in the case of region 1 'residues 120 to 180)

region 2 (residues360 to 480);

region 3 (residues 660 to 720);

region 4 (residues 817 to 900); and

region 5 (residues 900 to 960);

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises regions 1-5, together with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In an embodiment, each of regions 1-6, independently, have, 50%, 60%, 70%, or 80% homology with the corresponding residues of a Cas9 molecule or Cas9 polypeptide described herein, e.g., a sequence from Figs. 2A-2G or from Figs. 7A-7B.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 1 :

having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 1-180 (the numbering is according to the motif sequence in Figs. 2A-2G; 52% of residues in the four Cas9 sequences in Figs. 2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes;

differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua; or is identical to 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 1 ' :

having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 120-180 (55% of residues in the four Cas9 sequences in Figs. 2A- 2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S.

mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua ; or

is identical to 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 2:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 360-480 (52% of residues in the four Cas9 sequences in Figs. 2A- 2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S.

mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua; or

is identical to 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 3:

having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 660-720 (56% of residues in the four Cas9 sequences in Figs. 2A- 2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S.

mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua; or

is identical to 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 4:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 817-900 (55% of residues in the four Cas9 sequences in Figs. 2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua; or

is identical to 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 5:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 900-960 (60% of residues in the four Cas9 sequences in Figs. 2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua; or

is identical to 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S.

thermophilus, S. mutans or L. innocua.

Engineered or Altered Cas9 Molecules and Cas9 Polypeptides

Cas9 molecules and Cas9 polypeptides described herein, e.g., naturally occurring Cas9 molecules, can possess any of a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In an embodiment, a Cas9 molecule or Cas9 polypeptide can include all or a subset of these properties. In typical embodiments, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules and Cas9 polypeptides.

Cas9 molecules include engineered Cas9 molecules and engineered Cas9 polypeptides (engineered, as used in this context, means merely that the Cas9 molecule or Cas9 polypeptide differs from a reference sequences, and implies no process or origin limitation). An engineered Cas9 molecule or Cas9 polypeptide can comprise altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference Cas9 molecule) or altered helicase activity. As discussed herein, an engineered Cas9 molecule or Cas9 polypeptide can have nickase activity (as opposed to double strand nuclease activity). In an embodiment an engineered Cas9 molecule or Cas9 polypeptide can have an alteration that alters its size, e.g., a deletion of amino acid sequence that reduces its size, e.g., without significant effect on one or more, or any Cas9 activity. In an embodiment, an engineered Cas9 molecule or Cas9 polypeptide can comprise an alteration that affects PAM recognition. E.g., an engineered Cas9 molecule can be altered to recognize a PAM sequence other than that recognized by the endogenous wild-type PI domain. In an embodiment, a Cas9 molecule or Cas9 polypeptide can differ in sequence from a naturally occurring Cas9 molecule but not have significant alteration in one or more Cas9 activities.

Cas9 molecules or Cas9 polypeptides with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring, Cas9 molecules or Cas9 polypeptides, to provide an altered Cas9 molecule or Cas9 polypeptide having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule, can be introduced. Such mutations and differences comprise: substitutions (e.g., conservative substitutions or

substitutions of non-essential amino acids); insertions; or deletions. In an embodiment, a Cas9 molecule or Cas9 polypeptide can comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations, but less than 200, 100, or 80 mutations relative to a reference, e.g., a parental, Cas9 molecule.

In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein.

Non-Cleaving and Modified-Cleavage Cas9 Molecules and Cas9 Polypeptides

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule or Cas9 polypeptide can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded nucleic acid (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity) , e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

Modified Cleavage eaCas9 Molecules and eaCas9 Polypeptides

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following activities: cleavage activity associated with an N-terminal RuvC-like domain; cleavage activity associated with an HNH-like domain; cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an active, or cleavage competent, HNH-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO:21) and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N- terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figs. 2A-2G or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 molecule or eaCas9 polypeptide differs from wild type in the N-terminal RuvC-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or .1 % of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus . In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, N-terminal RuvC-like domain (e.g., an N-terminal RuvC-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine shown at position 856 of Figs. 2A-2G, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine shown at position 870 of Figs. 2A-2G and/or at position 879 of Figs. 2A-2G, e.g., can be substituted with an alanine. In an

embodiment, the eaCas9 differs from wild type in the HNH-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.

Alterations in the Ability to Cleave One or Both Strands of a Target Nucleic Acid

In an embodiment, exemplary Cas9 activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in one or more RuvC- like domain, e.g., an N-terminal RuvC-like domain; an HNH-like domain; a region outside the

RuvC-like domains and the HNH-like domain. In some embodiments, a mutation(s) is present in a RuvC-like domain, e.g., an N-terminal RuvC-like domain. In some embodiments, a mutation(s) is present in an HNH-like domain. In some embodiments, mutations are present in both a RuvC-like domain, e.g., an N-terminal RuvC-like domain, and an HNH-like domain.

Exemplary mutations that may be made in the RuvC domain or HNH domain with reference to the S. pyogenes sequence include: D10A, E762A, H840A, N854A, N863A and/or D986A.

In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide comprising one or more differences in a RuvC domain and/or in an HNH domain as compared to a reference Cas9 molecule, and the eiCas9 molecule or eiCas9 polypeptide does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wildype, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1% of a reference Cas9 molecule, as measured by an assay described herein.

Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc., can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative or by the method described in Section IV. In an embodiment, a "non-essential" amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an "essential" amino acid residue results in a substantial loss of activity (e.g., cleavage activity).

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule or Cas9 polypeptide can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S aureus, S. pyogenes, or C. jejuni as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S aureus, S.

pyogenes, or C. jejuni); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S aureus, S. pyogenes, or C. jejuni); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising one or more of the following activities: cleavage activity associated with a RuvC domain; cleavage activity associated with an HNH domain; cleavage activity associated with an HNH domain and cleavage activity associated with a RuvC domain.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule or eiCas9 polypeptide lacks substantial cleavage activity associated with a RuvC domain and cleavage activity associated with an HNH domain.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figs. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., has a substitution) at one or more residue (e.g., 2,

3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figs. 2A-2G or SEQ ID NO:7.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figs. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figs. 2A-2G;

the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figs. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figs. 2A-2G.

In an embodiment the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in

Figs. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figs. 2A-2G;

the sequence corresponding to the residues identified by "*"in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figs. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of S. mutans (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figs. 2A-2G. In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figs. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figs. 2A-2G;

the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutans Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutans Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of L. innocula shown in the consensus sequence disclosed in Figs. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of L. innocula (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-"in the consensus sequence disclosed in Figs. 2A-2G.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figs. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figs. 2A-2G;

the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule; and,

the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figs. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "-" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule, can be a fusion, e.g., of two of more different Cas9 molecules or Cas9 polypeptides, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of Cas9 molecule of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 molecule of a species other than S. pyogenes (e.g., S. thermophilus) comprising an HNH-like domain.

Cas9 Molecules and Cas9 Polypeptides with Altered PAM Recognition or No PAM

Recognition

Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example, the PAM recognition sequences described above for S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.

In an embodiment, a Cas9 molecule or Cas9 polypeptide has the same PAM specificities as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule or Cas9 polypeptide has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule or Cas9 polypeptide can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity, e.g., to decrease off target sites and increase specificity. In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules or Cas9 polypeptides that recognize different PAM sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt et al. NATURE 2011, 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section IV.

Alterations of the PI domain, which mediates PAM recognition, are discussed below. Synthetic Cas9 Molecules and Cas9 Polypeptides with Altered PI Domains Current genome-editing methods are limited in the diversity of target sequences that can be targeted by the PAM sequence that is recognized by the Cas9 molecule utilized. A synthetic Cas9 molecule (or Syn-Cas9 molecule), or synthetic Cas9 polypeptide (or Syn-Cas9

polypeptide), as that term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a Cas9 core domain from one bacterial species and a functional altered PI domain, i.e., a PI domain other than that naturally associated with the Cas9 core domain, e.g., from a different bacterial species.

In an embodiment, the altered PI domain recognizes a PAM sequence that is different from the PAM sequence recognized by the naturally- occurring Cas9 from which the Cas9 core domain is derived. In an embodiment, the altered PI domain recognizes the same PAM sequence recognized by the naturally- occurring Cas9 from which the Cas9 core domain is derived, but with different affinity or specificity. A Syn-Cas9 molecule or Syn-Cas9 polypetide can be, respectively, a Syn-eaCas9 molecule or Syn-eaCas9 polypeptide or a Syn-eiCas9 molecule Syn- eiCas9 polypeptide.

An exemplary Syn-Cas9 molecule or Syn-Cas9 polypetide comprises:

a) a Cas9 core domain, e.g., a Cas9 core domain from Table 11 or 12, e.g., a S. aureus, S. pyogenes, or C. jejuni Cas9 core domain; and

b) an altered PI domain from a species X Cas9 sequence selected from Tables 14 and 15.

In an embodiment, the RKR motif (the PAM binding motif) of said altered PI domain comprises: differences at 1, 2, or 3 amino acid residues; a difference in amino acid sequence at the first, second, or third position; differences in amino acid sequence at the first and second positions, the first and third positions, or the second and third positions; as compared with the sequence of the RKR motif of the native or endogenous PI domain associated with the Cas9 core domain.

In an embodiment, the Cas9 core domain comprises the Cas9 core domain from a species X Cas9 from Table 11 and said altered PI domain comprises a PI domain from a species Y Cas9 from Table 11.

In an embodiment, the RKR motif of the species X Cas9 is other than the RKR motif of the species Y Cas9. In an embodiment, the RKR motif of the altered PI domain is selected from XXY, XNG, and XNQ.

In an embodiment, the altered PI domain has at least 60, 70, 80, 90, 95, or 100% homology with the amino acid sequence of a naturally occurring PI domain of said species Y from Table 11.

In an embodiment, the altered PI domain differs by no more than 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from the amino acid sequence of a naturally occurring PI domain of said second species from Table 11.

In an embodiment, the Cas9 core domain comprises a S. aureus core domain and altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from

Table 15.

In an embodiment, the Cas9 core domain comprises a S. pyogenes core domain and the altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 15.

In an embodiment, the Cas9 core domain comprises a C. jejuni core domain and the altered PI domain comprises: an A. denitrificans PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 15.

In an embodiment, the Cas9 molecule or Cas9 polypeptide further comprises a linker disposed between said Cas9 core domain and said altered PI domain.

In an embodiment, the linker comprises: a linker described elsewhere herein disposed between the Cas9 core domain and the heterologous PI domain. Suitable linkers are further described in Section V.

Exemplary altered PI domains for use in Syn-Cas9 molecules are described in Tables 14 and 15. The sequences for the 83 Cas9 orthologs referenced in Tables 14 and 15 are provided in Table 11. Table 13 provides the Cas9 orthologs with known PAM sequences and the corresponding RKR motif.

In an embodiment, a Syn-Cas9 molecule or Syn-Cas9 polypeptide may also be size- optimized, e.g., the Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises one or more deletions, and optionally one or more linkers disposed between the amino acid residues flanking the deletions. In an embodiment, a Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises a REC deletion.

Size-Optimized Cas9 Molecules and Cas9 Polypeptides

Engineered Cas9 molecules and engineered Cas9 polypeptides described herein include a

Cas9 molecule or Cas9 polypeptide comprising a deletion that reduces the size of the molecule while still retaining desired Cas9 properties, e.g., essentially native conformation, Cas9 nuclease activity, and/or target nucleic acid molecule recognition. Provided herein are Cas9 molecules or Cas9 polypeptides comprising one or more deletions and optionally one or more linkers, wherein a linker is disposed between the amino acid residues that flank the deletion. Methods for identifying suitable deletions in a reference Cas9 molecule, methods for generating Cas9 molecules with a deletion and a linker, and methods for using such Cas9 molecules will be apparent to one of ordinary skill in the art upon review of this document.

A Cas9 molecule, e.g., a S. aureus, S. pyogenes, or C. jejuni, Cas9 molecule, having a deletion is smaller, e.g., has reduced number of amino acids, than the corresponding naturally- occurring Cas9 molecule. The smaller size of the Cas9 molecules allows increased flexibility for delivery methods, and thereby increases utility for genome-editing. A Cas9 molecule or Cas9 polypeptide can comprise one or more deletions that do not substantially affect or decrease the activity of the resultant Cas9 molecules or Cas9 polypeptides described herein. Activities that are retained in the Cas9 molecules or Cas9 polypeptides comprising a deletion as described herein include one or more of the following:

a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;

an endonuclease activity;

an exonuclease activity;

a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid;

and recognition activity of a nucleic acid molecule, e.g., a target nucleic acid or a gRNA. Activity of the Cas9 molecules or Cas9 polypeptides described herein can be assessed using the activity assays described herein or in the art.

Identifying regions suitable for deletion

Suitable regions of Cas9 molecules for deletion can be identified by a variety of methods.

Naturally- occurring orthologous Cas9 molecules from various bacterial species, e.g., any one of those listed in Table 11, can be modeled onto the crystal structure of S. pyogenes Cas9

(Nishimasu et al., Cell, 156:935-949, 2014) to examine the level of conservation across the selected Cas9 orthologs with respect to the three-dimensional conformation of the protein. Less conserved or unconserved regions that are spatially located distant from regions involved in Cas9 activity, e.g., interface with the target nucleic acid molecule and/or gRNA, represent regions or domains are candidates for deletion without substantially affecting or decreasing Cas9 activity.

REC-Optimized Cas9 Molecules and Cas9 Polypeptides

A REC-optimized Cas9 molecule, or a REC-optimized Cas9 polypeptide, as that term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a deletion in one or both of the REC2 domain and the REI _CT domain (collectively a REC deletion), wherein the deletion comprises at least 10% of the amino acid residues in the cognate domain. A REC- optimized Cas9 molecule or Cas9 polypeptide can be an eaCas9 molecule or eaCas9 polypetide, or an eiCas9 molecule or eiCas9 polypeptide. An exemplary REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises:

a) a deletion selected from:

i) a REC2 deletion;

ii) a REC I _CT deletion; or

iii) a REC 1 SU _B deletion.

Optionally, a linker is disposed between the amino acid residues that flank the deletion. In an embodiment, a Cas9 molecule or Cas9 polypeptide includes only one deletion, or only two deletions. A Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a RECI _CT deletion. A Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC I _SUB deletion. Generally, the deletion will contain at least 10% of the amino acids in the cognate domain, e.g., a REC2 deletion will include at least 10% of the amino acids in the REC2 domain.

A deletion can comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the amino acid residues of its cognate domain; all of the amino acid residues of its cognate domain; an amino acid residue outside its cognate domain; a plurality of amino acid residues outside its cognate domain; the amino acid residue immediately N terminal to its cognate domain; the amino acid residue immediately C terminal to its cognate domain; the amino acid residue immediately N terminal to its cognate and the amino acid residue immediately C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to to its cognate domain and a plurality of e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain.

In an embodiment, a deletion does not extend beyond: its cognate domain; the N terminal amino acid residue of its cognate domain; the C terminal amino acid residue of its cognate domain.

A REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide can include a linker disposed between the amino acid residues that flank the deletion. Suitable linkers for use between the amino acid resides that flank a REC deletion in a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide is disclosed in Section V.

In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associated linker, has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% homology with the amino acid sequence of a naturally occurring Cas 9, e.g., a Cas9 molecule described in Table 11, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

In an embodiment, a a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associated linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25, amino acid residues from the amino acid sequence of a naturally occurring Cas 9, e.g., a Cas9 molecule described in Table 11, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule. In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associate linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25% of the, amino acid residues from the amino acid sequence of a naturally occurring Cas 9, e.g., a Cas9 molecule described in Table 11, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Biol.

48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Sequence information for exemplary REC deletions are provided for 83 naturally- occurring Cas9 orthologs in Table 11.

The amino acid sequences of exemplary Cas9 molecules from different bacterial species are shown below.

Table 11. Amino Acid Sequence of Cas9 Orthologs

Finegoldia magna ATCC 29328 SEQ ID NO: 168 313 146 452 534 77 452 534 77 gill69823755lreflYP_001691366 315

.1

CoriobacteriumglomeransPW2 SEQ ID NO: 175 318 144 511 592 82 511 592 82 gil328956315lreflYP_004373648 316

.1

Eubacterium yurii ATCC 43715 SEQ ID NO: 169 310 142 552 633 76 552 633 76 gil306821691 lreflZP_07455288.1 317

Peptoniphilus duerdenii ATCC SEQ ID NO: 171 311 141 535 615 76 535 615 76 BAA- 1640 318

gil304438954lreflZP_07398877.1

Acidaminococcus sp. D21 SEQ ID NO: 167 306 140 511 591 75 511 591 75 gil227824983lreflZP_03989815.1 319

Lactobacillus farciminis KCTC SEQ ID NO: 171 310 140 542 621 85 542 621 85

3681 320

gil336394882lreflZP_08576281.1

Streptococcus sanguinis SK49 SEQ ID NO: 185 324 140 411 490 85 411 490 85 gil422884106lreflZP_l 6930555.1 321

Coprococcus catus GD-7 SEQ ID NO: 172 310 139 556 634 76 556 634 76 gil291520705lemblCBK78998.1 l 322

Streptococcus mutans UA159 SEQ ID NO: 176 314 139 392 470 84 392 470 84 gil24379809lreflNP_721764.11 323

Streptococcus pyogenes Ml SEQ ID NO: 176 314 139 523 600 82 523 600 82

GAS 324

gill3622193lgblAAK33936.1 l

Streptococcus thermophilus SEQ ID NO: 176 314 139 481 558 81 481 558 81 LMD-9 325

gil 1 16628213 Iref 1 YP_820832.11

Fusobacteriumnucleatum SEQ ID NO: 171 308 138 537 614 76 537 614 76 ATCC49256 326

gil34762592lreflZP_00143587.1 l

Planococcus antarcticus DSM SEQ ID NO: 162 299 138 538 614 94 538 614 94

14505 327

gil389815359lreflZP_10206685.1

Treponema denticola ATCC SEQ ID NO: 169 305 137 524 600 81 524 600 81

35405 328

gil42525843lreflNP_970941.11

Solobacterium moorei F0204 SEQ ID NO: 179 314 136 544 619 77 544 619 77 gil320528778lreflZP_08029929.1 329

Staphylococcus SEQ ID NO: 164 299 136 531 606 92 531 606 92 pseudintermedius ED99 330

gil323463801 lgblADX75954.11

Flavobacterium branchiophilum SEQ ID NO: 162 286 125 538 613 63 538 613 63

FL-15 331

gil347536497lreflYP_004843922

.1

Ignavibacterium album JCM SEQ ID NO: 223 329 107 357 432 90 357 432 90

16511 332

gil38581 1609lreflYP_005848005 .1

Bergeyella zoohelcum ATCC SEQ ID NO: 165 261 97 529 604 56 529 604 56

43767 333

gi 1423317190lref IZP_ 17295095.1

Nitrobacter hamburgensis X14 SEQ ID NO: 169 253 85 536 611 48 536 611 48 gil92109262lreflYP_571550.1 l 334

Odoribacter laneus YIT 12061 SEQ ID NO: 164 242 79 535 610 63 535 610 63 gil374384763lreflZP_09642280.1 335

Legionella pneumophila str. SEQ ID NO: 164 239 76 402 476 67 402 476 67

Paris 336

gil54296138lreflYP_122507.1 l

Bacteroides sp. 20 3 SEQ ID NO: 198 269 72 530 604 83 530 604 83 gi 130131 1869 Iref IZP_07217791.1 337

Akkermansia muciniphila ATCC SEQ ID NO: 136 202 67 348 418 62 348 418 62

BAA-835 338

gill87736489lreflYP_001878601

Prevotella sp. C561 SEQ ID NO: 184 250 67 357 425 78 357 425 78 gil345885718lreflZP_08837074.1 339

Wolinella succinogenes DSM SEQ ID NO: 157 218 36 401 468 60 401 468 60

1740 340

gil34557932lreflNP_907747.11

Alicyclobacillus hesperidum SEQ ID NO: 142 196 55 416 482 61 416 482 61 URH17-3-68 341

gil403744858lreflZP_10953934.1

Caenispirillum salinarum AK4 SEQ ID NO: 161 214 54 330 393 68 330 393 68 gil427429481 lreflZP_l 891951 1.1 342

Eubacterium rectale ATCC SEQ ID NO: 133 185 53 322 384 60 322 384 60 33656 343

gil238924075lreflYP_002937591

.1

Mycoplasma synoviae 53 SEQ ID NO: 187 239 53 319 381 80 319 381 80 gil71894592lref 1 YP_278700.11 344

Porphyromonas sp. oral taxon SEQ ID NO: 150 202 53 309 371 60 309 371 60 279 str. F0450 345

gil402847315 Iref IZP_10895610.1

Streptococcus thermophilus SEQ ID NO: 127 178 139 424 486 81 424 486 81 LMD-9 346

gil 1 16627542lref 1 YP_820161.11

Roseburia inulinivorans DSM SEQ ID NO: 154 204 51 318 380 69 318 380 69

16841 347

gil225377804lreflZP_03755025.1

Methylosinus trichosporium SEQ ID NO: 144 193 50 426 488 64 426 488 64

OB3b 348

gil296446027lreflZP_06887976.1

Ruminococcus albus 8 SEQ ID NO: 139 187 49 351 412 55 351 412 55 gil325677756lreflZP_08157403.1 349

Bifidobacterium longum SEQ ID NO: 183 230 48 370 431 44 370 431 44 DJO10A 350

gill89440764lreflYP_001955845 Enterococcus faecalis TX0012 SEQ ID NO: 123 170 48 327 387 60 327 387 60 gil315149830lgblEFT93846.11 351

Mycoplasma mobile 163K SEQ ID NO: 179 226 48 314 374 79 314 374 79 gil47458868lreflYP_015730.1 l 352

Actinomyces coleocanis DSM SEQ ID NO: 147 193 47 358 418 40 358 418 40

15436 353

gil227494853lreflZP_03925169.1

Dinoroseobacter shibae DFL 12 SEQ ID NO: 138 184 47 338 398 48 338 398 48 gil 159042956lref 1 YP_001531750 354

.1

Actinomyces sp. oral taxon 180 SEQ ID NO: 183 228 46 349 409 40 349 409 40 str. F0310 355

gil315605738lreflZP_07880770.1

Alcanivorax sp. Wl 1-5 SEQ ID NO: 139 183 45 344 404 61 344 404 61 gil407803669lreflZP_l 1150502.1 356

Aminomonas paucivorans DSM SEQ ID NO: 134 178 45 341 401 63 341 401 63

12260 357

gi 1312879015 Iref IZP_07738815.1

Mycoplasma canis PG 14 SEQ ID NO: 139 183 45 319 379 76 319 379 76 gil384393286lgblEIE39736.11 358

Lactobacillus coryniformis SEQ ID NO: 141 184 44 328 387 61 328 387 61 KCTC 3535 359

gil336393381 lreflZP_08574780.1

Elusimicrobium minutum Peil91 SEQ ID NO: 177 219 43 322 381 47 322 381 47 gill87250660lreflYP_001875142 360

.1

Neisseria meningitidis Z2491 SEQ ID NO: 147 189 43 360 419 61 360 419 61 gil218767588lreflYP_002342100 361

.1

Pasteurella multocida str. Pm70 SEQ ID NO: 139 181 43 319 378 61 319 378 61 gill5602992lreflNP_246064.11 362

Rhodovulum sp. PH10 SEQ ID NO: 141 183 43 319 378 48 319 378 48 gil402849997lreflZP_10898214.1 363

Eubacterium dolichum DSM SEQ ID NO: 131 172 42 303 361 59 303 361 59

3991 364

gill60915782lreflZP_02077990.1

Nitratifractor salsuginis DSM SEQ ID NO: 143 184 42 347 404 61 347 404 61

16511 365

gil319957206lreflYP_004168469

.1

Rhodospirillum rubrum ATCC SEQ ID NO: 139 180 42 314 371 55 314 371 55

11170 366

gil83591793 Irefl YP_425545.11

Clostridium cellulolyticum H10 SEQ ID NO: 137 176 40 320 376 61 320 376 61 gil220930482lreflYP_002507391 367

.1

Helicobacter mustelae 12198 SEQ ID NO: 148 187 40 298 354 48 298 354 48 gi 1291276265 Iref 1 YP_003516037 368

.1

Ilyobacter polytropus DSM 2926 SEQ ID NO: 134 173 40 462 517 63 462 517 63 gil310780384lref 1 YP_003968716 369 .1

Sphaerochaeta globus str. Buddy SEQ ID NO: 163 202 40 335 389 45 335 389 45 gil325972003lreflYP_004248194 370

.1

Staphylococcus lugdunensis SEQ ID NO: 128 167 40 337 391 57 337 391 57 M23590 371

gil315659848lreflZP_07912707.1

Treponema sp. JC4 SEQ ID NO: 144 183 40 328 382 63 328 382 63 gil384109266lreflZP_10010146.1 372

uncultured delta proteobacterium SEQ ID NO: 154 193 40 313 365 55 313 365 55 HF0070 07E19 373

gi 1297182908 Igb 1 ADI 19058.11

Alicycliphilus denitrificans K601 SEQ ID NO: 140 178 39 317 366 48 317 366 48 gil330822845lreflYP_004386148 374

.1

Azospirillum sp. B510 SEQ ID NO: 205 243 39 342 389 46 342 389 46 gil288957741 lreflYP_003448082 375

.1

Bradyrhizobium sp. BTAil SEQ ID NO: 143 181 39 323 370 48 323 370 48 gill48255343lreflYP_001239928 376

.1

Parvibaculum lavamentivorans SEQ ID NO: 138 176 39 327 374 58 327 374 58

DS-1 377

gill54250555lreflYP_001411379

.1

Prevotella timonensis CRIS 5C- SEQ ID NO: 170 208 39 328 375 61 328 375 61

B l 378

gil282880052lreflZP_06288774.1

Bacillus smithii 7 3 47FAA SEQ ID NO: 134 171 38 401 448 63 401 448 63 gil365156657lreflZP_09352959.1 379

Cand. Puniceispirillum marinum SEQ ID NO: 135 172 38 344 391 53 344 391 53

IMCC1322 380

gil29408611 l lreflYP_003552871

.1

Barnesiella intestinihominis YIT SEQ ID NO: 140 176 37 371 417 60 371 417 60

11860 381

gi|404487228lreflZP_l 1022414.1

Ralstonia syzygii R24 SEQ ID NO: 140 176 37 395 440 50 395 440 50 gil344171927lemblCCA84553.1 l 382

Wolinella succinogenes DSM SEQ ID NO: 145 180 36 348 392 60 348 392 60

1740 383

gil34557790lreflNP_907605.11

Mycoplasma gallisepticum str. F SEQ ID NO: 144 177 34 373 416 71 373 416 71 gi 128493171 Olgb IADC31648.11 384

Acidothermus cellulolyticus 1 IB SEQ ID NO: 150 182 33 341 380 58 341 380 58 gil 117929158 Irefl YP_873709.11 385

Mycoplasma ovipneumoniae SEQ ID NO: 156 184 29 381 420 62 381 420 62

SCOl 386

gil363542550lreflZP_09312133.1 Table 12. Amino Acid Sequence of Cas9 Core Domains

Table 13. Identified PAM sequences and corresponding RKR motifs.

PI domains are provided in Tables 14 and 15.

Table 14. Altered PI Domains

Table 15. Other Altered PI Domains

Flavobacterium branchiophilum FL-15 1182 1473 292 KQK

Prevotella timonensis CRIS 5C-B1 957 1218 262 KQQ

Methylosinus trichosporium OB3b 830 1082 253 KRP

Prevotella sp. C561 1099 1424 326 KRY

Mycoplasma gallisepticum str. F 911 1269 359 KTA

Lactobacillus rhamnosus GG 1077 1363 287 KYG

Wolinella succinogenes DSM 1740 811 1059 249 LPN

Streptococcus thermophilus LMD-9 1099 1388 290 MLA

Treponema denticola ATCC 35405 1092 1395 304 NDS

Bergeyella zoohelcum ATCC 43767 1098 1415 318 NEK

Veillonella atypica ACS-134-V-Col7a 1107 1398 292 NGF

Neisseria meningitidis Z2491 835 1082 248 NHN

Ignavibacterium album JCM 16511 1296 1688 393 NKK

Ruminococcus albus 8 853 1156 304 NNF

Streptococcus thermophilus LMD-9 811 1121 311 N K

Barnesiella intestinihominis YIT 11860 871 1153 283 NPV

Azospirillum sp. B510 911 1168 258 PFH

Rhodospirillum rubrum ATCC 11170 863 1173 311 PRG

Planococcus antarcticus DSM 14505 1087 1333 247 PYY

Staphylococcus pseudintermedius ED99 1073 1334 262 QIV

Alcanivorax sp. Wl l-5 843 1113 271 RIE

Bradyrhizobium sp. BTAil 811 1064 254 RIY

Streptococcus pyogenes Ml GAS 1099 1368 270 RKR

Streptococcus mutans UA159 1078 1345 268 RKR

Streptococcus Pyogenes 1099 1368 270 RKR

Bacteroides sp. 20 3 1147 1517 371 RNI

S. aureus 772 1053 282 RNK

Solobacterium moorei F0204 1062 1327 266 RSG

Finegoldia magna ATCC 29328 1081 1348 268 RTE uncultured delta proteobacterium HF0070 07E19 770 1011 242 SGG

Acidaminococcus sp. D21 1064 1358 295 SIG

Eubacterium rectale ATCC 33656 824 1114 291 SKK

Caenispirillum salinarum AK4 1048 1442 395 SLV

Acidothermus cellulolyticus 11B 830 1138 309 SPS

Catenibacterium mitsuokai DSM 15897 1068 1329 262 SPT

Parvibaculum lavamentivorans DS-1 827 1037 211 TGN

Staphylococcus lugdunensis M23590 772 1054 283 TKK

Streptococcus sanguinis SK49 1123 1421 299 TRM

Elusimicrobium minutum Peil91 910 1195 286 TTG Nitrobacter hamburgensis X14 914 1166 253 VAY

Mycoplasma synoviae 53 991 1314 324 VGF

Sphaerochaeta globus str. Buddy 877 1179 303 VKG

Ilyobacter polytropus DSM 2926 837 1092 256 VNG

Rhodovulum sp. PH10 821 1059 239 VPY

Bifidobacterium longum DJOIOA 904 1187 284 VRK

Amino acid sequences described in Table 11 (in order of appearance):

SEQ ID NO: 304

MKRNYILGLDIGITSVGYGI IDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEV EEDT GNELSTKEQI SRNSKALEEKYVAELQLERLKKDGEVRGS INRFKTSDYVKEAKQLLKVQKAYHQ LDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYN ADLY NALNDLNNLVITRDENEKLEYYEKFQI IENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK PEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQIS NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDF ILSP VVKRSFIQS IKVINAI IKKYGLPNDI I IELAREKNSKDAQKMINEMQKRNRQTNERIEEI IRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVK QEENSKKGNRTPFQYLSSSDSKI SYETFKKHILNLAKGKGRI SKTKKEYLLEERDINRFSVQKD FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFTSFLRRKWKFKKERNKGYKHHAED ALI IA ADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKD YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLL MYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLD ITDD YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKI SNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKT QS IKKYSTDILGNLYEVKSKKHPQI IKKG

SEQ ID NO: 305

MDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF KSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL EKMD GTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL PKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFD SVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINR LSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLS MPQV IVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQI SEFSKRV ILADANLDKVLSAYNKHRDKPIREQAE I IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQS ITGLYETRIDLSQLGGD

SEQ ID NO: 306

MARILAFDIGI SS IGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKAR LNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLI SPYELRFRALNELLSKQDFARVILHIAKR RGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKK ESYE RCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDE KRAP KNSPLAFMFVALTRI INLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYE FKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQN QIDS LSKLEFKDHL I SFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVT NPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDA ELEC EKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKI SDLQDEKMLEIDHIYPYSRSFDDSYMNKVL VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRN LNDT RYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKD RNNH LHHAIDAVI IAYANNS IVKAFSDFKKEQESNSAELYAKKI SELDYKNKRKFFEPFSGFRQKVLD KIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNG DMFR VDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDS LILI QTKDMQEPEFVYY AFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKS IGIQNLKVF EKYIVSALGEVTKAEFRQREDFKK

SEQ ID NO: 307

MKRILGLDLGTNS IGWALVNEAENKDERSS IVKLGVRVNPLTVDELTNFEKGKS ITTNADRTLK RGMRRNLQRYKLRRETLTEVLKEHKLITEDTILSENGNRTTFETYRLRAKAVTEEI SLEEFARV LLMINKKRGYKSSRKAKGVEEGTLIDGMDIARELYNNNLTPGELCLQLLDAGKKFLPDFY RSDL QNELDRIWEKQKEYYPEILTDVLKEELRGKKRDAVWAICAKYFVWKENYTEWNKEKGKTE QQER EHKLEGIYSKRKRDEAKRENLQWRVNGLKEKLSLEQLVIVFQEMNTQINNSSGYLGAI SDRSKE LYFNKQTVGQYQMEMLDKNPNASLRNMVFYRQDYLDEFNMLWEKQAVYHKELTEELKKEI RDI I IFYQRRLKSQKGLIGFCEFESRQIEVDIDGKKKIKTVGNRVI SRSSPLFQEFKIWQILNNIEVT VVGKKRKRRKLKENYSALFEELNDAEQLELNGSRRLCQEEKELLAQELFIRDKMTKSEVL KLLF DNPQELDLNFKTIDGNKTGYALFQAYSKMIEMSGHEPVDFKKPVEKVVEYIKAVFDLLNW NTDI LGFNSNEELDNQPYYKLWHLLYSFEGDNTPTGNGRLIQKMTELYGFEKEYATILANVSFQ DDYG SLSAKAIHKILPHLKEGNRYDVACVYAGYRHSESSLTREEIANKVLKDRLMLLPKNSLHN PVVE KILNQMVNVINVI IDIYGKPDEIRVELARELKKNAKEREELTKS IAQTTKAHEEYKTLLQTEFG LTNVSRTDILRYKLYKELESCGYKTLYSNTYI SREKLFSKEFDIEHI IPQARLFDDSFSNKTLE ARSVNIEKGNKTAYDFVKEKFGESGADNSLEHYLNNIEDLFKSGKI SKTKYNKLKMAEQDIPDG FIERDLRNTQYIAKKALSMLNEI SHRVVATSGSVTDKLREDWQLIDVMKELNWEKYKALGLVEY FEDRDGRQIGRIKDWTKRNDHRHHAMDALTVAFTKDVFIQYFNNKNASLDPNANEHAIKN KYFQ NGRAIAPMPLREFRAEAKKHLENTLI S IKAKNKVITGNINKTRKKGGVNKNMQQTPRGQLHLET IYGSGKQYLTKEEKVNASFDMRKIGTVSKSAYRDALLKRLYENDNDPKKAFAGKNSLDKQ PIWL DKEQMRKVPEKVKIVTLEAIYTIRKEI SPDLKVDKVIDVGVRKILIDRLNEYGNDAKKAFSNLD KNPIWLNKEKGI S IKRVTI SGI SNAQSLHVKKDKDGKPILDENGRNIPVDFVNTGNNHHVAVYY RPVIDKRGQLVVDEAGNPKYELEEVVVSFFEAVTRANLGLPI IDKDYKTTEGWQFLFSMKQNEY FVFPNEKTGFNPKEIDLLDVENYGLISPNLFRVQKFSLKNYVFRHHLETTIKDTSSILRG ITWI DFRSSKGLDTIVKVRVNHIGQIVSVGEY

SEQ ID NO: 308

MSRKNYVDDYAI SLDIGNASVGWSAFTPNYRLVRAKGHELIGVRLFDPADTAESRRMARTTRRR YSRRRWRLRLLDALFDQALSEIDPSFLARRKYSWVHPDDENNADCWYGSVLFDSNEQDKR FYEK YPTIYHLRKALMEDDSQHDIREIYLAIHHMVKYRGNFLVEGTLESSNAFKEDELLKLLGR ITRY EMSEGEQNSDIEQDDENKLVAPANGQLADALCATRGSRSMRVDNALEALSAVNDLSREQR AIVK AIFAGLEGNKLDLAKIFVSKEFSSENKKILGIYFNKSDYEEKCVQIVDSGLLDDEEREFL DRMQ GQYNAIALKQLLGRSTSVSDSKCASYDAHRANWNLIKLQLRTKENEKDINENYGILVGWK IDSG QRKSVRGESAYENMRKKANVFFKKMIETSDLSETDKNRLIHDIEEDKLFPIQRDSDNGVI PHQL HQNELKQI IKKQGKYYPFLLDAFEKDGKQINKIEGLLTFRVPYFVGPLVVPEDLQKSDNSENHW MVRKKKGEITPWNFDEMVDKDASGRKFIERLVGTDSYLLGEPTLPKNSLLYQEYEVLNEL NNVR LSVRTGNHWNDKRRMRLGREEKTLLCQRLFMKGQTVTKRTAENLLRKEYGRTYELSGLSD ESKF TSSLSTYGKMCRIFGEKYVNEHRDLMEKIVELQTVFEDKETLLHQLRQLEGI SEADCALLVNTH YTGWGRLSRKLLTTKAGECKI SDDFAPRKHS I IEIMRAEDRNLMEI ITDKQLGFSDWIEQENLG AENGSSLMEVVDDLRVSPKVKRGI IQS IRLIDDI SKAVGKRPSRIFLELADDIQPSGRTI SRKS RLQDLYRNANLGKEFKGIADELNACSDKDLQDDRLFLYYTQLGKDMYTGEELDLDRLSSA YDID HI IPQAVTQNDS IDNRVLVARAENARKTDSFTYMPQIADRMRNFWQILLDNGLI SRVKFERLTR QNEFSEREKERFVQRSLVETRQIMKNVATLMRQRYGNSAAVIGLNAELTKEMHRYLGFSH KNRD INDYHHAQDALCVGIAGQFAANRGFFADGEVSDGAQNSYNQYLRDYLRGYREKLSAEDRK QGRA FGFIVGSMRSQDEQKRVNPRTGEVVWSEEDKDYLRKVMNYRKMLVTQKVGDDFGALYDET RYAA TDPKGIKGIPFDGAKQDTSLYGGFSSAKPAYAVLIESKGKTRLVNVTMQEYSLLGDRPSD DELR KVLAKKKSEYAKANILLRHVPKMQLIRYGGGLMVIKSAGELNNAQQLWLPYEEYCYFDDL SQGK GSLEKDDLKKLLDS ILGSVQCLYPWHRFTEEELADLHVAFDKLPEDEKKNVITGIVSALHADAK TANLS IVGMTGSWRRMNNKSGYTFSDEDEFIFQSPSGLFEKRVTVGELKRKAKKEVNSKYRTNE KRLPTLSGASQP

SEQ ID NO: 309

METQTSNQLITSHLKDYPKQDYFVGLDIGTNSVGWAVTNTSYELLKFHSHKMWGSRL FEEGESA VTRRGFRSMRRRLERRKLRLKLLEELFADAMAQVDSTFFIRLHESKYHYEDKTTGHSSKH ILFI DEDYTDQDYFTEYPTIYHLRKDLMENGTDDIRKLFLAVHHILKYRGNFLYEGATFNSNAF TFED VLKQALVNITFNCFDTNSAI SS I SNILMESGKTKSDKAKAIERLVDTYTVFDEVNTPDKPQKEQ VKEDKKTLKAFANLVLGLSANLIDLFGSVEDIDDDLKKLQIVGDTYDEKRDELAKVWGDE IHI I DDCKSVYDAI ILMS IKEPGLTI SQSKVKAFDKHKEDLVILKSLLKLDRNVYNEMFKSDKKGLHN YVHYIKQGRTEETSCSREDFYKYTKKIVEGLADSKDKEYILNEIELQTLLPLQRIKDNGV IPYQ LHLEELKVILDKCGPKFPFLHTVSDGFSVTEKLIKMLEFRIPYYVGPLNTHHNIDNGGFS WAVR KQAGRVTPWNFEEKIDREKSAAAFIKNLTNKCTYLFGEDVLPKSSLLYSEFMLLNELNNV RIDG KALAQGVKQHLIDSIFKQDHKKMTKNRIELFLKDNNYITKKHKPEITGLDGEIKNDLTSY RDMV RILGNNFDVSMAEDI ITDITIFGESKKMLRQTLRNKFGSQLNDETIKKLSKLRYRDWGRLSKKL LKGIDGCDKAGNGAPKTI IELMRNDSYNLMEILGDKFSFMECIEEENAKLAQGQVVNPHDI IDE LALSPAVKRAVWQALRIVDEVAHIKKALPSRIFVEVARTNKSEKKKKDSRQKRLSDLYSA IKKD DVLQSGLQDKEFGALKSGLANYDDAALRSKKLYLYYTQMGRCAYTGNI IDLNQLNTDNYDIDHI YPRSLTKDDSFDNLVLCERTANAKKSDIYPIDNRIQTKQKPFWAFLKHQGLI SERKYERLTRIA PLTADDLSGFIARQLVETNQSVKATTTLLRRLYPDIDVVFVKAENVSDFRHNNNFIKVRS LNHH HHAKDAYLNIVVGNVYHEKFTRNFRLFFKKNGANRTYNLAKMFNYDVICTNAQDGKAWDV KTSM NTVKKMMASNDVRVTRRLLEQSGALADATIYKASVAAKAKDGAYIGMKTKYSVFADVTKY GGMT KIKNAYS I IVQYTGKKGEEIKEIVPLPIYLINRNATDIELIDYVKSVIPKAKDI S IKYRKLCIN QLVKVNGFYYYLGGKTNDKIYIDNAIELVVPHDIATYIKLLDKYDLLRKENKTLKASS ITTS IY INTSTVVSLNKVGIDVFDYFMSKLRTPLYMKMKGNKVDELSSTGRSKFIKMTLEEQS IYLLEV LNLLTNSKTTFDVKPLGITGSRSTIGVKIHNLDEFKI INES ITGLYSNEVTIV

SEQ ID NO: 310

MTKLNQPYGIGLDIGSNS IGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLS RTRWRLSFLRDFFAPHITKIDPDFFLRQKYSEI SPKDKDRFKYEKRLFNDRTDAEFYEDYPSMY HLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAAKDFNTDKVDLEDIFPALTEAYA QVYP DLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFAKAI TGLK TKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGI VPAG MSLSQAKVADYGQHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVK ALTK EVTAHPNEVSEQLLNRMGQANFMLKQRTKANGAIPIQLQQRELDQI IANQSKYYDWLAAPNPVE AHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGNITPYNFDEKV DREA SANTFIQRMKTTDTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREV FERH SSVTIKQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDI ENII TWSTVFEDHTIFETKLAEIEWLDPKKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQE LMLS NHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSNKKALRQVLRVVEDIKHA ANGQ DPSWLFIETADGTGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDR LVLY FMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTL FAGK MKATWRKWHEAGLI SGRKLRNLMLRPDEIDKFAKGFVARQLVETRQI IKLTEQIAAAQYPNTKI IAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAARIGTYLLKRYPKLAPFFTYGEFAK VDVK KFREFNFIGALTHAKK I IAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFETADLFKQTI YAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKI SAQNASKI ASANLKSREKGKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMV NSDT YYRNYQELWLSRENQKLLKKLFS IKYEKTQMNHDALQVYKAI IDQVEKFFKLYDINQFRAKLSD AIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDLGLLQVGSGIKLD KDTQ IVYQSPSGLFKRRIPLADL SEQ ID NO: 311

MTKEYYLGLDVGTNSVGWAVTDSQYNLCKFKKKDMWGIRLFESANTAKDRRLQRGNRRRL ERKK QRIDLLQEIFSPEICKIDPTFFIRLNESRLHLEDKSNDFKYPLFIEKDYSDIEYYKEFPT IFHL RKHLIESEEKQDIRLIYLALHNI IKTRGHFLIDGDLQSAKQLRPILDTFLLSLQEEQNLSVSLS ENQKDEYEEILKNRS IAKSEKVKKLKNLFEI SDELEKEEKKAQSAVIENFCKFIVGNKGDVCKF LRVSKEELEIDSFSFSEGKYEDDIVKNLEEKVPEKVYLFEQMKAMYDWNILVDILETEEY I SFA KVKQYEKHKTNLRLLRDI ILKYCTKDEYNRMFNDEKEAGSYTAYVGKLKKNNKKYWIEKKRNPE EFYKSLGKLLDKIEPLKEDLEVLTMMIEECKNHTLLPIQKNKDNGVIPHQVHEVELKKIL ENAK KYYSFLTETDKDGYSVVQKIES IFRFRIPYYVGPLSTRHQEKGSNVWMVRKPGREDRIYPWNME EI IDFEKSNENFITRMTNKCTYLIGEDVLPKHSLLYSKYMVLNELNNVKVRGKKLPTSLKQK VF EDLFENKSKVTGKNLLEYLQIQDKDIQIDDLSGFDKDFKTSLKSYLDFKKQIFGEEIEKE S IQN MIEDI IKWITIYGNDKEMLKRVIRANYSNQLTEEQMKKITGFQYSGWGNFSKMFLKGI SGSDVS TGETFDI ITAMWETDNNLMQILSKKFTFMDNVEDFNSGKVGKIDKITYDSTVKEMFLSPENKRA VWQTIQVAEEIKKVMGCEPKKIFIEMARGGEKVKKRTKSRKAQLLELYAACEEDCRELIK EIED RDERDFNSMKLFLYYTQFGKCMYSGDDIDINELIRGNSKWDRDHIYPQSKIKDDS IDNLVLVNK TYNAKKSNELLSEDIQKKMHSFWLSLLNKKLITKSKYDRLTRKGDFTDEELSGFIARQLV ETRQ STKAIADIFKQIYSSEVVYVKSSLVSDFRKKPLNYLKSRRVNDYHHAKDAYLNIVVGNVY NKKF TSNPIQWMKKNRDTNYSLNKVFEHDVVINGEVIWEKCTYHEDTNTYDGGTLDRIRKIVER DNIL YTEYAYCEKGELFNATIQNKNGNSTVSLKKGLDVKKYGGYFSANTSYFSLIEFEDKKGDR ARHI IGVPIYIANMLEHSPSAFLEYCEQKGYQNVRILVEKIKKNSLLI INGYPLRIRGENEVDTSFKR AIQLKLDQKNYELVRNIEKFLEKYVEKKGNYPIDENRDHITHEKMNQLYEVLLSKMKKFN KKGM ADPSDRIEKSKPKFIKLEDLIDKINVINKMLNLLRCDNDTKADLSLIELPKNAGSFVVKK NTIG KSKI ILVNQSVTGLYENRREL

SEQ ID NO: 312

MARDYSVGLDIGTSSVGWAAIDNKYHLIRAKSKNLIGVRLFDSAVTAEKRRGYRTTRRRL SRRH WRLRLLNDIFAGPLTDFGDENFLARLKYSWVHPQDQSNQAHFAAGLLFDSKEQDKDFYRK YPTI YHLRLALMNDDQKHDLREVYLAIHHLVKYRGHFLIEGDVKADSAFDVHTFADAIQRYAES NNSD ENLLGKIDEKKLSAALTDKHGSKSQRAETAETAFDILDLQSKKQIQAILKSVVGNQANLM AIFG LDSSAI SKDEQKNYKFSFDDADIDEKIADSEALLSDTEFEFLCDLKAAFDGLTLKMLLGDDKTV SAAMVRRFNEHQKDWEYIKSHIRNAKNAGNGLYEKSKKFDGINAAYLALQSDNEDDRKKA KKIF QDEI SSADIPDDVKADFLKKIDDDQFLPIQRTKNNGTIPHQLHRNELEQI IEKQGIYYPFLKDT YQENSHELNKITALINFRVPYYVGPLVEEEQKIADDGKNIPDPTNHWMVRKSNDTITPWN LSQV VDLDKSGRRFIERLTGTDTYLIGEPTLPKNSLLYQKFDVLQELNNIRVSGRRLDIRAKQD AFEH LFKVQKTVSATNLKDFLVQAGYI SEDTQIEGLADVNGKNFN ALTTYNYLVSVLGREFVENPSN EELLEEITELQTVFEDKKVLRRQLDQLDGLSDHNREKLSRKHYTGWGRI SKKLLTTKIVQNADK IDNQTFDVPRMNQS I IDTLYNTKMNLMEI IN AEDDFGVRAWIDKQNTTDGDEQDVYSLIDELA GPKEIKRGIVQSFRILDDITKAVGYAPKRVYLEFARKTQESHLTNSRKNQLSTLLKNAGL SELV TQVSQYDAAALQNDRLYLYFLQQGKDMYSGEKLNLDNLSNYDIDHI IPQAYTKDNSLDNRVLVS NITNRRKSDSSNYLPALIDKMRPFWSVLSKQGLLSKHKFANLTRTRDFDDMEKERFIARS LVET RQIIKNVASLIDSHFGGETKAVAIRSSLTADMRRYVDIPKNRDINDYHHAFDALLFSTVG QYTE NSGLMKKGQLSDSAGNQYNRYIKEWIHAARLNAQSQRVNPFGFVVGSMRNAAPGKLNPET GEIT PEENADWS IADLDYLHKVMNFRKITVTRRLKDQKGQLYDESRYPSVLHDAKSKAS INFDKHKPV DLYGGFSSAKPAYAALIKFKNKFRLVNVLRQWTYSDKNSEDYILEQIRGKYPKAEMVLSH IPYG QLVKKDGALVTI SSATELHNFEQLWLPLADYKLINTLLKTKEDNLVDILHNRLDLPEMTIESAF YKAFDS ILSFAFNRYALHQNALVKLQAHRDDFNALNYEDKQQTLERILDALHASPASSDLKKIN LSSGFGRLFSPSHFTLADTDEFIFQSVTGLFSTQKTVAQLYQETK

SEQ ID NO: 313

MVYDVGLDIGTGSVGWVALDENGKLARAKGKNLVGVRLFDTAQTAADRRGFRTTRRRLSR RKWR LRLLDELFSAEINEIDSSFFQRLKYSYVHPKDEENKAHYYGGYLFPTEEETKKFHRSYPT IYHL RQELMAQPNKRFDIREIYLAIHHLVKYRGHFLSSQEKITIGSTYNPEDLANAIEVYADEK GLSW ELNNPEQLTEI I SGEAGYGLNKSMKADEALKLFEFDNNQDKVAIKTLLAGLTGNQIDFAKLFGK DISDKDEAKLWKLKLDDEALEEKSQTILSQLTDEEIELFHAVVQAYDGFVLIGLLNGADS VSAA MVQLYDQHREDRKLLKSLAQKAGLKHKRFSEIYEQLALATDEATIKNGI STARELVEESNLSKE VKEDTLRRLDENEFLPKQRTKANSVIPHQLHLAELQKILQNQGQYYPFLLDTFEKEDGQD NKIE ELLRFRIPYYVGPLVTKKDVEHAGGDADNHWVERNEGFEKSRVTPWNFDKVFNRDKAARD FIER LTGNDTYLIGEKTLPQNSLRYQLFTVLNELNNVRVNGKKFDSKTKADLINDLFKARKTVS LSAL KDYLKAQGKGDVTITGLADESKFNSSLSSYNDLKKTFDAEYLENEDNQETLEKI IEIQTVFEDS KIASRELSKLPLDDDQVKKLSQTHYTGWGRLSEKLLDSKI IDERGQKVS ILDKLKSTSQNFMS I INNDKYGVQAWITEQNTGSSKLTFDEKVNELTTSPANKRGIKQSFAVLNDIKKAMKEEPR RVYL EFAREDQTSVRSVPRYNQLKEKYQSKSLSEEAKVLKKTLDGNKNKMSDDRYFLYFQQQGK DMYT GRPINFERLSQDYDIDHI IPQAFTKDDSLDNRVLVSRPENARKSDSFAYTDEVQKQDGSLWTSL LKSGFINRKKYERLTKAGKYLDGQKTGFIARQLVETRQI IKNVASLIEGEYENSKAVAIRSEIT ADMRLLVGIKKHREINSFHHAFDALLITAAGQYMQNRYPDRDSTNVYNEFDRYTNDYLKN LRQL SSRDEVRRLKSFGFVVGTMRKGNEDWSEENTSYLRKVMMFKNILTTKKTEKDRGPLNKET IFSP KSGKKLIPLNSKRSDTALYGGYSNVYSAYMTLVRANGKNLLIKIPI S IANQIEVGNLKINDYIV NNPAIKKFEKILI SKLPLGQLVNEDGNLIYLASNEYRHNAKQLWLSTTDADKIAS I SENSSDEE LLEAYDILTSENVKNRFPFFKKDIDKLSQVRDEFLDSDKRIAVIQTILRGLQIDAAYQAP VKI I SKKVSDWHKLQQSGGIKLSDNSEMIYQSATGIFETRVKI SDLL

SEQ ID NO: 314

IVDYCIGLDLGTGSVGWAVVDMNHRLMKRNGKHLWGSRLFSNAETAANRRASRS IRRRYNKRRE RIRLLRAILQDMVLEKDPTFFIRLEHTSFLDEEDKAKYLGTDYKDNYNLFIDEDFNDYTY YHKY PTIYHLRKALCESTEKADPRLIYLALHHIVKYRGNFLYEGQKFNMDASNIEDKLSDIFTQ FTSF NNIPYEDDEKKNLEILEILKKPLSKKAKVDEVMTLIAPEKDYKSAFKELVTGIAGNKMNV TKMI LCEPIKQGDSEIKLKFSDSNYDDQFSEVEKDLGEYVEFVDALHNVYSWVELQTIMGATHT DNAS I SEAMVSRYNKHHDDLKLLKDCIKNNVPNKYFDMFRNDSEKSKGYYNYINRPSKAPVDEFY KYV KKCIEKVDTPEAKQILNDIELENFLLKQNSRTNGSVPYQMQLDEMIKI IDNQAEYYPILKEKRE QLLS ILTFRIPYYFGPLNETSEHAWIKRLEGKENQRILPWNYQDIVDVDATAEGFIKRMRSYCT YFPDEEVLPKNSLIVSKYEVYNELNKIRVDDKLLEVDVKNDIYNELFMKNKTVTEKKLKN WLVN NQCCSKDAEIKGFQKENQFSTSLTPWIDFTNIFGKIDQSNFDLIENI IYDLTVFEDKKIMKRRL KKKYALPDDKVKQILKLKYKDWSRLSKKLLDGIVADNRFGSSVTVLDVLEMSRLNLMEI INDKD LGYAQMIEEATSCPEDGKFTYEEVERLAGSPALKRGIWQSLQIVEEITKVMKCRPKYIYI EFER SEEAKERTESKIKKLENVYKDLDEQTKKEYKSVLEELKGFDNTKKI SSDSLFLYFTQLGKCMYS GKKLDIDSLDKYQIDHIVPQSLVKDDSFDNRVLVVPSENQRKLDDLVVPFDIRDKMYRFW KLLF DHELI SPKKFYSLIKTEYTERDEERFINRQLVETRQITKNVTQI IEDHYSTTKVAAIRANLSHE FRVKNHIYKNRDINDYHHAHDAYIVALIGGFMRDRYPNMHDSKAVYSEYMKMFRKNKNDQ KRWK DGFVINSMNYPYEVDGKLIWNPDLINEIKKCFYYKDCYCTTKLDQKSGQLFNLTVLSNDA HADK GVTKAVVPVNKNRSDVHKYGGFSGLQYTIVAIEGQKKKGKKTELVKKI SGVPLHLKAAS INEKI NYIEEKEGLSDVRI IKDNIPVNQMIEMDGGEYLLTSPTEYVNARQLVLNEKQCALIADIYNAIY KQDYDNLDDILMIQLYIELTNKMKVLYPAYRGIAEKFESMNENYVVI SKEEKANI IKQMLIVMH RGPQNGNIVYDDFKI SDRIGRLKTKNHNLNNIVFI SQSPTGIYTKKYKL SEQ ID NO: 315

MKSEKKYYIGLDVGTNSVGWAVTDEFYNILRAKGKDLWGVRLFEKADTAANTRIFRSGRR RNDR KGMRLQILREIFEDEIKKVDKDFYDRLDESKFWAEDKKVSGKYSLFNDKNFSDKQYFEKF PTIF HLRKYLMEEHGKVDIRYYFLAINQMMKRRGHFLIDGQI SHVTDDKPLKEQLILLINDLLKIELE EELMDS IFEILADVNEKRTDKKNNLKELIKGQDFNKQEGNILNS IFES IVTGKAKIKNI I SDED ILEKIKEDNKEDFVLTGDSYEENLQYFEEVLQENITLFNTLKSTYDFLILQS ILKGKSTLSDAQ VERYDEHKKDLEILKKVIKKYDEDGKLFKQVFKEDNGNGYVSYIGYYLNKNKKITAKKKI SNIE FTKYVKGILEKQCDCEDEDVKYLLGKIEQENFLLKQI SS INSVIPHQIHLFELDKILENLAKNY PSFNNKKEEFTKIEKIRKTFTFRIPYYVGPLNDYHKNNGGNAWIFRNKGEKIRPWNFEKI VDLH KSEEEFIKRMLNQCTYLPEETVLPKSS ILYSEYMVLNELNNLRINGKPLDTDVKLKLIEELFKK KTKVTLKS IRDYMVRNNFADKEDFDNSEKNLEIASNMKSYIDFNNILEDKFDVEMVEDLIEKIT IHTGNKKLLKKYIEETYPDLSSSQIQKI INLKYKDWGRLSRKLLDGIKGTKKETEKTDTVINFL RNSSDNLMQI IGSQNYSFNEYIDKLRKKYIPQEI SYEVVENLYVSPSVKKMIWQVIRVTEEITK VMGYDPDKIFIEMAKSEEEKKTTI SRKNKLLDLYKAIKKDERDSQYEKLLTGLNKLDDSDLRSR KLYLYYTQMGRDMYTGEKIDLDKLFDSTHYDKDHI IPQSMKKDDS I INNLVLVNKNANQTTKGN IYPVPSS IRNNPKIYNYWKYLMEKEFI SKEKYNRLIRNTPLTNEELGGFINRQLVETRQSTKAI KELFEKFYQKSKI IPVKASLASDLRKDMNTLKSREVNDLHHAHDAFLNIVAGDVWNREFTSNPI NYVKENREGDKVKYSLSKDFTRPRKSKGKVIWTPEKGRKLIVDTLNKPSVLI SNESHVKKGELF NATIAGKKDYKKGKIYLPLKKDDRLQDVSKYGGYKAINGAFFFLVEHTKSKKRIRS IELFPLHL LSKFYEDKNTVLDYAINVLQLQDPKI I IDKINYRTEI I IDNFSYLISTKSNDGSITVKPNEQMY WRVDEI SNLKKIENKYKKDAILTEEDRKIMESYIDKIYQQFKAGKYKNRRTTDTI IEKYEI IDL DTLDNKQLYQLLVAFI SLSYKTSNNAVDFTVIGLGTECGKPRITNLPDNTYLVYKS ITGIYEKR IRIK

SEQ ID NO: 316

MKLRGIEDDYS IGLDMGTSSVGWAVTDERGTLAHFKRKPTWGSRLFREAQTAAVARMPRGQRRR YVRRRWRLDLLQKLFEQQMEQADPDFFIRLRQSRLLRDDRAEEHADYRWPLFNDCKFTER DYYQ RFPTIYHVRSWLMETDEQADIRLIYLALHNIVKHRGNFLREGQSLSAKSARPDEALNHLR ETLR VWSSERGFECS IADNGS ILAMLTHPDLSPSDRRKKIAPLFDVKSDDAAADKKLGIALAGAVIGL KTEFKNIFGDFPCEDSS IYLSNDEAVDAVRSACPDDCAELFDRLCEVYSAYVLQGLLSYAPGQT I SANMVEKYRRYGEDLALLKKLVKIYAPDQYRMFFSGATYPGTGIYDAAQARGYTKYNLGP KKS EYKPSESMQYDDFRKAVEKLFAKTDARADERYRMMMDRFDKQQFLRRLKTSDNGS IYHQLHLEE LKAIVENQGRFYPFLKRDADKLVSLVSFRIPYYVGPLSTRNARTDQHGENRFAWSERKPG MQDE PIFPWNWES I IDRSKSAEKFILRMTGMCTYLQQEPVLPKSSLLYEEFCVLNELNGAHWS IDGDD EHRFDAADREGI IEELFRRKRTVSYGDVAGWMERERNQIGAHVCGGQGEKGFESKLGSYIFFCK DVFKVERLEQSDYPMIERI ILWNTLFEDRKILSQRLKEEYGSRLSAEQIKTICKKRFTGWGRLS EKFLTGITVQVDEDSVS IMDVLREGCPVSGKRGRAMVMMEILRDEELGFQKKVDDFNRAFFAEN AQALGVNELPGSPAVRRSLNQS IRIVDEIAS IAGKAPANIFIEVTRDEDPKKKGRRTKRRYNDL KDALEAFKKEDPELWRELCETAPNDMDERLSLYFMQRGKCLYSGRAIDIHQLSNAGIYEV DHI I PRTYVKDDSLENKALVYREENQRKTDMLLIDPEIRRRMSGYWRMLHEAKLIGDKKFRNLL RSRI DDKALKGFIARQLVETGQMVKLVRSLLEARYPETNI I SVKAS I SHDLRTAAELVKCREANDFHH AHDAFLACRVGLFIQKRHPCVYENPIGLSQVVRNYVRQQADIFKRCRTIPGSSGFIVNSF MTSG FDKETGEIFKDDWDAEAEVEGIRRSLNFRQCFI SRMPFEDHGVFWDATIYSPRAKKTAALPLKQ GLNPSRYGSFSREQFAYFFIYKARNPRKEQTLFEFAQVPVRLSAQIRQDENALERYAREL AKDQ GLEFIRIERSKILKNQLIEIDGDRLCITGKEEVRNACELAFAQDEMRVIRMLVSEKPVSR ECVI SLFNRILLHGDQASRRLSKQLKLALLSEAFSEASDNVQRNVVLGLIAIFNGSTNMVNLSD IGGS KFAGNVRIKYKKELASPKVNVHLIDQSVTGMFERRTKIGL

SEQ ID NO: 317

MENKQYYIGLDVGTNSVGWAVTDTSYNLLRAKGKDMWGARLFEKANTAAERRTKRTSRRR SERE KARKAMLKELFADEINRVDPSFFIRLEESKFFLDDRSENNRQRYTLFNDATFTDKDYYEK YKTI FHLRSALINSDEKFDVRLVFLAILNLFSHRGHFLNASLKGDGDIQGMDVFYNDLVESCEY FEIE LPRITNIDNFEKILSQKGKSRTKILEELSEELS I SKKDKSKYNLIKLI SGLEASVVELYNIEDI QDENKKIKIGFRESDYEESSLKVKEI IGDEYFDLVERAKSVHDMGLLSNI IGNSKYLCEARVEA YENHHKDLLKIKELLKKYDKKAYNDMFRKMTDKNYSAYVGSVNSNIAKERRSVDKRKIED LYKY IEDTALKNIPDDNKDKIEILEKIKLGEFLKKQLTASNGVIPNQLQSRELRAILKKAENYL PFLK EKGEKNLTVSEMI IQLFEFQIPYYVGPLDKNPKKDNKANSWAKIKQGGRILPWNFEDKVDVKGS RKEFIEKMVRKCTYI SDEHTLPKQSLLYEKFMVLNEINNIKIDGEKI SVEAKQKIYNDLFVKGK KVSQKDIKKELI SLNIMDKDSVLSGTDTVCNAYLSS IGKFTGVFKEEINKQS IVDMIEDI IFLK TVYGDEKRFVKEEIVEKYGDEIDKDKIKRILGFKFSNWGNLSKSFLELEGADVGTGEVRS HQS LWETNFNLMELLSSRFTYMDELEKRVKKLEKPLSEWTIEDLDDMYLSSPVKRMIWQSMKI VDEI QTVIGYAPKRIFVEMTRSEGEKVRTKSRKDRLKELYNGIKEDSKQWVKELDSKDESYFRS KKMY LYYLQKGRCMYSGEVIELDKLMDDNLYDIDHIYPRSFVKDDSLDNLVLVKKEINNRKQND PITP QIQASCQGFWKILHDQGFMSNEKYSRLTRKTQEFSDEEKLSFINRQIVETGQATKCMAQI LQKS MGEDVDVVFSKARLVSEFRHKFELFKSRLINDFHHANDAYLNIVVGNSYFVKFTRNPANF IKDA RKNPDNPVYKYHMDRFFERDVKSKSEVAWIGQSEGNSGTIVIVKKTMAKNSPLITKKVEE GHGS ITKETIVGVKEIKFGRNKVEKADKTPKKPNLQAYRPIKTSDERLC ILRYGGRTS I S I SGYCLV EYVKKRKTIRSLEAIPVYLGRKDSLSEEKLLNYFRYNLNDGGKDSVSDIRLCLPFI STNSLVKI DGYLYYLGGKNDDRIQLYNAYQLKMKKEEVEYIRKIEKAVSMSKFDEIDREKNPVLTEEK NIEL YNKIQDKFENTVFSKRMSLVKYNKKDLSFGDFLKNKKSKFEEIDLEKQCKVLY I IFNLSNLKE VDLSDIGGSKSTGKCRCKKNITNYKEFKLIQQS ITGLYSCEKDLMTI

SEQ ID NO: 318

MKNLKEYYIGLDIGTASVGWAVTDESYNIPKFNGKKMWGVRLFDDAKTAEERRTQRGSRR RLNR RKERINLLQDLFATEI SKVDPNFFLRLDNSDLYREDKDEKLKSKYTLFNDKDFKDRDYHKKYPT IHHLIMDLIEDEGKKDIRLLYLACHYLLKNRGHFIFEGQKFDTKNSFDKS INDLKIHLRDEY I DLEFNNEDLIEI ITDTTLNKTNKKKELKNIVGDTKFLKAI SAIMIGSSQKLVDLFEDGEFEETT VKSVDFSTTAFDDKYSEYEEALGDTI SLLNILKS IYDSS ILENLLKDADKSKDGNKYI SKAFVK KFNKHGKDLKTLKRI IKKYLPSEYA IFRNKS INDNYVAYTKS ITSNKRTKASKFTKQEDFYK FIKKHLDTIKETKLNSSENEDLKLIDEMLTDIEFKTFIPKLKSSDNGVIPYQLKLMELKK ILDN QSKYYDFLNESDEYGTVKDKVES IMEFRIPYYVGPLNPDSKYAWIKRENTKITPWNFKDIVDLD SSREEFIDRLIGRCTYLKEEKVLPKASLIYNEFMVLNELNNLKLNEFLITEEMKKAIFEE LFKT KKKVTLKAVSNLLKKEFNLTGDILLSGTDGDFKQGLNSYIDFKNI IGDKVDRDDYRIKIEEI IK LIVLYEDDKTYLKKKIKSAYKNDFTDDEIKKIAALNYKDWGRLSKRFLTGIEGVDKTTGE KGS I IYFMREYNLNLMELMSGHYTFTEEVEKLNPVENRELCYEMVDELYLSPSVKRMLWQSLRV VDEI KRIIGKDPKKIFIEMARAKEAKNSRKESRKNKLLEFYKFGKKAFINEIGEERYNYLLNEI NSEE ESKFRWDNLYLYYTQLGRCMYSLEPIDLADLKSN IYDQDHIYPKSKIYDDSLENRVLVKKNLN HEKGNQYPIPEKVLNKNAYGFWKILFDKGLIGQKKYTRLTRRTPFEERELAEFIERQIVE TRQA TKETANLLK ICQDSEIVYSKAENASRFRQEFDI IKCRTVNDLHHMHDAYL IVVGNVYNTKFT KNPLNFIKDKDNVRSYNLENMFKYDVVRGSYTAWIADDSEGNVKAATIKKVKRELEGKNY RFTR MSYIGTGGLYDQNLMRKGKGQIPQKENTNKSNIEKYGGYNKASSAYFALIESDGKAGRER TLET IPIMVYNQEKYGNTEAVDKYLKDNLELQDPKILKDKIKINSLIKLDGFLYNIKGKTGDSL SIAG SVQLIVNKEEQKLIKKMDKFLVKKKDNKDIKVTSFD IKEEELIKLYKTLSDKLNNGIYSNKRN NQAKNI SEALDKFKEI S IEEKIDVLNQI ILLFQSYNNGCNLKS IGLSAKTGVVFIPKKLNYKEC KLINQSITGLFENEVDLLNL

SEQ ID NO: 319

MGKMYYLGLDIGTNSVGYAVTDPSYHLLKFKGEPMWGAHVFAAGNQSAERRSFRTSRRRL DRRQ QRVKLVQEIFAPVI SPIDPRFFIRLHESALWRDDVAETDKHIFFNDPTYTDKEYYSDYPTIHHL IVDLMESSEKHDPRLVYLAVAWLVAHRGHFLNEVDKDNIGDVLSFDAFYPEFLAFLSDNG VSPW VCESKALQATLLSRNSVNDKYKALKSLIFGSQKPEDNFDANI SEDGLIQLLAGKKVKVNKLFPQ ESNDASFTLNDKEDAIEEILGTLTPDECEWIAHIRRLFDWAIMKHALKDGRTI SESKVKLYEQH HHDLTQLKYFVKTYLAKEYDDIFRNVDSETTKNYVAYSYHVKEVKGTLPKNKATQEEFCK YVLG KVKNIECSEADKVDFDEMIQRLTDNSFMPKQVSGENRVIPYQLYYYELKTILNKAASYLP FLTQ CGKDAI SNQDKLLS IMTFRIPYFVGPLRKDNSEHAWLERKAGKIYPWNFNDKVDLDKSEEAFIR RMTNTCTYYPGEDVLPLDSLIYEKFMILNEINNIRIDGYPI SVDVKQQVFGLFEKKRRVTVKDI QNLLLSLGALDKHGKLTGIDTTIHSNYNTYHHFKSLMERGVLTRDDVERIVERMTYSDDT KRVR LWLNNNYGTLTADDVKHI SRLRKHDFGRLSKMFLTGLKGVHKETGERAS ILDFMWNTNDNLMQL LSECYTFSDEITKLQEAYYAKAQLSLNDFLDSMYI SNAVKRPIYRTLAVVNDIRKACGTAPKRI FIEMARDGESKKKRSVTRREQIKNLYRS IRKDFQQEVDFLEKILENKSDGQLQSDALYLYFAQL GRDMYTGDPIKLEHIKDQSFYNIDHIYPQSMVKDDSLDNKVLVQSEINGEKSSRYPLDAA IRNK MKPLWDAYYNHGLI SLKKYQRLTRSTPFTDDEKWDFINRQLVETRQSTKALAILLKRKFPDTEI VYSKAGLSSDFRHEFGLVKSRNINDLHHAKDAFLAIVTGNVYHERFNRRWFMVNQPYSVK TKTL FTHS IKNGNFVAWNGEEDLGRIVKMLKQNKNTIHFTRFSFDRKEGLFDIQPLKASTGLVPRKAG LDVVKYGGYDKSTAAYYLLVRFTLEDKKTQHKLMMIPVEGLYKARIDHDKEFLTDYAQTT I SEI LQKDKQKVI IMFPMGTRHIKLNSMI S IDGFYLS IGGKSSKGKSVLCHAMVPLIVPHKIECYIK AMESFARKFKENNKLRIVEKFDKITVEDNLNLYELFLQKLQHNPYNKFFSTQFDVLTNGR STFT KLSPEEQVQTLLNILS IFKTCRSSGCDLKS INGSAQAARIMI SADLTGLSKKYSDIRLVEQSAS GLFVSKSQNLLEYL

SEQ ID NO: 320

MTKKEQPYNIGLDIGTSSVGWAVTNDNYDLLNIKKKNLWGVRLFEEAQTAKETRLNR STRRRYR RRKNRINWLNEIFSEELAKTDPSFLIRLQNSWVSKKDPDRKRDKYNLFIDGPYTDKEYYR EFPT IFHLRKELILNKDKADIRLIYLALH ILKYRGNFTYEHQKF I SNLNNNLSKELIELNQQLIKY DI SFPDDCDWNHI SDILIGRGNATQKSSNILKDFTLDKETKKLLKEVINLILGNVAHLNTIFKT SLTKDEEKLNFSGKDIESKLDDLDS ILDDDQFTVLDAANRIYSTITLNEILNGESYFSMAKVNQ YENHAIDLCKLRDMWHTTKNEEAVEQSRQAYDDYINKPKYGTKELYTSLKKFLKVALPTN LAKE AEEKI SKGTYLVKPRNSENGVVPYQLNKIEMEKI IDNQSQYYPFLKENKEKLLS ILSFRIPYYV GPLQSAEKNPFAWMERKSNGHARPWNFDEIVDREKSSNKFIRRMTVTDSYLVGEPVLPKN SLIY QRYEVLNELN IRITENLKTNPIGSRLTVETKQRIYNELFKKYKKVTVKKLTKWLIAQGYYKNP ILIGLSQKDEFNSTLTTYLDMKKIFGSSFMEDNKNYDQIEELIEWLTIFEDKQILNEKLH SSKY SYTPDQIKKI SNMRYKGWGRLSKKILMDITTETNTPQLLQLSNYS ILDLMWATNNNFI S IMSND KYDFKNYIENHNLNKNEDQNI SDLVNDIHVSPALKRGITQS IKIVQEIVKFMGHAPKHIFIEVT RETKKSEITTSREKRIKRLQSKLLNKANDFKPQLREYLVPNKKIQEELKKHKNDLSSERI MLYF LQNGKSLYSEESLNINKLSDYQVDHILPRTYIPDDSLENKALVLAKENQRKADDLLLNSN VIDR NLERWTYMLNNNMIGLKKFKNLTRRVITDKDKLGFIHRQLVQTSQMVKGVANILDNMYKN QGTT CIQARANLSTAFRKALSGQDDTYHFKHPELVKNRNVNDFHHAQDAYLASFLGTYRLRRFP TNEM LLMNGEYNKFYGQVKELYSKKKKLPDSRKNGFI I SPLVNGTTQYDRNTGEI IWNVGFRDKILKI FNYHQCNVTRKTEIKTGQFYDQTIYSPKNPKYKKLIAQKKDMDPNIYGGFSGDNKSSITI VKID NNKIKPVAIPIRLINDLKDKKTLQNWLEENVKHKKS IQI IKNNVPIGQI IYSKKVGLLSLNSDR EVANRQQLILPPEHSALLRLLQIPDEDLDQILAFYDKNILVEILQELITKMKKFYPFYKG EREF LIANIENFNQATTSEKVNSLEELITLLHANSTSAHLIFNNIEKKAFGRKTHGLTLNNTDF IYQS VTGLYETRIHIE

SEQ ID NO: 321

MTKFNKNYS IGLDIGVSSVGYAVVTEDYRVPAFKFKVLGNTEKEKIKKNLIGSTTFVSAQPAKG TRVFRVNRRRIDRRNHRITYLRDIFQKEIEKVDKNFYRRLDESFRVLGDKSEDLQIKQPF FGDK ELETAYHKKYPTIYHLRKHLADADKNSPVADIREVYMAI SHILKYRGHFLTLDKINPNNINMQN SWIDFIESCQEVFDLEI SDESKNIADIFKSSENRQEKVKKILPYFQQELLKKDKS IFKQLLQLL FGLKTKFKDCFELEEEPDLNFSKENYDENLENFLGSLEEDFSDVFAKLKVLRDTILLSGM LTYT GATHARFSATMVERYEEHRKDLQRFKFFIKQNLSEQDYLDIFGRKTQNGFDVDKETKGYV GYIT NKMVLTNPQKQKTIQQNFYDYI SGKITGIEGAEYFLNKI SDGTFLRKLRTSDNGAIPNQIHAYE LEKI IERQGKDYPFLLENKDKLLS ILTFKIPYYVGPLAKGSNSRFAWIKRATSSDILDDNDEDT RNGKIRPWNYQKLINMDETRDAFITNLIGNDI ILLNEKVLPKRSLIYEEVMLQNELTRVKYKDK YGKAHFFDSELRQNI INGLFKNNSKRVNAKSLIKYLSDNHKDLNAIEIVSGVEKGKSFNSTLKT YNDLKTIFSEELLDSEIYQKELEEI IKVITVFDDKKSIKNYLTKFFGHLEILDEEKINQLSKLR YSGWGRYSAKLLLDIRDEDTGFNLLQFLRNDEENRNLTKLI SDNTLSFEPKIKDIQSKSTIEDD IFDEIKKLAGSPAIKRGILNS IKIVDELVQI IGYPPHNIVIEMARENMTTEEGQKKAKTRKTKL ESALKNIENSLLENGKVPHSDEQLQSEKLYLYYLQNGKDMYTLDKTGSPAPLYLDQLDQY EVDH I IPYSFLPIDS IDNKVLTHRENNQQKLNNIPDKETVANMKPFWEKLYNAKLI SQTKYQRLTTSE RTPDGVLTESMKAGFIERQLVETRQI IKHVARILDNRFSDTKI ITLKSQLITNFRNTFHIAKIR ELNDYHHAHDAYLAVVVGQTLLKVYPKLAPELIYGHHAHFNRHEENKATLRKHLYS IMRFFNN PDSKVSKDIWDCNRDLPI IKDVIYNSQINFVKRTMIKKGAFYNQNPVGKFNKQLAANNRYPLKT KALCLDTS IYGGYGPMNSALS I I I IAERFNEKKGKIETVKEFHDIFI IDYEKFNNNPFQFLNDT SENGFLKKNNINRVLGFYRIPKYSLMQKIDGTRMLFESKSNLHKATQFKLTKTQNELFFH MKRL LTKSNLMDLKSKSAIKESQNFILKHKEEFD I SNQLSAFSQKMLGNTTSLKNLIKGYNERKIKE IDIRDETIKYFYDNFIKMFSFVKSGAPKDINDFFDNKCTVARMRPKPDKKLLNATLIHQS ITGL YETRIDLSKLGED

SEQ ID NO: 322

MKQEYFLGLDMGTGSLGWAVTDSTYQVMRKHGKALWGTRLFESASTAEERRMFRTARRRL DRRN WRIQVLQEIFSEEI SKVDPGFFLRMKESKYYPEDKRDAEGNCPELPYALFVDDNYTDKNYHKDY PTIYHLRKMLMETTEIPDIRLVYLVLHHMMKHRGHFLLSGDI SQIKEFKSTFEQLIQNIQDEEL EWHI SLDDAAIQFVEHVLKDRNLTRSTKKSRLIKQLNAKSACEKAILNLLSGGTVKLSDIFNNK ELDESERPKVSFADSGYDDYIGIVEAELAEQYYI IASAKAVYDWSVLVEILGNSVS I SEAKIKV YQKHQADLKTLKKIVRQYMTKEDYKRVFVDTEEKLNNYSAYIGMTKKNGKKVDLKSKQCT QADF YDFLKKNVIKVIDHKEITQEIESEIEKENFLPKQVTKDNGVIPYQVHDYELKKILDNLGT RMPF IKENAEKIQQLFEFRIPYYVGPLNRVDDGKDGKFTWSVRKSDARIYPWNFTEVIDVEASA EKFI RRMTNKCTYLVGEDVLPKDSLVYSKFMVLNELNNLRLNGEKI SVELKQRIYEELFCKYRKVTRK KLERYLVIEGIAKKGVEITGIDGDFKASLTAYHDFKERLTDVQLSQRAKEAIVLNVVLFG DDKK LLKQRLSKMYPNLTTGQLKGICSLSYQGWGRLSKTFLEEITVPAPGTGEVWNIMTALWQT NDNL MQLLSRNYGFTNEVEEFNTLKKETDLSYKTVDELYVSPAVKRQIWQTLKVVKEIQKVMGN APKR VFVEMAREKQEGKRSDSRKKQLVELYRACKNEERDWITELNAQSDQQLRSDKLFLYYIQK GRCM YSGETIQLDELWDNTKYDIDHIYPQSKTMDDSLNNRVLVKKNYNAIKSDTYPLSLDIQKK MMSF WKMLQQQGFITKEKYVRLVRSDELSADELAGFIERQIVETRQSTKAVATILKEALPDTEI VYVK AGNVSNFRQTYELLKVREMNDLHHAKDAYLNIVVGNAYFVKFTKNAAWFIRNNPGRSYNL KRMF EFDIERSGEIAWKAGNKGS IVTVKKVMQKN ILVTRKAYEVKGGLFDQQIMKKGKGQVPIKGND ERLADIEKYGGYNKAAGTYFMLVKSLDKKGKEIRTIEFVPLYLKNQIEINHESAIQYLAQ ERGL NSPEILLSKIKIDTLFKVDGFKMWLSGRTGNQLIFKGANQLILSHQEAAILKGVVKYVNR KNEN KDAKLSERDGMTEEKLLQLYDTFLDKLSNTVYS IRLSAQIKTLTEKRAKFIGLSNEDQCIVLNE ILHMFQCQSGSANLKLIGGPGSAGILVMNN ITACKQI SVINQSPTGIYEKEIDLIKL

SEQ ID NO: 323

MKKPYS IGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKSHIEKNLLGALLFDSGNTAEDRRL KRTARRRYTRRRNRILYLQEIFSEEMGKVDDSFFHRLEDSFLVTEDKRGERHPIFGNLEE EVKY HENFPTIYHLRQYLADNPEKVDLRLVYLALAHI IKFRGHFLIEGKFDTRNNDVQRLFQEFLAVY DNTFENSSLQEQNVQVEEILTDKI SKSAKKDRVLKLFPNEKSNGRFAEFLKLIVGNQADFKKHF ELEEKAPLQFSKDTYEEELEVLLAQIGDNYAELFLSAKKLYDS ILLSGILTVTDVGTKAPLSAS MIQRYNEHQMDLAQLKQFIRQKLSDKYNEVFSDVSKDGYAGYIDGKTNQEAFYKYLKGLL NKIE GSGYFLDKIEREDFLRKQRTFDNGS IPHQIHLQEMRAI IRRQAEFYPFLADNQDRIEKLLTFRI PYYVGPLARGKSDFAWLSRKSADKITPWNFDEIVDKESSAEAFINRMTNYDLYLPNQKVL PKHS LLYEKFTVYNELTKVKYKTEQGKTAFFDANMKQEIFDGVFKVYRKVTKDKLMDFLEKEFD EFRI VDLTGLDKENKVFNASYGTYHDLCKILDKDFLDNSKNEKILEDIVLTLTLFEDREMIRKR LENY SDLLTKEQVKKLERRHYTGWGRLSAELIHGIRNKESRKTILDYLIDDGNSNRNFMQLIND DALS FKEEIAKAQVIGETDNLNQVVSDIAGSPAIKKGILQSLKIVDELVKIMGHQPENIVVEMA RENQ FTNQGRRNSQQRLKGLTDS IKEFGSQILKEHPVENSQLQNDRLFLYYLQNGRDMYTGEELDIDY LSQYDIDHI IPQAFIKDNS IDNRVLTSSKENRGKSDDVPSKDVVRKMKSYWSKLLSAKLITQRK FDNLTKAERGGLTDDDKAGFIKRQLVETRQITKHVARILDERFNTETDENNKKIRQVKIV TLKS NLVSNFRKEFELYKVREINDYHHAHDAYLNAVIGKALLGVYPQLEPEFVYGDYPHFHGHK ENKA TAKKFFYS IMNFFKKDDVRTDKNGEI IWKKDEHI S IKKVLSYPQV IVKKVEEQTGGFSKES ILPKGNSDKLIPRKTKKFYWDTKKYGGFDSPIVAYS ILVIADIEKGKSKKLKTVKALVGVTIME KMTFERDPVAFLERKGYRNVQEE I IKLPKYSLFKLENGRKRLLASARELQKGNEIVLPNHLGT LLYHAKNIHKVDEPKHLDYVDKHKDEFKELLDVVSNFSKKYTLAEGNLEKIKELYAQNNG EDLK ELASSFINLLTFTAIGAPATFKFFDKNIDRKRYTSTTEILNATLIHQSITGLYETRIDLN KLGG D

SEQ ID NO: 324

MDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF KSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL EKMD GTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL PKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFD SVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINR LSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLS MPQV IVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQI SEFSKRV ILADANLDKVLSAYNKHRDKPIREQAE I IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQS ITGLYETRIDLSQLGGD SEQ ID NO: 325

MTKPYS IGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRL KRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVE EKAY HDEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDF LDTY NAIFESDLSLENSKQLEEIVKDKI SKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCF NLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAP LSSA MIKRYNEHKEDLALLKEYIRNI SLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKKLLAEFE GADYFLEKIDREDFLRKQRTFDNGS IPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRI PYYVGPLARGNSDFAWS IRKRNEKITPWNFEDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHS LLYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDKDI IEYLHAIYGYDG IELKGIEKQFNSSLSTYHDLLNI INDKEFLDDSSNEAI IEEI IHTLTIFEDREMIKQRLSKFEN IFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDGI SNRNFMQLIHDDALSFK KKIQKAQI IGDEDKGNIKEVVKSLPGSPAIKKGILQS IKIVDELVKVMGGRKPES IVVEMAREN QYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKD MYTG DDLDIDRLSNYDIDHI IPQAFLKDNS IDNKVLVSSASNRGKSDDVPSLEVVKKRKTFWYQLLKS KLISQRKFDNLTKAERGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRA VRTV KIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVVASALLKKYPKLEPEFVYGDY PKYN SFRERKSATEKVYFYS IM IFKKS I SLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLS YPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGI SNSF TVLVKGTIEKGAKKKITNVLEFQGI S ILDRINYRKDKLNFLLEKGYKDIELI IELPKYSLFELS DGSRRMLAS ILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRI SNTINENHRKYVENHKKEFEEL FYYILEFNENYVGAKKNGKLLNSAFQSWQNHS IDELCSSFIGPTGSERKGLFELTSRGSAADFE FLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG

SEQ ID NO: 326

MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKKDMWGSRLFDEAKTAAERRVQRNS RRRL KRRKWRLNLLEEIFSDEIMKIDSNFFRRLKESSLWLEDKNSKEKFTLFNDDNYKDYDFYK QYPT IFHLRDELIKNPEKKDIRLIYLALHS IFKSRGHFLFEGQNLKEIKNFETLYNNLI SFLEDNGIN KS IDKD IEKLEKI ICDSGKGLKDKEKEFKGIFNSDKQLVAIFKLSVGSSVSLNDLFDTDEYKK EEVEKEKI SFREQIYEDDKPIYYS ILGEKIELLDIAKSFYDFMVLNNILSDSNYI SEAKVKLYE EHKKDLKNLKYI IRKYNKENYDKLFKDKNENNYPAYIGLNKEKDKKEVVEKSRLKIDDLIKVIK GYLPKPERIEEKDKTIFNEILNKIELKTILPKQRI SDNGTLPYQIHEVELEKILENQSKYYDFL NYEENGVSTKDKLLKTFKFRIPYYVGPLNSYHKDKGGNSWIVRKEEGKILPWNFEQKVDI EKSA EEFIKRMTNKCTYLNGEDVIPKDSFLYSEYI ILNELNKVQVNDEFLNEENKRKI IDELFKENKK VSEKKFKEYLLVNQIANRTVELKGIKDSFNSNYVSYIKFKDIFGEKLNLDIYKEI SEKS ILWKC LYGDDKKIFEKKIKNEYGDILNKDEIKKINSFKFNTWGRLSEKLLTGIEFINLETGECYS SVME ALRRTNYNLMELLSSKFTLQES IDNENKEMNEVSYRDLIEESYVSPSLKRAILQTLKIYEEIKK ITGRVPKKVFIEMARGGDESMKNKKIPARQEQLKKLYDSCGNDIANFS IDIKEMKNSLSSYDNN SLRQKKLYLYYLQFGKCMYTGREIDLDRLLQNNDTYDIDHIYPRSKVIKDDSFDNLVLVL KNEN AEKSNEYPVKKEIQEKMKSFWRFLKEKNFI SDEKYKRLTGKDDFELRGFMARQLVNVRQTTKEV GKILQQIEPEIKIVYSKAEIASSFREMFDFIKVRELNDTHHAKDAYLNIVAGNVYNTKFT EKPY RYLQEIKENYDVKKIYNYDIKNAWDKENSLEIVKKNMEKNTV ITRFIKEEKGELFNLNPIKKG ETSNEI I S IKPKLYDGKDNKLNEKYGYYTSLKAAYFIYVEHEKKNKKVKTFERITRIDSTLIKN EKNLIKYLVSQKKLLNPKI IKKIYKEQTLI IDSYPYTFTGVDSNKKVELKNKKQLYLEKKYEQI LKNALKFVEDNQGETEENYKFIYLKKRNNNEKNETIDAVKERY IEFNEMYDKFLEKLSSKDYK NYINNKLYTNFLNSKEKFKKLKLWEKSLILREFLKIFNKNTYGKYEIKDSQTKEKLFSFP EDTG RIRLGQSSLGNNKELLEESVTGLFVKKIKL

SEQ ID NO: 327

MKNYTIGLDIGVASVGWVCIDENYKILNYNNRHAFGVHEFESAESAAGRRLKRGMRRRYN RRKK RLQLLQSLFDSYITDSGFFSKTDSQHFWKNNNEFENRSLTEVLSSLRISSRKYPTIYHLR SDLI ESNKKMDLRLVYLALHNLVKYRGHFLQEGNWSEAASAEGMDDQLLELVTRYAELENLSPL DLSE SQWKAAETLLLNRNLTKTDQSKELTAMFGKEYEPFCKLVAGLGVSLHQLFPSSEQALAYK ETKT KVQLSNENVEEVMELLLEEESALLEAVQPFYQQVVLYELLKGETYVAKAKVSAFKQYQKD MASL KNLLDKTFGEKVYRSYFI SDKNSQREYQKSHKVEVLCKLDQFNKEAKFAETFYKDLKKLLEDKS KTS IGTTEKDEMLRI IKAIDSNQFLQKQKGIQNAAIPHQNSLYEAEKILRNQQAHYPFITTEWI EKVKQILAFRIPYYIGPLVKDTTQSPFSWVERKGDAPITPWNFDEQIDKAASAEAFI SRMRKTC TYLKGQEVLPKSSLTYERFEVLNELNGIQLRTTGAESDFRHRLSYEMKCWI IDNVFKQYKTVST KRLLQELKKSPYADELYDEHTGEIKEVFGTQKENAFATSLSGYI SMKS ILGAVVDDNPAMTEEL IYWIAVFEDREILHLKIQEKYPS ITDVQRQKLALVKLPGWGRFSRLLIDGLPLDEQGQSVLDHM EQYSSVFMEVLKNKGFGLEKKIQKMNQHQVDGTKKIRYEDIEELAGSPALKRGIWRSVKI VEEL VSIFGEPANIVLEVAREDGEKKRTKSRKDQWEELTKTTLKNDPDLKSFIGEIKSQGDQRF NEQR FWLYVTQQGKCLYTGKALDIQNLSMYEVDHILPQNFVKDDSLDNLALVMPEANQRKNQVG QNKM PLEI IEANQQYAMRTLWERLHELKLI SSGKLGRLKKPSFDEVDKDKFIARQLVETRQI IKHVRD LLDERFSKSDIHLVKAGIVSKFRRFSEIPKIRDYNNKHHAMDALFAAALIQS ILGKYGKNFLAF DLSKKDRQKQWRSVKGSNKEFFLFKNFGNLRLQSPVTGEEVSGVEYMKHVYFELPWQTTK MTQT GDGMFYKES IFSPKVKQAKYVSPKTEKFVHDEVKNHS ICLVEFTFMKKEKEVQETKFIDLKVIE HHQFLKEPESQLAKFLAEKETNSPI IHARI IRTIPKYQKIWIEHFPYYFI STRELHNARQFEI S YELMEKVKQLSERSSVEELKIVFGLLIDQMNDNYPIYTKSS IQDRVQKFVDTQLYDFKSFEIGF EELKKAVAANAQRSDTFGSRI SKKPKPEEVAIGYES ITGLKYRKPRSVVGTKR

SEQ ID NO: 328

MKKEIKDYFLGLDVGTGSVGWAVTDTDYKLLKANRKDLWGMRCFETAETAEVRRLHRGAR RRIE RRKKRIKLLQELFSQEIAKTDEGFFQRMKESPFYAEDKTILQENTLFNDKDFADKTYHKA YPTI NHLIKAWIENKVKPDPRLLYLACH I IKKRGHFLFEGDFDSENQFDTS IQALFEYLREDMEVDI DADSQKVKEILKDSSLKNSEKQSRLNKILGLKPSDKQKKAITNLI SGNKINFADLYDNPDLKDA EKNS I SFSKDDFDALSDDLAS ILGDSFELLLKAKAVYNCSVLSKVIGDEQYLSFAKVKIYEKHK TDLTKLKNVIKKHFPKDYKKVFGYNKNEKNNNNYSGYVGVCKTKSKKLI INNSVNQEDFYKFLK TILSAKSEIKEVNDILTEIETGTFLPKQI SKSNAEIPYQLRKMELEKILSNAEKHFSFLKQKDE KGLSHSEKI IMLLTFKIPYYIGPINDNHKKFFPDRCWVVKKEKSPSGKTTPWNFFDHIDKEKTA EAFITSRTNFCTYLVGESVLPKSSLLYSEYTVLNEINNLQI I IDGK ICDIKLKQKIYEDLFKK YKKITQKQISTFIKHEGICNKTDEVI ILGIDKECTSSLKSYIELK IFGKQVDEISTKNMLEEI IRWATIYDEGEGKTILKTKIKAEYGKYCSDEQIKKILNLKFSGWGRLSRKFLETVTSEMP GFSE PVNI ITAMRETQNNLMELLSSEFTFTENIKKINSGFEDAEKQFSYDGLVKPLFLSPSVKKMLWQ TLKLVKEISHITQAPPKKIFIEMAKGAELEPARTKTRLKILQDLYNNCKNDADAFSSEIK DLSG KIENEDNLRLRSDKLYLYYTQLGKCMYCGKPIEIGHVFDTSNYDIDHIYPQSKIKDDS I SNRVL VCSSCNKNKEDKYPLKSEIQSKQRGFWNFLQRNNFI SLEKLNRLTRATPI SDDETAKFIARQLV ETRQATKVAAKVLEKMFPETKIVYSKAETVSMFRNKFDIVKCREINDFHHAHDAYLNIVV GNVY NTKFTNNPWNFIKEKRDNPKIADTYNYYKVFDYDVKRNNITAWEKGKTI ITVKDMLKRNTPIYT RQAACKKGELFNQTIMKKGLGQHPLKKEGPFS I SKYGGYNKVSAAYYTLIEYEEKGNKIRSLE TIPLYLVKDIQKDQDVLKSYLTDLLGKKEFKILVPKIKINSLLKINGFPCHITGKTNDSF LLRP AVQFCCSNNEVLYFKKI IRFSEIRSQREKIGKTI SPYEDLSFRSYIKENLWKKTKNDEIGEKEF YDLLQKKNLEIYDMLLTKHKDTIYKKRPNSATIDILVKGKEKFKSLI IENQFEVILEILKLFSA TRNVSDLQHIGGSKYSGVAKIGNKI SSLDNCILIYQS ITGIFEKRIDLLKV

SEQ ID NO: 329

MEGQMKNNGNNLQQGNYYLGLDVGTSSVGWAVTDTDYNVLKFRGKSMWGARLFDEASTAE ERRT HRGNRRRLARRKYRLLLLEQLFEKEIRKIDDNFFVRLHESNLWADDKSKPSKFLLFNDTN FTDK DYLKKYPTIYHLRSDLIHNSTEHDIRLVFLALHHLIKYRGHFIYDNSANGDVKTLDEAVS DFEE YLNENDIEFNIENKKEFINVLSDKHLTKKEKKI SLKKLYGDITDSENINI SVLIEMLSGSS I SL SNLFKDIEFDGKQNLSLDSDIEETLNDVVDILGDNIDLLIHAKEVYDIAVLTSSLGKHKY LCDA KVELFEKNKKDLMILKKYIKKNHPEDYKKIFSSPTEKKNYAAYSQTNSKNVCSQEEFCLF IKPY IRDMVKSENEDEVRIAKEVEDKSFLTKLKGTNNSVVPYQIHERELNQILKNIVAYLPFMN DEQE DI SVVDKIKLIFKFKIPYYVGPLNTKSTRSWVYRSDEKIYPWNFSNVIDLDKTAHEFMNRLI GR CTYTNDPVLPMDSLLYSKYNVLNEINPIKVNGKAIPVEVKQAIYTDLFENSKKKVTRKS IYIYL LKNGYIEKEDIVSGIDIEIKSKLKSHHDFTQIVQENKCTPEEIERI IKGILVYSDDKSMLRRWL KNNIKGLSENDVKYLAKLNYKEWGRLSKTLLTDIYTINPEDGEACS ILDIMWNTNATLMEILSN EKYQFKQ IENYKAENYDEKQNLHEELDDMYI SPAARRS IWQALRIVDEIVDIKKSAPKKIFIE MAREKKSAMKKKRTESRKDTLLELYKSCKSQADGFYDEELFEKLSNESNSRLRRDQLYLY YTQM GRSMYTGKRIDFDKLINDKNTYDIDHIYPRSKIKDDS ITNRVLVEKDINGEKTDIYPI SEDIRQ KMQPFWKILKEKGLINEEKYKRLTRNYELTDEELSSFVARQLVETQQSTKALATLLKKEY PSAK IVYSKAGNVSEFRNRKDKELPKFREINDLHHAKDAYLNIVVGNVYDTKFTEKFFNNIRNE NYSL KRVFDFSVPGAWDAKGSTFNTIKKYMAKNNPI IAFAPYEVKGELFDQQIVPKGKGQFPIKQGKD IEKYGGYNKLSSAFLFAVEYKGKKARERSLETVYIKDVELYLQDPIKYCESVLGLKEPQI IKPK ILMGSLFS INNKKLVVTGRSGKQYVCHHIYQLS INDEDSQYLK IAKYLQEEPDG IERQ ILN ITSVN IKLFDVLCTKFNSNTYEI ILNSLKNDVNEGREKFSELDILEQC ILLQLLKAFKCNRE SSNLEKLNNKKQAGVIVIPHLFTKCSVFKVIHQS ITGLFEKEMDLLK

SEQ ID NO: 330

MGRKPYILSLDIGTGSVGYACMDKGFNVLKYHDKDALGVYLFDGALTAQERRQFRTSRRR KNRR IKRLGLLQELLAPLVQNPNFYQFQRQFAWKNDNMDFKNKSLSEVLSFLGYESKKYPTIYH LQEA LLLKDEKFDPELIYMALYHLVKYRGHFLFDHLKIENLTNNDNMHDFVELIETYENLNNIK LNLD YEKTKVIYEILKDNEMTKNDRAKRVKNMEKKLEQFS IMLLGLKFNEGKLFNHADNAEELKGANQ SHTFADNYEENLTPFLTVEQSEFIERANKIYLSLTLQDILKGKKSMAMSKVAAYDKFRNE LKQV KDIVYKADSTRTQFKKIFVSSKKSLKQYDATPNDQTFSSLCLFDQYLIRPKKQYSLLIKE LKKI IPQDSELYFEAENDTLLKVLNTTDNAS IPMQINLYEAETILRNQQKYHAEITDEMIEKVLSLIQ FRIPYYVGPLVNDHTASKFGWMERKSNES IKPWNFDEVVDRSKSATQFIRRMTNKCSYLINEDV LPKNSLLYQEMEVLNELNATQIRLQTDPKNRKYRMMPQIKLFAVEHIFKKYKTVSHSKFL EIML NSNHRENFMNHGEKLS IFGTQDDKKFASKLSSYQDMTKIFGDIEGKRAQIEEI IQWITIFEDKK ILVQKLKECYPELTSKQINQLKKLNYSGWGRLSEKLLTHAYQGHS I IELLRHSDENFMEILTND VYGFQNFIKEENQVQSNKIQHQDIANLTTSPALKKGIWSTIKLVRELTS IFGEPEKI IMEFATE DQQKGKKQKSRKQLWDD IKKNKLKSVDEYKYI IDVANKLNNEQLQQEKLWLYLSQNGKCMYSG QS IDLDALLSPNATKHYEVDHIFPRSFIKDDS IDNKVLVIKKMNQTKGDQVPLQFIQQPYERIA YWKSLNKAGLI SDSKLHKLMKPEFTAMDKEGFIQRQLVETRQI SVHVRDFLKEEYPNTKVIPMK AKMVSEFRKKFDIPKIRQMNDAHHAIDAYLNGVVYHGAQLAYPNVDLFDFNFKWEKVREK WKAL GEFNTKQKSRELFFFKKLEKMEVSQGERLI SKIKLDMNHFKINYSRKLA IPQQFYNQTAVSPK TAELKYESNKSNEVVYKGLTPYQTYVVAIKSVNKKGKEKMEYQMIDHYVFDFYKFQNGNE KELA LYLAQRENKDEVLDAQIVYSLNKGDLLYINNHPCYFVSRKEVINAKQFELTVEQQLSLYN VMNN KETNVEKLLIEYDFIAEKVINEYHHYLNSKLKEKRVRTFFSESNQTHEDFIKALDELFKV VTAS ATRSDKIGSRKNSMTHRAFLGKGKDVKIAYTS I SGLKTTKPKSLFKLAESRNEL SEQ ID NO: 331

MAKILGLDLGTNS IGWAVVERENIDFSLIDKGVRIFSEGVKSEKGIESSRAAERTGYRSARKIK YRRKLRKYETLKVLSLNRMCPLS IEEVEEWKKSGFKDYPLNPEFLKWLSTDEESNVNPYFFRDR ASKHKVSLFELGRAFYHIAQRRGFLSNRLDQSAEGILEEHCPKIEAIVEDLI S IDEI STNITDY FFETGILDSNEKNGYAKDLDEGDKKLVSLYKSLLAILKKNESDFENCKSEI IERLNKKDVLGKV KGKIKDI SQAMLDGNYKTLGQYFYSLYSKEKIRNQYTSREEHYLSEFITICKVQGIDQINEEEK INEKKFDGLAKDLYKAIFFQRPLKSQKGLIGKCSFEKSKSRCAI SHPDFEEYRMWTYLNTIKIG TQSDKKLRFLTQDEKLKLVPKFYRKNDFNFDVLAKELIEKGSSFGFYKSSKKNDFFYWFN YKPT DTVAACQVAASLKNAIGEDWKTKSFKYQTINSNKEQVSRTVDYKDLWHLLTVATSDVYLY EFAI DKLGLDEKNAKAFSKTKLKKDFASLSLSAINKILPYLKEGLLYSHAVFVANIENIVDENI WKDE KQRDYIKTQI SEI IENYTLEKSRFEI INGLLKEYKSENEDGKRVYYSKEAEQSFENDLKKKLVL FYKSNEIENKEQQETIFNELLPIFIQQLKDYEFIKIQRLDQKVLIFLKGKNETGQIFCTE EKGT AEEKEKKIKNRLKKLYHPSDIEKFKKKI IKDEFGNEKIVLGSPLTPS IKNPMAMRALHQLRKVL NALILEGQIDEKTI IHIEMARELNDANKRKGIQDYQNDNKKFREDAIKEIKKLYFEDCKKEVEP TEDDILRYQLWMEQNRSEIYEEGKNI S ICDI IGSNPAYDIEHTIPRSRSQDNSQMNKTLCSQRF NREVKKQSMPIELNNHLEILPRIAHWKEEADNLTREIEI I SRS IKAAATKEIKDKKIRRRHYLT LKRDYLQGKYDRFIWEEPKVGFKNSQIPDTGI ITKYAQAYLKSYFKKVESVKGGMVAEFRKIWG IQESFIDENGMKHYKVKDRSKHTHHTIDAITIACMTKEKYDVLAHAWTLEDQQNKKEARS I IEA SKPWKTFKEDLLKIEEEILVSHYTPDNVKKQAKKIVRVRGKKQFVAEVERDVNGKAVPKK AASG KTIYKLDGEGKKLPRLQQGDTIRGSLHQDS IYGAIKNPLNTDEIKYVIRKDLES IKGSDVES IV DEVVKEKIKEAIANKVLLLSSNAQQKNKLVGTVWMNEEKRIAINKVRIYANSVKNPLHIK EHSL LSKSKHVHKQKVYGQNDENYAMAIYELDGKRDFELI IFNLAKLIKQGQGFYPLHKKKEIKGKI VFVPIEKRNKRDVVLKRGQQVVFYDKEVENPKDI SEIVDFKGRIYI IEGLS IQRIVRPSGKVDE YGVIMLRYFKEARKADDIKQDNFKPDGVFKLGENKPTRKMNHQFTAFVEGIDFKVLPSGK FEKI

SEQ ID NO: 332

MEFKKVLGLDIGTNS IGCALLSLPKS IQDYGKGGRLEWLTSRVIPLDADYMKAFIDGKNGLPQV ITPAGKRRQKRGSRRLKHRYKLRRSRLIRVFKTLNWLPEDFPLDNPKRIKETI STEGKFSFRI S DYVPI SDESYREFYREFGYPENEIEQVIEEINFRRKTKGKNKNPMIKLLPEDWVVYYLRKKALI KPTTKEELIRI IYLFNQRRGFKSSRKDLTETAILDYDEFAKRLAEKEKYSAENYETKFVS ITKV KEVVELKTDGRKGKKRFKVILEDSRIEPYEIERKEKPDWEGKEYTFLVTQKLEKGKFKQN KPDL PKEEDWALCTTALDNRMGSKHPGEFFFDELLKAFKEKRGYKIRQYPVNRWRYKKELEFIW TKQC QLNPELNNL INKEILRKLATVLYPSQSKFFGPKIKEFENSDVLHI I SEDI IYYQRDLKSQKSL I SECRYEKRKGIDGEIYGLKCIPKSSPLYQEFRIWQDIH IKVIRKESEVNGKKKI IDETQLY INENIKEKLFELFNSKDSLSEKDILELI SLNI INSGIKI SKKEEETTHRINLFANRKELKGNET KSRYRKVFKKLGFDGEYILNHPSKLNRLWHSDYSNDYADKEKTEKS ILSSLGWKNRNGKWEKSK NYDVFNLPLEVAKAIANLPPLKKEYGSYSALAIRKMLVVMRDGKYWQHPDQIAKDQENTS LMLF DKNLIQLTNNQRKVLNKYLLTLAEVQKRSTLIKQKLNEIEHNPYKLELVSDQDLEKQVLK SFLE KKNESDYLKGLKTYQAGYLIYGKHSEKDVPIVNSPDELGEYIRKKLPNNSLRNPIVEQVI RETI FIVRDVWKSFGI IDEIHIELGRELKNNSEERKKTSESQEKNFQEKERARKLLKELLNSSNFEHY DENGNKIFSSFTVNPNPDSPLDIEKFRIWKNQSGLTDEELNKKLKDEKIPTEIEVKKYIL WLTQ KCRSPYTGKI IPLSKLFDSNVYEIEHI IPRSKMKNDSTNNLVICELGVNKAKGDRLAANFI SES NGKCKFGEVEYTLLKYGDYLQYCKDTFKYQKAKYKNLLATEPPEDFIERQINDTRYIGRK LAEL LTPVVKDSKNI IFTIGS ITSELKITWGLNGVWKDILRPRFKRLES I INKKLIFQDEDDPNKYHF DLS INPQLDKEGLKRLDHRHHALDATI IAATTREHVRYLNSLNAADNDEEKREYFLSLCNHKIR DFKLPWENFTSEVKSKLLSCVVSYKESKPILSDPFNKYLKWEYKNGKWQKVFAIQIKNDR WKAV RRSMFKEPIGTVWIKKIKEVSLKEAIKIQAIWEEVKNDPVRKKKEKYIYDDYAQKVIAKI VQEL GLSSSMRKQDDEKLNKFINEAKVSAGVNKNLNTTNKTIYNLEGRFYEKIKVAEYVLYKAK RMPL NKKEYIEKLSLQKMFNDLPNFILEKS ILDNYPEILKELESDNKYI IEPHKKNNPVNRLLLEHIL EYHNNPKEAFSTEGLEKLNKKAINKIGKPIKYITRLDGDINEEEIFRGAVFETDKGSNVY FVMY ENNQTKDREFLKPNPS I SVLKAIEHKNKIDFFAPNRLGFSRI ILSPGDLVYVPTNDQYVLIKDN SSNETI INWDDNEFI SNRIYQVKKFTGNSCYFLKNDIASLILSYSASNGVGEFGSQNI SEYSVD DPPIRIKDVCIKIRVDRLGNVRPL

SEQ ID NO: 333

MKHILGLDLGTNS IGWALIERNIEEKYGKI IGMGSRIVPMGAELSKFEQGQAQTKNADRRTNRG ARRLNKRYKQRRNKLIYILQKLDMLPSQIKLKEDFSDPNKIDKITILPI SKKQEQLTAFDLVSL RVKALTEKVGLEDLGKI IYKYNQLRGYAGGSLEPEKEDIFDEEQSKDKKNKSFIAFSKIVFLGE PQEEIFKNKKLNRRAI IVETEEGNFEGSTFLENIKVGDSLELLINISASKSGDTITIKLPNKTN WRKKMENIENQLKEKSKEMGREFYI SEFLLELLKENRWAKIRNNTILRARYESEFEAIWNEQVK HYPFLENLDKKTLIEIVSFIFPGEKESQKKYRELGLEKGLKYI IKNQVVFYQRELKDQSHLI SD CRYEPNEKAIAKSHPVFQEYKVWEQINKLIVNTKIEAGTNRKGEKKYKYIDRPIPTALKE WIFE ELQNKKEITFSAIFKKLKAEFDLREGIDFLNGMSPKDKLKGNETKLQLQKSLGELWDVLG LDSI NRQIELW ILYNEKGNEYDLTSDRTSKVLEFINKYGN IVDDNAEETAIRI SKIKFARAYSSLS LKAVERILPLVRAGKYFNNDFSQQLQSKILKLLNENVEDPFAKAAQTYLDNNQSVLSEGG VGNS IATILVYDKHTAKEYSHDELYKSYKEINLLKQGDLRNPLVEQI INEALVLIRDIWKNYGIKPNE IRVELARDLKNSAKERATIHKRNKDNQTINNKIKETLVKNKKELSLANIEKVKLWEAQRH LSPY TGQPIPLSDLFDKEKYDVDHI IPI SRYFDDSFTNKVI SEKSVNQEKANRTAMEYFEVGSLKYS I FTKEQFIAHVNEYFSGVKRKNLLATS IPEDPVQRQIKDTQYIAIRVKEELNKIVGNENVKTTTG SITDYLRNHWGLTDKFKLLLKERYEALLESEKFLEAEYDNYKKDFDSRKKEYEEKEVLFE EQEL TREEFIKEYKENYIRYKKNKLI IKGWSKRIDHRHHAIDALIVACTEPAHIKRLNDLNKVLQDWL VEHKSEFMPNFEGSNSELLEEILSLPENERTEIFTQIEKFRAIEMPWKGFPEQVEQKLKE I I I S HKPKDKLLLQYNKAGDRQIKLRGQLHEGTLYGI SQGKEAYRIPLTKFGGSKFATEK IQKIVSP FLSGFIANHLKEYNNKKEEAFSAEGIMDLNNKLAQYRNEKGELKPHTPI STVKIYYKDPSKNKK KKDEEDLSLQKLDREKAFNEKLYVKTGDNYLFAVLEGEIKTKKTSQIKRLYDI I SFFDATNFLK EEFRNAPDKKTFDKDLLFRQYFEERNKAKLLFTLKQGDFVYLPNENEEVILDKESPLYNQ YWGD LKERGK IYVVQKFSKKQIYFIKHTIADI IKKDVEFGSQNCYETVEGRS IKENCFKLEIDRLGN IVKVIKR SEQ ID NO: 334

MHVEIDFPHFSRGDSHLAMNKNEILRGSSVLYRLGLDLGSNSLGWFVTHLEKRGDRHEPV ALGP GGVRIFPDGRDPQSGTSNAVDRRMARGARKRRDRFVERRKELIAALIKYNLLPDDARERR ALEV LDPYALRKTALTDTLPAHHVGRALFHLNQRRGFQSNRKTDSKQSEDGAIKQAASRLATDK GNET LGVFFADMHLRKSYEDRQTAIRAELVRLGKDHLTGNARKKIWAKVRKRLFGDEVLPRADA PHGV RARATITGTKASYDYYPTRDMLRDEFNAIWAGQSAHHATITDEARTEIEHI IFYQRPLKPAIVG KCTLDPATRPFKEDPEGYRAPWSHPLAQRFRILSEARNLEIRDTGKGSRRLTKEQSDLVV AALL ANREVKFDKLRTLLKLPAEARFNLESDRRAALDGDQTAARLSDKKGFNKAWRGFPPERQI AIVA RLEETEDENELIAWLEKECALDGAAAARVANTTLPDGHCRLGLRAIKKIVPIMQDGLDED GVAG AGYHIAAKRAGYDHAKLPTGEQLGRLPYYGQWLQDAVVGSGDARDQKEKQYGQFPNPTVH IGLG QLRRVVNDLIDKYGPPTEI S IEFTRALKLSEQQKAERQREQRRNQDKNKARAEELAKFGRPANP RNLLKMRLWEELAHDPLDRKCVYTGEQI S IERLLSDEVDIDHILPVAMTLDDSPANKI ICMRYA NRHKRKQTPSEAFGSSPTLQGHRYNWDDIAARATGLPRNKRWRFDANAREEFDKRGGFLA RQLN ETGWLARLAKQYLGAVTDPNQIWVVPGRLTSMLRGKWGLNGLLPSDNYAGVQDKAEEFLA STDD MEFSGVKNRADHRHHAIDGLVTALTDRSLLWKMANAYDEEHEKFVIEPPWPTMRDDLKAA LEKM VVSHKPDHGIEGKLHEDSAYGFVKPLDATGLKEEEAGNLVYRKAIESLNENEVDRIRDIQ LRTI VRDHVNVEKTKGVALADALRQLQAPSDDYPQFKHGLRHVRILKKEKGDYLVPIANRASGV AYKA YSAGENFCVEVFETAGGKWDGEAVRRFDANKKNAGPKIAHAPQWRDANEGAKLVMRIHKG DLIR LDHEGRARIMVVHRLDAAAGRFKLADHNETGNLDKRHATNNDIDPFRWLMASYNTLKKLA AVPV RVDELGRVWRVMPN

SEQ ID NO: 335

METTLGIDLGTNS IGLALVDQEEHQILYSGVRIFPEGINKDTIGLGEKEESRNATRRAKRQMRR QYFRKKLRKAKLLELLIAYDMCPLKPEDVRRWKNWDKQQKSTVRQFPDTPAFREWLKQNP YELR KQAVTEDVTRPELGRILYQMIQRRGFLSSRKGKEEGKIFTGKDRMVGIDETRKNLQKQTL GAYL YDIAPKNGEKYRFRTERVRARYTLRDMYIREFEI IWQRQAGHLGLAHEQATRKKNIFLEGSATN VRNSKLITHLQAKYGRGHVLIEDTRITVTFQLPLKEVLGGKIEIEEEQLKFKSNESVLFW QRPL RSQKSLLSKCVFEGRNFYDPVHQKWI IAGPTPAPLSHPEFEEFRAYQFINNI IYGKNEHLTAIQ REAVFELMCTESKDFNFEKIPKHLKLFEKFNFDDTTKVPACTTI SQLRKLFPHPVWEEKREEIW HCFYFYDDNTLLFEKLQKDYALQTNDLEKIKKIRLSESYGNVSLKAIRRINPYLKKGYAY STAV LLGGIRNSFGKRFEYFKEYEPEIEKAVCRILKEKNAEGEVIRKIKDYLVHNRFGFAKNDR AFQK LYHHSQAITTQAQKERLPETGNLRNPIVQQGLNELRRTVNKLLATCREKYGPSFKFDHIH VEMG RELRSSKTEREKQSRQIRENEKKNEAAKVKLAEYGLKAYRDNIQKYLLYKEIEEKGGTVC CPYT GKTLNI SHTLGSDNSVQIEHI IPYS I SLDDSLANKTLCDATFNREKGELTPYDFYQKDPSPEKW GASSWEEIEDRAFRLLPYAKAQRFIRRKPQESNEFI SRQLNDTRYI SKKAVEYLSAICSDVKAF PGQLTAELRHLWGLNNILQSAPDITFPLPVSATENHREYYVITNEQNEVIRLFPKQGETP RTEK GELLLTGEVERKVFRCKGMQEFQTDVSDGKYWRRIKLSSSVTWSPLFAPKPI SADGQIVLKGRI EKGVFVCNQLKQKLKTGLPDGSYWI SLPVI SQTFKEGESVNNSKLTSQQVQLFGRVREGIFRCH NYQCPASGADGNFWCTLDTDTAQPAFTPIKNAPPGVGGGQI ILTGDVDDKGIFHADDDLHYELP ASLPKGKYYGIFTVESCDPTLIPIELSAPKTSKGENLIEGNIWVDEHTGEVRFDPKKNRE DQRH HAIDAIVIALSSQSLFQRLSTYNARRENKKRGLDSTEHFPSPWPGFAQDVRQSVVPLLVS YKQN PKTLCKI SKTLYKDGKKIHSCGNAVRGQLHKETVYGQRTAPGATEKSYHIRKDIRELKTSKHIG KVVDITIRQMLLKHLQENYHIDITQEFNIPSNAFFKEGVYRIFLPNKHGEPVPIKKIRMK EELG NAERLKD INQYVNPRNNHHVMIYQDADGNLKEEIVSFWSVIERQNQGQPIYQLPREGR IVS I LQINDTFLIGLKEEEPEVYRNDLSTLSKHLYRVQKLSGMYYTFRHHLASTLNNEREEFRI QSLE AWKRANPVKVQIDEIGRITFLNGPLC SEQ ID NO: 336

MESSQILSPIGIDLGGKFTGVCLSHLEAFAELPNHANTKYSVILIDHNNFQLSQAQRRAT RHRV RNKKRNQFVKRVALQLFQHILSRDLNAKEETALCHYLNNRGYTYVDTDLDEYIKDETTIN LLKE LLPSESEHNFIDWFLQKMQSSEFRKILVSKVEEKKDDKELKNAVKNIKNFITGFEKNSVE GHRH RKVYFENIKSDITKDNQLDS IKKKIPSVCLSNLLGHLSNLQWKNLHRYLAKNPKQFDEQTFGNE FLRMLKNFRHLKGSQESLAVRNLIQQLEQSQDYI S ILEKTPPEITIPPYEARTNTGMEKDQSLL LNPEKLNNLYPNWRNLIPGI IDAHPFLEKDLEHTKLRDRKRI I SPSKQDEKRDSYILQRYLDLN KKIDKFKIKKQLSFLGQGKQLPANLIETQKEMETHFNSSLVSVLIQIASAYNKEREDAAQ GIWF DNAFSLCELSNINPPRKQKILPLLVGAILSEDFINNKDKWAKFKIFWNTHKIGRTSLKSK CKEI EEARKNSGNAFKIDYEEALNHPEHSNNKALIKI IQTIPDI IQAIQSHLGHNDSQALIYHNPFSL SQLYTILETKRDGFHKNCVAVTCENYWRSQKTEIDPEI SYASRLPADSVRPFDGVLARMMQRLA YEIAMAKWEQIKHIPDNSSLLIPIYLEQNRFEFEESFKKIKGSSSDKTLEQAIEKQNIQW EEKF QRI INASM ICPYKGAS IGGQGEIDHIYPRSLSKKHFGVIFNSEVNLIYCSSQGNREKKEEHYL LEHLSPLYLKHQFGTDNVSDIKNFI SQNVA IKKYI SFHLLTPEQQKAARHALFLDYDDEAFKT ITKFLMSQQKARVNGTQKFLGKQIMEFLSTLADSKQLQLEFS IKQITAEEVHDHRELLSKQEPK LVKSRQQSFPSHAIDATLTMS IGLKEFPQFSQELDNSWFINHLMPDEVHLNPVRSKEKYNKP I SSTPLFKDSLYAERFIPVWVKGETFAIGFSEKDLFEIKPSNKEKLFTLLKTYSTKNPGES LQEL QAKSKAKWLYFPINKTLALEFLHHYFHKEIVTPDDTTVCHFINSLRYYTKKES ITVKILKEPMP VLSVKFESSKKNVLGSFKHTIALPATKDWERLFNHPNFLALKANPAPNPKEFNEFIRKYF LSDN NPNSDIPNNGHNIKPQKHKAVRKVFSLPVIPGNAGTMMRIRRKDNKGQPLYQLQTIDDTP SMGI QINEDRLVKQEVLMDAYKTRNLSTIDGINNSEGQAYATFDNWLTLPVSTFKPEI IKLEMKPHSK TRRYIRITQSLADFIKTIDEALMIKPSDS IDDPLNMPNEIVCKNKLFGNELKPRDGKMKIVSTG KIVTYEFESDSTPQWIQTLYVTQLKKQP

SEQ ID NO: 337

MKKIVGLDLGTNS IGWALINAYINKEHLYGIEACGSRI IPMDAAILGNFDKGNS I SQTADRTSY RGIRRLRERHLLRRERLHRILDLLGFLPKHYSDSLNRYGKFLNDIECKLPWVKDETGSYK FIFQ ESFKEMLANFTEHHPILIANNKKVPYDWTIYYLRKKALTQKI SKEELAWILLNFNQKRGYYQLR GEEEETPNKLVEYYSLKVEKVEDSGERKGKDTWYNVHLENGMIYRRTS IPLDWEGKTKEFIVT TDLEADGSPKKDKEG IKRSFRAPKDDDWTLIKKKTEADIDKIKMTVGAYIYDTLLQKPDQKIR GKLVRTIERKYYKNELYQILKTQSEFHEELRDKQLYIACLNELYPNNEPRRNS I STRDFCHLFI EDI IFYQRPLKSKKSLIDNCPYEENRYIDKESGEIKHAS IKCIAKSHPLYQEFRLWQFIVNLRI YRKETDVDVTQELLPTEADYVTLFEWLNEKKEIDQKAFFKYPPFGFKKTTSNYRWNYVED KPYP CNETHAQI IARLGKAHIPKAFLSKEKEETLWHILYS IEDKQEIEKALHSFANKNNLSEEFIEQF KNFPPFKKEYGSYSAKAIKKLLPLMRMGKYWS IE IDNGTRIRINKI IDGEYDE IRERVRQKA INLTDITHFRALPLWLACYLVYDRHSEVKDIVKWKTPKDIDLYLKSFKQHSLRNPIVEQV ITET LRTVRDIWQQVGHIDEIHIELGREMKNPADKRARMSQQMIKNENTNLRIKALLTEFLNPE FGIE NVRPYSPSQQDLLRIYEEGVLNS ILELPEDIGI ILGKFNQTDTLKRPTRSEILRYKLWLEQKYR SPYTGEMIPLSKLFTPAYEIEHI IPQSRYFDDSLSNKVICESEINKLKDRSLGYEFIKNHHGEK VELAFDKPVEVLSVEAYEKLVHESYSHNRSKMKKLLMEDIPDQFIERQLNDSRYI SKVVKSLLS IVREENEQEAI SKNVIPCTGGITDRLKKDWGINDVWNKIVLPRFIRLNELTESTRFTS INTNN TMIPSMPLELQKGFNKKRIDHRHHAMDAI I IACANR IVNYLNNVSASKNTKITRRDLQTLLCH KDKTDNNGNYKWVIDKPWETFTQDTLTALQKITVSFKQNLRVINKTTNHYQHYENGKKIV SNQS KGDSWAIRKSMHKETVHGEVNLRMIKTVSFNEALKKPQAIVEMDLKKKILAMLELGYDTK RIKN YFEENKDTWQDINPSKIKVYYFTKETKDRYFAVRKPIDTSFDKKKIKES ITDTGIQQIMLRHLE TKDNDPTLAFSPDGIDEMNRNILILNKGKKHQPIYKVRVYEKAEKFTVGQKGNKRTKFVE AAKG TNLFFAIYETEEIDKDTKKVIRKRSYSTIPLNVVIERQKQGLSSAPEDENGNLPKYILSP NDLV YVPTQEEINKGEVVMPIDRDRIYKMVDSSGITANFIPASTANLIFALPKATAEIYCNGEN CIQN EYGIGSPQSKNQKAITGEMVKEICFPIKVDRLGNI IQVGSCILTN

SEQ ID NO: 338

MSRSLTFSFDIGYAS IGWAVIASASHDDADPSVCGCGTVLFPKDDCQAFKRREYRRLRR IRSR RVRIERIGRLLVQAQI ITPEMKETSGHPAPFYLASEALKGHRTLAPIELWHVLRWYAHNRGYDN NASWSNSLSEDGGNGEDTERVKHAQDLMDKHGTATMAETICRELKLEEGKADAPMEVSTP AYKN LNTAFPRLIVEKEVRRILELSAPLIPGLTAEI IELIAQHHPLTTEQRGVLLQHGIKLARRYRGS LLFGQLIPRFDNRI I SRCPVTWAQVYEAELKKGNSEQSARERAEKLSKVPTANCPEFYEYRMAR ILC IRADGEPLSAEIRRELMNQARQEGKLTKASLEKAI SSRLGKETETNVSNYFTLHPDSEEA LYLNPAVEVLQRSGIGQILSPSVYRIAANRLRRGKSVTPNYLLNLLKSRGESGEALEKKI EKES KKKEADYADTPLKPKYATGRAPYARTVLKKVVEEILDGEDPTRPARGEAHPDGELKAHDG CLYC LLDTDSSVNQHQKERRLDTMTNNHLVRHRMLILDRLLKDLIQDFADGQKDRI SRVCVEVGKELT TFSAMDSKKIQRELTLRQKSHTDAVNRLKRKLPGKALSANLIRKCRIAMDMNWTCPFTGA TYGD HELENLELEHIVPHSFRQSNALSSLVLTWPGVNRMKGQRTGYDFVEQEQENPVPDKPNLH ICSL NNYRELVEKLDDKKGHEDDRRRKKKRKALLMVRGLSHKHQSQNHEAMKEIGMTEGMMTQS SHLM KLACKS IKTSLPDAHIDMIPGAVTAEVRKAWDVFGVFKELCPEAADPDSGKILKENLRSLTHLH HALDACVLGLIPYI IPAHHNGLLRRVLAMRRIPEKLIPQVRPVANQRHYVLNDDGRMMLRDLSA SLKENIREQLMEQRVIQHVPADMGGALLKETMQRVLSVDGSGEDAMVSLSKKKDGKKEKN QVKA SKLVGVFPEGPSKLKALKAAIEIDGNYGVALDPKPVVIRHIKVFKRIMALKEQNGGKPVR ILKK GMLIHLTSSKDPKHAGVWRIES IQDSKGGVKLDLQRAHCAVPKNKTHECNWREVDLI SLLKKYQ MKRYPTSYTGTPR

SEQ ID NO: 339

MTQKVLGLDLGTNS IGSAVRNLDLSDDLQWQLEFFSSDIFRSSVNKESNGREYSLAAQRSAHRR SRGLNEVRRRRLWATLNLLIKHGFCPMSSESLMRWCTYDKRKGLFREYPIDDKDFNAWIL LDFN GDGRPDYSSPYQLRRELVTRQFDFEQPIERYKLGRALYHIAQHRGFKSSKGETLSQQETN SKPS STDEIPDVAGAMKASEEKLSKGLSTYMKEHNLLTVGAAFAQLEDEGVRVRNNNDYRAIRS QFQH EIETIFKFQQGLSVESELYERLI SEKKNVGTIFYKRPLRSQRGNVGKCTLERSKPRCAIGHPLF EKFRAWTLINNIKVRMSVDTLDEQLPMKLRLDLYNECFLAFVRTEFKFEDIRKYLEKRLG IHFS YNDKTINYKDSTSVAGCPITARFRKMLGEEWESFRVEGQKERQAHSKNNI SFHRVSYS IEDIWH FCYDAEEPEAVLAFAQETLRLERKKAEELVRIWSAMPQGYAMLSQKAIRNINKILMLGLK YSDA VILAKVPELVDVSDEELLS IAKDYYLVEAQVNYDKRINS IVNGLIAKYKSVSEEYRFADHNYEY LLDESDEKDI IRQIENSLGARRWSLMDANEQTDILQKVRDRYQDFFRSHERKFVESPKLGESFE NYLTKKFPMVEREQWKKLYHPSQITIYRPVSVGKDRSVLRLGNPDIGAIKNPTVLRVLNT LRRR VNQLLDDGVI SPDETRVVVETARELNDANRKWALDTYNRIRHDENEKIKKILEEFYPKRDGI ST DDIDKARYVIDQREVDYFTGSKTYNKDIKKYKFWLEQGGQCMYTGRTINLSNLFDPNAFD IEHT IPESLSFDSSDMNLTLCDAHYNRFIKKNHIPTDMPNYDKAITIDGKEYPAITSQLQRWVE RVER LNRNVEYWKGQARRAQNKDRKDQCMREMHLWKMELEYWKKKLERFTVTEVTDGFKNSQLV DTRV ITRHAVLYLKS IFPHVDVQRGDVTAKFRKILGIQSVDEKKDRSLHSHHAIDATTLTI IPVSAKR DRMLELFAKIEEINKMLSFSGSEDRTGLIQELEGLKNKLQMEVKVCRIGHNVSEIGTFIN DNII VNHHIKNQALTPVRRRLRKKGYIVGGVDNPRWQTGDALRGEIHKASYYGAITQFAKDDEG KVLM KEGRPQVNPTIKFVIRRELKYKKSAADSGFASWDDLGKAIVDKELFALMKGQFPAETSFK DACE QGIYMIKKGKNGMPDIKLHHIRHVRCEAPQSGLKIKEQTYKSEKEYKRYFYAAVGDLYAM CCYT NGKIREFRIYSLYDVSCHRKSDIEDIPEFITDKKGNRLMLDYKLRTGDMILLYKDNPAEL YDLD NVNLSRRLYKINRFESQSNLVLMTHHLSTSKERGRSLGKTVDYQNLPES IRSSVKSLNFLIMGE NRDFVIKNGKI IFNHR

SEQ ID NO: 340

MLVSPI SVDLGGKNTGFFSFTDSLDNSQSGTVIYDESFVLSQVGRRSKRHSKRNNLRNKLVKRL FLLILQEHHGLS IDVLPDEIRGLFNKRGYTYAGFELDEKKKDALESDTLKEFLSEKLQS IDRDS DVEDFLNQIASNAESFKDYKKGFEAVFASATHSPNKKLELKDELKSEYGENAKELLAGLR VTKE ILDEFDKQENQGNLPRAKYFEELGEYIATNEKVKSFFDSNSLKLTDMTKLIG I SNYQLKELRR YFNDKEMEKGDIWIPNKLHKITERFVRSWHPKNDADRQRRAELMKDLKSKEIMELLTTTE PVMT IPPYDDMNNRGAVKCQTLRLNEEYLDKHLPNWRDIAKRLNHGKFNDDLADSTVKGYSEDS TLLH RLLDTSKEIDIYELRGKKPNELLVKTLGQSDANRLYGFAQNYYELIRQKVRAGIWVPVKN KDDS LNLEDNSNMLKRCNHNPPHKKNQIHNLVAGILGVKLDEAKFAEFEKELWSAKVGNKKLSA YCKN IEELRKTHGNTFKIDIEELRKKDPAELSKEEKAKLRLTDDVILNEWSQKIANFFDIDDKH RQRF NNLFSMAQLHTVIDTPRSGFSSTCKRCTAENRFRSETAFYNDETGEFHKKATATCQRLPA DTQR PFSGKIERYIDKLGYELAKIKAKELEGMEAKEIKVPI ILEQNAFEYEESLRKSKTGSNDRVINS KKDRDGKKLAKAKENAEDRLKDKDKRIKAFSSGICPYCGDTIGDDGEIDHILPRSHTLKI YGTV FNPEGNLIYVHQKCNQAKADS IYKLSDIKAGVSAQWIEEQVANIKGYKTFSVLSAEQQKAFRYA LFLQNDNEAYKKVVDWLRTDQSARVNGTQKYLAKKIQEKLTKMLPNKHLSFEFILADATE VSEL RRQYARQNPLLAKAEKQAPSSHAIDAVMAFVARYQKVFKDGTPPNADEVAKLAMLDSWNP ASNE PLTKGLSTNQKIEKMIKSGDYGQKNMREVFGKS IFGENAIGERYKPIVVQEGGYYIGYPATVKK GYELKNCKVVTSKNDIAKLEKI IKNQDLI SLKENQYIKIFS INKQTI SELSNRYFNMNYKNLVE RDKEIVGLLEFIVENCRYYTKKVDVKFAPKYIHETKYPFYDDWRRFDEAWRYLQENQNKT SSKD RFVIDKSSLNEYYQPDKNEYKLDVDTQPIWDDFCRWYFLDRYKTANDKKS IRIKARKTFSLLAE SGVQGKVFRAKRKIPTGYAYQALPMDNNVIAGDYANILLEANSKTLSLVPKSGI S IEKQLDKKL DVIKKTDVRGLAIDNNSFFNADFDTHGIRLIVENTSVKVGNFPI SAIDKSAKRMIFRALFEKEK GKRKKKTTI SFKESGPVQDYLKVFLKKIVKIQLRTDGS I S IVVRKNAADFTLSFRSEHIQKLL K

SEQ ID NO: 341 MAYRLGLDIGITSVGWAVVALEKDESGLKPVRIQDLGVRIFDKAEDSKTGASLALPRREA RSAR RRTRRRRHRLWRVKRLLEQHGILSMEQIEALYAQRTSSPDVYALRVAGLDRCLIAEEIAR VLIH IAHRRGFQSNRKSEIKDSDAGKLLKAVQENENLMQSKGYRTVAEMLVSEATKTDAEGKLV HGKK HGYVSNVRNKAGEYRHTVSRQAIVDEVRKIFAAQRALGNDVMSEELEDSYLKILCSQRNF DDGP GGDSPYGHGSVSPDGVRQS IYERMVGSCTFETGEKRAPRSSYSFERFQLLTKVVNLRIYRQQED GGRYPCELTQTERARVIDCAYEQTKITYGKLRKLLDMKDTESFAGLTYGLNRSRNKTEDT VFVE MKFYHEVRKALQRAGVFIQDLS IETLDQIGWILSVWKSDDNRRKKLSTLGLSDNVIEELLPLNG SKFGHLSLKAIRKILPFLEDGYSYDVACELAGYQFQGKTEYVKQRLLPPLGEGEVTNPVV RRAL SQAIKVVNAVIRKHGSPES IHIELARELSKNLDERRKIEKAQKENQKNNEQIKDEIREILGSAH VTGRDIVKYKLFKQQQEFCMYSGEKLDVTRLFEPGYAEVDHI IPYGI SFDDSYDNKVLVKTEQN RQKGNRTPLEYLRDKPEQKAKFIALVES IPLSQKKKNHLLMDKRAIDLEQEGFRERNLSDTRYI TRALMNHIQAWLLFDETASTRSKRVVCVNGAVTAYMRARWGLTKDRDAGDKHHAADAVVV ACIG DSLIQRVTKYDKFKRNALADRNRYVQQVSKSEGITQYVDKETGEVFTWESFDERKFLPNE PLEP WPFFRDELLARLSDDPSKNIRAIGLLTYSETEQIDPIFVSRMPTRKVTGAAHKETIRSPR IVKV DDNKGTEIQVVVSKVALTELKLTKDGEIKDYFRPEDDPRLYNTLRERLVQFGGDAKAAFK EPVY KI SKDGSVRTPVRKVKIQEKLTLGVPVHGGRGIAENGGMVRIDVFAKGGKYYFVPIYVADVL KR ELPNRLATAHKPYSEWRVVDDSYQFKFSLYPNDAVMIKPSREVDITYKDRKEPVGCRIMY FVSA NIASAS I SLRTHDNSGELEGLGIQGLEVFEKYVVGPLGDTHPVYKERRMPFRVERKMN SEQ ID NO: 342

MPVLSPLSPNAAQGRRRWSLALDIGEGS IGWAVAEVDAEGRVLQLTGTGVTLFPSAWSNENGTY VAHGAADRAVRGQQQRHDSRRRRLAGLARLCAPVLERSPEDLKDLTRTPPKADPRAIFFL RADA ARRPLDGPELFRVLHHMAAHRGIRLAELQEVDPPPESDADDAAPAATEDEDGTRRAAADE RAFR RLMAEHMHRHGTQPTCGEIMAGRLRETPAGAQPVTRARDGLRVGGGVAVPTRALIEQEFD AIRA IQAPRHPDLPWDSLRRLVLDQAPIAVPPATPCLFLEELRRRGETFQGRTITREAIDRGLT VDPL IQALRIRETVGNLRLHERITEPDGRQRYVPRAMPELGLSHGELTAPERDTLVRALMHDPD GLAA KDGRIPYTRLRKLIGYDNSPVCFAQERDTSGGGITVNPTDPLMARWIDGWVDLPLKARSL YVRD VVARGADSAALARLLAEGAHGVPPVAAAAVPAATAAILESDIMQPGRYSVCPWAAEAILD AWAN APTEGFYDVTRGLFGFAPGEIVLEDLRRARGALLAHLPRTMAAARTPNRAAQQRGPLPAY ESVI PSQLITSLRRAHKGRAADWSAADPEERNPFLRTWTGNAATDHILNQVRKTANEVITKYGN RRGW DPLPSRITVELAREAKHGVIRRNEIAKENRENEGRRKKESAALDTFCQDNTVSWQAGGLP KERA ALRLRLAQRQEFFCPYCAERPKLRATDLFSPAETEIDHVIERRMGGDGPDNLVLAHKDCN NAKG KKTPHEHAGDLLDSPALAALWQGWRKENADRLKGKGHKARTPREDKDFMDRVGWRFEEDA RAKA EENQERRGRRMLHDTARATRLARLYLAAAVMPEDPAEIGAPPVETPPSPEDPTGYTAIYR TI SR VQPVNGSVTHMLRQRLLQRDKNRDYQTHHAEDACLLLLAGPAVVQAFNTEAAQHGADAPD DRPV DLMPTSDAYHQQRRARALGRVPLATVDAALADIVMPESDRQDPETGRVHWRLTRAGRGLK RRID DLTRNCVILSRPRRPSETGTPGALHNATHYGRREITVDGRTDTVVTQRMNARDLVALLDN AKIV PAARLDAAAPGDTILKEICTEIADRHDRVVDPEGTHARRWI SARLAALVPAHAEAVARDIAELA DLDALADADRTPEQEARRSALRQSPYLGRAI SAKKADGRARAREQEILTRALLDPHWGPRGLRH LIMREARAPSLVRIRANKTDAFGRPVPDAAVWVKTDGNAVSQLWRLTSVVTDDGRRIPLP KPIE KRIEI SNLEYARLNGLDEGAGVTGNNAPPRPLRQDIDRLTPLWRDHGTAPGGYLGTAVGELEDK ARSALRGKAMRQTLTDAGITAEAGWRLDSEGAVCDLEVAKGDTVKKDGKTYKVGVITQGI FGMP VDAAGSAPRTPEDCEKFEEQYGIKPWKAKGIPLA SEQ ID NO: 343

MNYTEKEKLFMKYILALDIGIASVGWAILDKESETVIEAGSNIFPEASAADNQLRRDMRG AKRN NRRLKTRINDFIKLWENNNLSIPQFKSTEIVGLKVRAITEEITLDELYLILYSYLKHRGI SYLE DALDDTVSGSSAYANGLKLNAKELETHYPCEIQQERLNTIGKYRGQSQI INENGEVLDLSNVFT IGAYRKEIQRVFEIQKKYHPELTDEFCDGYMLIFNRKRKYYEGPGNEKSRTDYGRFTTKL DANG NYITEDNIFEKLIGKCSVYPDELRAAAASYTAQEYNVLNDLNNLTINGRKLEENEKHEIV ERIK SSNTINMRKI I SDCMGENIDDFAGARIDKSGKEIFHKFEVYNKMRKALLEIGIDI SNYSREELD EIGYIMTINTDKEAMMEAFQKSWIDLSDDVKQCLINMRKTNGALFNKWQSFSLKIMNELI PEMY AQPKEQMTLLTEMGVTKGTQEEFAGLKYIPVDVVSEDIFNPVVRRSVRI SFKILNAVLKKYKAL DTIVIEMPRDRNSEEQKKRINDSQKLNEKEMEYIEKKLAVTYGIKLSPSDFSSQKQLSLK LKLW NEQDGICLYSGKTIDPNDI INNPQLFEIDHI IPRS I SFDDARSNKVLVYRSENQKKGNQTPYYY LTHSHSEWSFEQYKATVMNLSKKKEYAI SRKKIQNLLYSEDITKMDVLKGFINR INDTSYASR LVLNTIQNFFMANEADTKVKVIKGSYTHQMRCNLKLDKNRDESYSHHAVDAMLIGYSELG YEAY HKLQGEFIDFETGEILRKDMWDENMSDEVYADYLYGKKWANIRNEVVKAEKNVKYWHYVM RKSN RGLCNQTIRGTREYDGKQYKINKLDIRTKEGIKVFAKLAFSKKDSDRERLLVYLNDRRTF DDLC KIYEDYSDAANPFVQYEKETGDI IRKYSKKHNGPRIDKLKYKDGEVGACIDI SHKYGFEKGSKK VILESLVPYRMDVYYKEENHSYYLVGVKQSDIKFEKGRNVIDEEAYARILVNEKMIQPGQ SRAD LENLGFKFKLSFYKNDI IEYEKDGKIYTERLVSRTMPKQRNYIETKPIDKAKFEKQNLVGLGKT KFIKKYRYDILGNKYSCSEEKFTSFC

SEQ ID NO: 344

MLRLYCANNLVLNNVQNLWKYLLLLIFDKKI IFLFKIKVILIRRYMENNNKEKIVIGFDLGVAS VGWS IVNAETKEVIDLGVRLFSEPEKADYRRAKRTTRRLLRRKKFKREKFHKLILKNAEIFGLQ SRNEILNVYKDQSSKYRNILKLKINALKEEIKPSELVWILRDYLQNRGYFYKNEKLTDEF VSNS FPSKKLHEHYEKYGFFRGSVKLDNKLDNKKDKAKEKDEEEESDAKKESEELIFSNKQWIN EIVK VFENQSYLTESFKEEYLKLFNYVRPFNKGPGSKNSRTAYGVFSTDIDPETNKFKDYSNIW DKTI GKCSLFEEEIRAPKNLPSALIFNLQNEICTIKNEFTEFKNWWLNAEQKSEILKFVFTELF NWKD KKYSDKKFNKNLQDKIKKYLLNFALENFNLNEEILKNRDLENDTVLGLKGVKYYEKSNAT ADAA LEFSSLKPLYVFIKFLKEKKLDLNYLLGLENTEILYFLDS IYLAI SYSSDLKERNEWFKKLLKE LYPKIKNNNLEI IENVEDIFEITDQEKFESFSKTHSLSREAFNHI IPLLLSNNEGKNYESLKHS NEELKKRTEKAELKAQQNQKYLKDNFLKEALVPLSVKTSVLQAIKIFNQI IKNFGKKYEI SQVV IEMARELTKPNLEKLLNNATNSNIKILKEKLDQTEKFDDFTKKKFIDKIENSVVFRNKLF LWFE QDRKDPYTQLDIKINEIEDETEIDHVIPYSKSADDSWFNKLLVKKSTNQLKKNKTVWEYY QNES DPEAKWNKFVAWAKRIYLVQKSDKESKDNSEKNS IFKNKKPNLKFK ITKKLFDPYKDLGFLAR NLNDTRYATKVFRDQLNNYSKHHSKDDENKLFKVVCMNGS ITSFLRKSMWRKNEEQVYRFNFWK KDRDQFFHHAVDAS I IAIFSLLTKTLYNKLRVYESYDVQRREDGVYLINKETGEVKKADKDYWK DQHNFLKIRENAIEIKNVLNNVDFQNQVRYSRKANTKLNTQLFNETLYGVKEFENNFYKL EKVN LFSRKDLRKFILEDLNEESEKNKKNENGSRKRILTEKYIVDEILQILENEEFKDSKSDIN ALNK YMDSLPSKFSEFFSQDFINKCKKENSLILTFDAIKHNDPKKVIKIKNLKFFREDATLKNK QAVH KDSKNQIKSFYESYKCVGFIWLKNKNDLEES IFVPINSRVIHFGDKDKDIFDFDSYNKEKLLNE INLKRPENKKFNS INEIEFVKFVKPGALLLNFENQQIYYI STLESSSLRAKIKLLNKMDKGKAV SMKKITNPDEYKI IEHVNPLGINLNWTKKLENNN

SEQ ID NO: 345

MLMSKHVLGLDLGVGS IGWCLIALDAQGDPAEILGMGSRVVPLNNATKAIEAFNAGAAFTASQE RTARRTMRRGFARYQLRRYRLRRELEKVGMLPDAALIQLPLLELWELRERAATAGRRLTL PELG RVLCHINQKRGYRHVKSDAAAIVGDEGEKKKDSNSAYLAGIRANDEKLQAEHKTVGQYFA EQLR QNQSESPTGGI SYRIKDQIFSRQCYIDEYDQIMAVQRVHYPDILTDEFIRMLRDEVIFMQRPLK SCKHLVSLCEFEKQERVMRVQQDDGKGGWQLVERRVKFGPKVAPKSSPLFQLCCIYEAVN NIRL TRPNGSPCDITPEERAKIVAHLQSSASLSFAALKKLLKEKALIADQLTSKSGLKGNSTRV ALAS ALQPYPQYHHLLDMELETRMMTVQLTDEETGEVTEREVAVVTDSYVRKPLYRLWHILYS IEERE AMRRALITQLGMKEEDLDGGLLDQLYRLDFVKPGYGNKSAKFICKLLPQLQQGLGYSEAC AAVG YRHSNSPTSEEITERTLLEKIPLLQRNELRQPLVEKILNQMINLVNALKAEYGIDEVRVE LARE LKMSREERERMARNNKDREERNKGVAAKIRECGLYPTKPRIQKYMLWKEAGRQCLYCGRS IEEE QCLREGGMEVEHI IPKSVLYDDSYGNKTCACRRCNKEKGNRTALEYIRAKGREAEYMKRINDLL KEKKI SYSKHQRLRWLKEDIPSDFLERQLRLTQYI SRQAMAILQQGIRRVSASEGGVTARLRSL WGYGKILHTLNLDRYDSMGETERVSREGEATEELHITNWSKRMDHRHHAIDALVVACTRQ SYIQ RLNRLSSEFGREDKKKEDQEAQEQQATETGRLSNLERWLTQRPHFSVRTVSDKVAEILI SYRPG QRVVTRGRNIYRKKMADGREVSCVQRGVLVPRGELMEASFYGKILSQGRVRIVKRYPLHD LKGE VVDPHLRELITTYNQELKSREKGAPIPPLCLDKDKKQEVRSVRCYAKTLSLDKAIPMCFD EKGE PTAFVKSASNHHLALYRTPKGKLVES IVTFWDAVDRARYGIPLVITHPREVMEQVLQRGDIPEQ VLSLLPPSDWVFVDSLQQDEMVVIGLSDEELQRALEAQNYRKI SEHLYRVQKMSSSYYVFRYHL ETSVADDKNTSGRIPKFHRVQSLKAYEERNIRKVRVDLLGRI SLL SEQ ID NO: 346

MSDLVLGLDIGIGSVGVGILNKVTGEI IHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRV RLNRLFEESGLITDFTKI S INLNPYQLRVKGLTDELSNEELFIALKNMVKHRGI SYLDDASDDG NSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSA YRSE ALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIF GILI GKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQI INYVKNEKAMGPAKLF KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLT LNTE REGIQEALEHEFADGSFSQKQVDELVQFRKANSS IFGKGWHNFSVKLMMELIPELYETSEEQMT ILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVI EMAR ETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQ GERC LYTGKTI S IHDLINNSNQFEVDHILPLS ITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA WSFRELKAFVRESKTLSNKKKEYLLTEEDI SKFDVRKKFIERNLVDTRYASRVVLNALQEHFRA HKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALI IAASSQLNLWKKQKNTLVSYSEDQ LLDIETGELI SDDEYKESVFKAPYQHFVDTLKSKEFEDS ILFSYQVDSKFNRKI SDATIYATRQ AKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILEN YPNK QINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNK VVLQ SVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKI SQEKYNDIKKKEGVDSDSEFKFTLY KNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQ CKKG LGKSNI S IYKVRTDVLGNQHI IKNEGDKPKLDF SEQ ID NO: 347

MNAEHGKEGLLIMEENFQYRIGLDIGITSVGWAVLQNNSQDEPVRITDLGVRIFDVAENP KNGD ALAAPRRDARTTRRRLRRRRHRLERIKFLLQENGLIEMDSFMERYYKGNLPDVYQLRYEG LDRK LKDEELAQVLIHIAKHRGFRSTRKAETKEKEGGAVLKATTENQKIMQEKGYRTVGEMLYL DEAF HTECLWNEKGYVLTPRNRPDDYKHTILRSMLVEEVHAIFAAQRAHGNQKATEGLEEAYVE IMTS QRSFDMGPGLQPDGKPSPYAMEGFGDRVGKCTFEKDEYRAPKATYTAELFVALQKINHTK LIDE FGTGRFFSEEERKTI IGLLLSSKELKYGTIRKKLNIDPSLKFNSLNYSAKKEGETEEERVLDTE KAKFASMFWTYEYSKCLKDRTEEMPVGEKADLFDRIGEILTAYKNDDSRSSRLKELGLSG EEID GLLDLSPAKYQRVSLKAMRKMQPYLEDGLIYDKACEAAGYDFRALNDGNKKHLLKGEEIN AIVN DITNPVVKRSVSQTIKVINAI IQKYGSPQAVNIELAREMSKNFQDRTNLEKEMKKRQQENERAK QQI IELGKQNPTGQDILKYRLWNDQGGYCLYSGKKIPLEELFDGGYDIDHILPYS ITFDDSYRN KVLVTAQENRQKGNRTPYEYFGADEKRWEDYEASVRLLVRDYKKQQKLLKKNFTEEERKE FKER NLNDTKYITRVVYNMIRQNLELEPFNHPEKKKQVWAVNGAVTSYLRKRWGLMQKDRSTDR HHAM DAVVIACCTDGMIHKI SRYMQGRELAYSRNFKFPDEETGEILNRDNFTREQWDEKFGVKVPLPW NSFRDELDIRLLNEDPKNFLLTHADVQRELDYPGWMYGEEESPIEEGRYINYIRPLFVSR MPNH KVTGSAHDATIRSARDYETRGVVITKVPLTDLKLNKDNEIEGYYDKDSDRLLYQALVRQL LLHG NDGKKAFAEDFHKPKADGTEGPVVRKVKIEKKQTSGVMVRGGTGIAANGEMVRIDVFREN GKYY FVPVYTADVVRKVLPNRAATHTKPYSEWRVMDDANFVFSLYSRDLIHVKSKKDIKTNLVN GGLL LQKEIFAYYTGADIATAS IAGFANDSNFKFRGLGIQSLEIFEKCQVDILGNI SVVRHENRQEFH

SEQ ID NO: 348

MRVLGLDAGIASLGWALIEIEESNRGELSQGTI IGAGTWMFDAPEEKTQAGAKLKSEQRRTFRG QRRVVRRRRQRMNEVRRILHSHGLLPSSDRDALKQPGLDPWRIRAEALDRLLGPVELAVA LGHI ARHRGFKSNSKGAKTNDPADDTSKMKRAVNETREKLARFGSAAKMLVEDESFVLRQTPTK NGAS EIVRRFRNREGDYSRSLLRDDLAAEMRALFTAQARFQSAIATADLQTAFTKAAFFQRPLQ DSEK LVGPCPFEVDEKRAPKRGYSFELFRFLSRLNHVTLRDGKQERTLTRDELALAAADFGAAA KVSF TALRKKLKLPETTVFVGVKADEESKLDVVARSGKAAEGTARLRSVIVDALGELAWGALLC SPEK LDKIAEVI SFRSDIGRI SEGLAQAGCNAPLVDALTAAASDGRFDPFTGAGHI SSKAARNILSGL RQGMTYDKACCAADYDHTASRERGAFDVGGHGREALKRILQEERI SRELVGSPTARKALIES IK QVKAIVERYGVPDRIHVELARDVGKS IEEREEITRGIEKRNRQKDKLRGLFEKEVGRPPQDGAR GKEELLRFELWSEQMGRCLYTDDYI SPSQLVATDDAVQVDHILPWSRFADDSYANKTLCMAKAN QDKKGRTPYEWFKAEKTDTEWDAFIVRVEALADMKGFKKRNYKLRNAEEAAAKFRNRNLN DTRW ACRLLAEALKQLYPKGEKDKDGKERRRVFSRPGALTDRLRRAWGLQWMKKSTKGDRIPDD RHHA LDAIVIAATTESLLQRATREVQEIEDKGLHYDLVKNVTPPWPGFREQAVEAVEKVFVARA ERRR ARGKAHDATIRHIAVREGEQRVYERRKVAELKLADLDRVKDAERNARLIEKLRNWIEAGS PKDD PPLSPKGDPIFKVRLVTKSKVNIALDTGNPKRPGTVDRGEMARVDVFRKASKKGKYEYYL VPIY PHDIATMKTPPIRAVQAYKPEDEWPEMDSSYEFCWSLVPMTYLQVI SSKGEIFEGYYRGMNRSV GAIQLSAHSNSSDVVQGIGARTLTEFKKFNVDRFGRKHEVERELRTWRGETWRGKAYI

SEQ ID NO: 349

MGNYYLGLDVGIGS IGWAVINIEKKRIEDFNVRIFKSGEIQEKNRNSRASQQCRRSRGLRRLYR RKSHRKLRLKNYLS I IGLTTSEKIDYYYETADNNVIQLRNKGLSEKLTPEEIAACLIHICNNRG YKDFYEVNVEDIEDPDERNEYKEEHDS IVLI SNLMNEGGYCTPAEMICNCREFDEPNSVYRKFH NSAASKNHYLITRHMLVKEVDLILENQSKYYGILDDKTIAKIKDI IFAQRDFEIGPGKNERFRR FTGYLDS IGKCQFFKDQERGSRFTVIADIYAFVNVLSQYTYTNNRGESVFDTSFANDLINSALK NGSMDKRELKAIAKSYHIDI SDKNSDTSLTKCFKYIKVVKPLFEKYGYDWDKLIENYTDTDNNV LNRIGIVLSQAQTPKRRREKLKALNIGLDDGLINELTKLKLSGTANVSYKYMQGS IEAFCEGDL YGKYQAKFNKEIPDIDENAKPQKLPPFKNEDDCEFFKNPVVFRS INETRKLINAI IDKYGYPAA VNIETADELNKTFEDRAIDTKRNNDNQKENDRIVKEI IECIKCDEVHARHLIEKYKLWEAQEGK CLYSGETITKEDMLRDKDKLFEVDHIVPYSLILDNTINNKALVYAEENQKKGQRTPLMYM NEAQ AADYRVRVNTMFKSKKCSKKKYQYLMLPDLNDQELLGGWRSRNLNDTRYICKYLVNYLRK NLRF DRSYESSDEDDLKIRDHYRVFPVKSRFTSMFRRWWLNEKTWGRYDKAELKKLTYLDHAAD AI I I ANCRPEYVVLAGEKLKLNKMYHQAGKRITPEYEQSKKACIDNLYKLFRMDRRTAEKLLSG HGRL TPI IPNLSEEVDKRLWDKNIYEQFWKDDKDKKSCEELYRENVASLYKGDPKFASSLSMPVI SLK PDHKYRGTITGEEAIRVKEIDGKLIKLKRKSISEITAESINSIYTDDKILIDSLKTIFEQ ADYK DVGDYLKKTNQHFFTTSSGKRVNKVTVIEKVPSRWLRKEIDDNNFSLLNDSSYYCIELYK DSKG DNNLQGIAMSDIVHDRKTKKLYLKPDFNYPDDYYTHVMYIFPGDYLRIKSTSKKSGEQLK FEGY FI SVKNVNENSFRFI SDNKPCAKDKRVS ITKKDIVIKLAVDLMGKVQGENNGKGI SCGEPLSLL KEKN SEQ ID NO: 350

MLSRQLLGASHLARPVSYSYNVQDNDVHCSYGERCFMRGKRYRIGIDVGLNSVGLAAVEV SDEN SPVRLLNAQSVIHDGGVDPQKNKEAITRKNMSGVARRTRRMRRRKRERLHKLDMLLGKFG YPVI EPESLDKPFEEWHVRAELATRYIEDDELRRES I S IALRHMARHRGWRNPYRQVDSLI SDNPYSK QYGELKEKAKAYNDDATAAEEESTPAQLVVAMLDAGYAEAPRLRWRTGSKKPDAEGYLPV RLMQ EDNANELKQIFRVQRVPADEWKPLFRSVFYAVSPKGSAEQRVGQDPLAPEQARALKASLA FQEY RIANVITNLRIKDASAELRKLTVDEKQS IYDQLVSPSSEDITWSDLCDFLGFKRSQLKGVGSLT EDGEERI SSRPPRLTSVQRIYESDNKIRKPLVAWWKSASDNEHEAMIRLLSNTVDIDKVREDVA YASAIEFIDGLDDDALTKLDSVDLPSGRAAYSVETLQKLTRQMLTTDDDLHEARKTLFNV TDSW RPPADPIGEPLGNPSVDRVLKNVNRYLMNCQQRWGNPVSVNIEHVRSSFSSVAFARKDKR EYEK NNEKRS IFRSSLSEQLRADEQMEKVRESDLRRLEAIQRQNGQCLYCGRTITFRTCEMDHIVPRK GVGSTNTRTNFAAVCAECNRMKSNTPFAIWARSEDAQTRGVSLAEAKKRVTMFTFNPKSY APRE VKAFKQAVIARLQQTEDDAAIDNRS IESVAWMADELHRRIDWYFNAKQYVNSAS IDDAEAETMK TTVSVFQGRVTASARRAAGIEGKIHFIGQQSKTRLDRRHHAVDASVIAMMNTAAAQTLME RESL RESQRLIGLMPGERSWKEYPYEGTSRYESFHLWLDNMDVLLELLNDALDNDRIAVMQSQR YVLG NSIAHDATIHPLEKVPLGSAMSADLIRRASTPALWCALTRLPDYDEKEGLPEDSHREIRV HDTR YSADDEMGFFASQAAQIAVQEGSADIGSAIHHARVYRCWKTNAKGVRKYFYGMIRVFQTD LLRA CHDDLFTVPLPPQS I SMRYGEPRVVQALQSGNAQYLGSLVVGDEIEMDFSSLDVDGQIGEYLQF FSQFSGGNLAWKHWVVDGFFNQTQLRIRPRYLAAEGLAKAFSDDVVPDGVQKIVTKQGWL PPVN TASKTAVRIVRRNAFGEPRLSSAHHMPCSWQWRHE

SEQ ID NO: 351

MYS IGLDLGI SSVGWSVIDERTGNVIDLGVRLFSAKNSEKNLERRTNRGGRRLIRRKTNRLKDA KKILAAVGFYEDKSLKNSCPYQLRVKGLTEPLSRGEIYKVTLHILKKRGI SYLDEVDTEAAKES QDYKEQVRKNAQLLTKYTPGQIQLQRLKENNRVKTGINAQGNYQLNVFKVSAYANELATI LKTQ QAFYPNELTDDWIALFVQPGIAEEAGLIYRKRPYYHGPGNEANNSPYGRWSDFQKTGEPA TNIF DKLIGKDFQGELRASGLSLSAQQYNLLNDLTNLKIDGEVPLSSEQKEYILTELMTKEFTR FGVN DVVKLLGVKKERLSGWRLDKKGKPEIHTLKGYRNWRKIFAEAGIDLATLPTETIDCLAKV LTLN TEREGIENTLAFELPELSESVKLLVLDRYKELSQS I STQSWHRFSLKTLHLLIPELMNATSEQN TLLEQFQLKSDVRKRYSEYKKLPTKDVLAEIYNPTVNKTVSQAFKVIDALLVKYGKEQIR YITI EMPRDDNEEDEKKRIKELHAKNSQRKNDSQSYFMQKSGWSQEKFQTTIQKNRRFLAKLLY YYEQ DGICAYTGLPI SPELLVSDSTEIDHI IPI S I SLDDS INNKVLVLSKANQVKGQQTPYDAWMDGS FKKINGKFSNWDDYQKWVESRHFSHKKENNLLETRNIFDSEQVEKFLARNLNDTRYASRL VLNT LQSFFTNQETKVRVVNGSFTHTLRKKWGADLDKTRETHHHHAVDATLCAVTSFVKVSRYH YAVK EETGEKVMREIDFETGEIVNEMSYWEFKKSKKYERKTYQVKWPNFREQLKPVNLHPRIKF SHQV DRKANRKLSDATIYSVREKTEVKTLKSGKQKITTDEYTIGKIKDIYTLDGWEAFKKKQDK LLMK DLDEKTYERLLS IAETTPDFQEVEEKNGKVKRVKRSPFAVYCEENDIPAIQKYAKKNNGPLIRS LKYYDGKLNKHINITKDSQGRPVEKTKNGRKVTLQSLKPYRYDIYQDLETKAYYTVQLYY SDLR FVEGKYGITEKEYMKKVAEQTKGQVVRFCFSLQKNDGLEIEWKDSQRYDVRFYNFQSANS INFK GLEQEMMPAENQFKQKPYNNGAINLNIAKYGKEGKKLRKFNTDILGKKHYLFYEKEPKNI IK

SEQ ID NO: 352

MYFYKNKENKLNKKVVLGLDLGIASVGWCLTDI SQKEDNKFPI ILHGVRLFETVDDSDDKLLNE TRRKKRGQRRRNRRLFTRKRDFIKYLIDNNI IELEFDKNPKILVRNFIEKYINPFSKNLELKYK SVTNLPIGFHNLRKAAINEKYKLDKSELIVLLYFYLSLRGAFFDNPEDTKSKEMNKNEIE IFDK NES IKNAEFPIDKI IEFYKI SGKIRSTINLKFGHQDYLKEIKQVFEKQNIDFMNYEKFAMEEKS FFSRIRNYSEGPGNEKSFSKYGLYANENGNPELI INEKGQKIYTKIFKTLWESKIGKCSYDKKL YRAPKNSFSAKVFDITNKLTDWKHKNEYI SERLKRKILLSRFLNKDSKSAVEKILKEE IKFEN LSEIAYNKDDNKINLPI INAYHSLTTIFKKHLINFENYLI SNENDLSKLMSFYKQQSEKLFVPN EKGSYEINQNNNVLHIFDAISNILNKFSTIQDRIRILEGYFEFSNLKKDVKSSEIYSEIA KLRE FSGTSSLSFGAYYKFIPNLISEGSKNYSTISYEEKALQNQKNNFSHSNLFEKTWVEDLIA SPTV KRSLRQTMNLLKEIFKYSEKNNLEIEKIVVEVTRSSNNKHERKKIEGINKYRKEKYEELK KVYD LPNENTTLLKKLWLLRQQQGYDAYSLRKIEANDVINKPWNYDIDHIVPRS I SFDDSFSNLVIVN KLDNAKKSNDLSAKQFIEKIYGIEKLKEAKENWGNWYLRNANGKAFNDKGKFIKLYTIDN LDEF DNSDFINRNLSDTSYITNALVNHLTFSNSKYKYSVVSVNGKQTSNLRNQIAFVGIKNNKE TERE WKRPEGFKSINSNDFLIREEGKNDVKDDVLIKDRSFNGHHAEDAYFITI ISQYFRSFKRIERLN VNYRKETRELDDLEKNNIKFKEKASFDNFLLINALDELNEKLNQMRFSRMVITKKNTQLF NETL YSGKYDKGKNTIKKVEKLNLLDNRTDKIKKIEEFFDEDKLKENELTKLHIFNHDKNLYET LKII WNEVKIEIKNKNLNEKNYFKYFVNKKLQEGKI SFNEWVPILDNDFKI IRKIRYIKFSSEEKETD EI IFSQSNFLKIDQRQNFSFHNTLYWVQIWVYKNQKDQYCFI S IDARNSKFEKDEIKINYEKLK TQKEKLQI INEEPILKINKGDLFENEEKELFYIVGRDEKPQKLEIKYILGKKIKDQKQIQKPVK KYFPNWKKVNLTYMGEIFKK

SEQ ID NO: 353

MDNKNYRIGIDVGLNS IGFCAVEVDQHDTPLGFLNLSVYRHDAGIDPNGKKTNTTRLAMSGVAR RTRRLFRKRKRRLAALDRFIEAQGWTLPDHADYKDPYTPWLVRAELAQTPIRDENDLHEK LAIA VRHIARHRGWRSPWVPVRSLHVEQPPSDQYLALKERVEAKTLLQMPEGATPAEMVVALDL SVDV NLRPKNREKTDTRPENKKPGFLGGKLMQSDNANELRKIAKIQGLDDALLRELIELVFAAD SPKG ASGELVGYDVLPGQHGKRRAEKAHPAFQRYRIAS IVSNLRIRHLGSGADERLDVETQKRVFEYL LNAKPTADITWSDVAEEIGVERNLLMGTATQTADGERASAKPPVDVTNVAFATCKIKPLK EWWL NADYEARCVMVSALSHAEKLTEGTAAEVEVAEFLQNLSDEDNEKLDSFSLPIGRAAYSVD SLER LTKRMIENGEDLFEARVNEFGVSEDWRPPAEPIGARVGNPAVDRVLKAVNRYLMAAEAEW GAPL SV IEHVREGFI SKRQAVEIDRENQKRYQRNQAVRSQIADHINATSGVRGSDVTRYLAIQRQNG ECLYCGTAITFVNSEMDHIVPRAGLGSTNTRDNLVATCERCNKSKSNKPFAVWAAECGIP GVSV AEALKRVDFWIADGFASSKEHRELQKGVKDRLKRKVSDPEIDNRSMESVAWMARELAHRV QYYF DEKHTGTKVRVFRGSLTSAARKASGFESRVNFIGGNGKTRLDRRHHAMDAATVAMLRNSV AKTL VLRGNIRASERAIGAAETWKSFRGENVADRQIFESWSENMRVLVEKFNLALYNDEVS IFSSLRL QLGNGKAHDDTITKLQMHKVGDAWSLTEIDRASTPALWCALTRQPDFTWKDGLPANEDRT IIVN GTHYGPLDKVGIFGKAAASLLVRGGSVDIGSAIHHARIYRIAGKKPTYGMVRVFAPDLLR YRNE DLFNVELPPQSVSMRYAEPKVREAIREGKAEYLGWLVVGDELLLDLSSETSGQIAELQQD FPGT THWTVAGFFSPSRLRLRPVYLAQEGLGEDVSEGSKS I IAGQGWRPAVNKVFGSAMPEVIRRDGL GRKRRFSYSGLPVSWQG

SEQ ID NO: 354

MRLGLDIGTSS IGWWLYETDGAGSDARITGVVDGGVRIFSDGRDPKSGASLAVDRRAARAMRRR RDRYLRRRATLMKVLAETGLMPADPAEAKALEALDPFALRAAGLDEPLPLPHLGRALFHL NQRR GFKSNRKTDRGDNESGKIKDATARLDMEMMANGARTYGEFLHKRRQKATDPRHVPSVRTR LSIA NRGGPDGKEEAGYDFYPDRRHLEEEFHKLWAAQGAHHPELTETLRDLLFEKIFFQRPLKE PEVG LCLFSGHHGVPPKDPRLPKAHPLTQRRVLYETVNQLRVTADGREARPLTREERDQVIHAL DNKK PTKSLSSMVLKLPALAKVLKLRDGERFTLETGVRDAIACDPLRASPAHPDRFGPRWS ILDADAQ WEVI SRIRRVQSDAEHAALVDWLTEAHGLDRAHAEATAHAPLPDGYGRLGLTATTRILYQLTAD VVTYADAVKACGWHHSDGRTGECFDRLPYYGEVLERHVIPGSYHPDDDDITRFGRITNPT VHIG LNQLRRLVNRI IETHGKPHQIVVELARDLKKSEEQKRADIKRIRDTTEAAKKRSEKLEELEIED NGRNRMLLRLWEDLNPDDAMRRFCPYTGTRI SAAMIFDGSCDVDHILPYSRTLDDSFPNRTLCL REANRQKRNQTPWQAWGDTPHWHAIAANLKNLPENKRWRFAPDAMTRFEGENGFLDRALK DTQY LARI SRSYLDTLFTKGGHVWVVPGRFTEMLRRHWGLNSLLSDAGRGAVKAKNRTDHRHHAIDAA VIAATDPGLLNRI SRAAGQGEAAGQSAELIARDTPPPWEGFRDDLRVRLDRI IVSHRADHGRID HAARKQGRDSTAGQLHQETAYS IVDDIHVASRTDLLSLKPAQLLDEPGRSGQVRDPQLRKALRV ATGGKTGKDFENALRYFASKPGPYQAIRRVRI IKPLQAQARVPVPAQDPIKAYQGGSNHLFEIW RLPDGEIEAQVITSFEAHTLEGEKRPHPAAKRLLRVHKGDMVALERDGRRVVGHVQKMDI ANGL FIVPHNEANADTRNNDKSDPFKWIQIGARPAIASGIRRVSVDEIGRLRDGGTRPI

SEQ ID NO: 355

MLHCIAVIRVPPSEEPGFFETHADSCALCHHGCMTYAANDKAIRYRVGIDVGLRS IGFCAVEVD DEDHPIRILNSVVHVHDAGTGGPGETESLRKRSGVAARARRRGRAEKQRLKKLDVLLEEL GWGV SSNELLDSHAPWHIRKRLVSEYIEDETERRQCLSVAMAHIARHRGWRNSFSKVDTLLLEQ APSD RMQGLKERVEDRTGLQFSEEVTQGELVATLLEHDGDVTIRGFVRKGGKATKVHGVLEGKY MQSD LVAELRQICRTQRVSETTFEKLVLS IFHSKEPAPSAARQRERVGLDELQLALDPAAKQPRAERA HPAFQKFKVVATLANMRIREQSAGERSLTSEELNRVARYLLNHTESESPTWDDVARKLEV PRHR LRGSSRASLETGGGLTYPPVDDTTVRVMSAEVDWLADWWDCANDESRGHMIDAI SNGCGSEPDD VEDEEVNELI SSATAEDMLKLELLAKKLPSGRVAYSLKTLREVTAAILETGDDLSQAITRLYGV DPGWVPTPAPIEAPVGNPSVDRVLKQVARWLKFASKRWGVPQTVNIEHTREGLKSASLLE EERE RWERFEARREIRQKEMYKRLGI SGPFRRSDQVRYEILDLQDCACLYCGNEINFQTFEVDHI IPR VDASSDSRRTNLAAVCHSCNSAKGGLAFGQWVKRGDCPSGVSLENAIKRVRSWSKDRLGL TEKA MGKRKSEVI SRLKTEMPYEEFDGRSMESVAWMAIELKKRIEGYFNSDRPEGCAAVQVNAYSGRL TACARRAAHVDKRVRLIRLKGDDGHHKNRFDRRNHAMDALVIALMTPAIARTIAVREDRR EAQQ LTRAFESWKNFLGSEERMQDRWESWIGDVEYACDRLNELIDADKIPVTENLRLRNSGKLH ADQP ESLKKARRGSKRPRPQRYVLGDALPADVINRVTDPGLWTALVRAPGFDSQLGLPADLNRG LKLR GKRI SADFPIDYFPTDSPALAVQGGYVGLEFHHARLYRI IGPKEKVKYALLRVCAIDLCGIDCD DLFEVELKPSS I SMRTADAKLKEAMGNGSAKQIGWLVLGDEIQIDPTKFPKQS IGKFLKECGPV SSWRVSALDTPSKITLKPRLLSNEPLLKTSRVGGHESDLVVAECVEKIMKKTGWVVEINA LCQS GLIRVIRRNALGEVRTSPKSGLPI SLNLR SEQ ID NO: 356

MRYRVGLDLGTASVGAAVFSMDEQGNPMELIWHYERLFSEPLVPDMGQLKPKKAARRLAR QQRR QIDRRASRLRRIAIVSRRLGIAPGRNDSGVHGNDVPTLRAMAVNERIELGQLRAVLLRMG KKRG YGGTFKAVRKVGEAGEVASGASRLEEEMVALASVQNKDSVTVGEYLAARVEHGLPSKLKV AANN EYYAPEYALFRQYLGLPAIKGRPDCLPNMYALRHQIEHEFERIWATQSQFHDVMKDHGVK EEIR NAIFFQRPLKSPADKVGRCSLQTNLPRAPRAQIAAQNFRIEKQMADLRWGMGRRAEMLND HQKA VIRELLNQQKELSFRKIYKELERAGCPGPEGKGLNMDRAALGGRDDLSGNTTLAAWRKLG LEDR WQELDEVTQIQVINFLADLGSPEQLDTDDWSCRFMGKNGRPRNFSDEFVAFMNELRMTDG FDRL SKMGFEGGRSSYS IKALKALTEWMIAPHWRETPETHRVDEEAAIRECYPESLATPAQGGRQSKL EPPPLTGNEVVDVALRQVRHTINMMIDDLGSVPAQIVVEMAREMKGGVTRRNDIEKQNKR FASE RKKAAQS IEENGKTPTPARILRYQLWIEQGHQCPYCESNI SLEQALSGAYTNFEHILPRTLTQI GRKRSELVLAHRECNDEKGNRTPYQAFGHDDRRWRIVEQRANALPKKSSRKTRLLLLKDF EGEA LTDES IDEFADRQLHESSWLAKVTTQWLSSLGSDVYVSRGSLTAELRRRWGLDTVIPQVRFESG MPVVDEEGAEITPEEFEKFRLQWEGHRVTREMRTDRRPDKRIDHRHHLVDAIVTALTSRS LYQQ YAKAWKVADEKQRHGRVDVKVELPMPILTIRDIALEAVRSVRI SHKPDRYPDGRFFEATAYGIA QRLDERSGEKVDWLVSRKSLTDLAPEKKS IDVDKVRANI SRIVGEAIRLHI SNIFEKRVSKGMT PQQALREPIEFQGNILRKVRCFYSKADDCVRIEHSSRRGHHYKMLLNDGFAYMEVPCKEG ILYG VPNLVRPSEAVGIKRAPESGDFIRFYKGDTVKNIKTGRVYTIKQILGDGGGKLILTPVTE TKPA DLLSAKWGRLKVGGRNIHLLRLCAE

SEQ ID NO: 357

MIGEHVRGGCLFDDHWTPNWGAFRLPNTVRTFTKAENPKDGSSLAEPRRQARGLRRR LRRKTQR LEDLRRLLAKEGVLSLSDLETLFRETPAKDPYQLRAEGLDRPLSFPEWVRVLYHITKHRG FQSN RRNPVEDGQERSRQEEEGKLLSGVGENERLLREGGYRTAGEMLARDPKFQDHRRNRAGDY SHTL SRSLLLEEARRLFQSQRTLGNPHASSNLEEAFLHLVAFQNPFASGEDIRNKAGHCSLEPD QIRA PRRSASAETFMLLQKTGNLRLIHRRTGEERPLTDKEREQIHLLAWKQEKVTHKTLRRHLE IPEE WLFTGLPYHRSGDKAEEKLFVHLAGIHEIRKALDKGPDPAVWDTLRSRRDLLDS IADTLTFYKN EDEILPRLESLGLSPENARALAPLSFSGTAHLSLSALGKLLPHLEEGKSYTQARADAGYA APPP DRHPKLPPLEEADWRNPVVFRALTQTRKVVNALVRRYGPPWCIHLETARELSQPAKVRRR IETE QQANEKKKQQAEREFLDIVGTAPGPGDLLKMRLWREQGGFCPYCEEYLNPTRLAEPGYAE MDHI LPYSRSLDNGWHNRVLVHGKDNRDKGNRTPFEAFGGDTARWDRLVAWVQASHLSAPKKRN LLRE DFGEEAERELKDRNLTDTRFITKTAATLLRDRLTFHPEAPKDPVMTLNGRLTAFLRKQWG LHKN RKNGDLHHALDAAVLAVASRSFVYRLSSHNAAWGELPRGREAENGFSLPYPAFRSEVLAR LCPT REEILLRLDQGGVGYDEAFRNGLRPVFVSRAPSRRLRGKAHMETLRSPKWKDHPEGPRTA SRIP LKDLNLEKLERMVGKDRDRKLYEALRERLAAFGGNGKKAFVAPFRKPCRSGEGPLVRSLR IFDS GYSGVELRDGGEVYAVADHESMVRVDVYAKKNRFYLVPVYVADVARGIVKNRAIVAHKSE EEWD LVDGSFDFRFSLFPGDLVEIEKKDGAYLGYYKSCHRGDGRLLLDRHDRMPRESDCGTFYV STRK DVLSMSKYQVDPLGEIRLVGSEKPPFVL

SEQ ID NO: 358

MEKKRKVTLGFDLGIASVGWAIVDSETNQVYKLGSRLFDAPDTNLERRTQRGTRRLLRRR KYRN QKFYNLVKRTEVFGLSSREAIENRFRELS IKYPNI IELKTKALSQEVCPDEIAWILHDYLKNRG YFYDEKETKEDFDQQTVESMPSYKLNEFYKKYGYFKGALSQPTESEMKDNKDLKEAFFFD FSNK EWLKEINYFFNVQKNILSETFIEEFKKIFSFTRDI SKGPGSDNMPSPYGIFGEFGDNGQGGRYE HIWDKNIGKCS IFTNEQRAPKYLPSALIFNFLNELANIRLYSTDKKNIQPLWKLSSVDKLNILL NLFNLPI SEKKKKLTSTNINDIVKKES IKS IMI SVEDIDMIKDEWAGKEPNVYGVGLSGLNIEE SAKENKFKFQDLKILNVLINLLDNVGIKFEFKDRNDI IKNLELLDNLYLFLIYQKESNNKDSS I DLFIAKNESLNIENLKLKLKEFLLGAGNEFENHNSKTHSLSKKAIDEILPKLLDNNEGWN LEAI KNYDEEIKSQIEDNSSLMAKQDKKYLNDNFLKDAILPPNVKVTFQQAILIFNKI IQKFSKDFEI DKVVIELAREMTQDQENDALKGIAKAQKSKKSLVEERLEANNIDKSVFNDKYEKLIYKIF LWI S QDFKDPYTGAQI SVNEIVNNKVEIDHI IPYSLCFDDSSANKVLVHKQSNQEKSNSLPYEYIKQG HSGWNWDEFTKYVKRVFVNNVDS ILSKKERLKKSENLLTASYDGYDKLGFLARNLNDTRYATIL FRDQLNNYAEHHLIDNKKMFKVIAMNGAVTSFIRKNMSYDNKLRLKDRSDFSHHAYDAAI IALF SNKTKTLYNLIDPSLNGI I SKRSEGYWVIEDRYTGEIKELKKEDWTS IKNNVQARKIAKEIEEY LIDLDDEVFFSRKTKRKTNRQLYNETIYGIATKTDEDGITNYYKKEKFS ILDDKDIYLRLLRER EKFVINQSNPEVIDQI IEI IESYGKENNIPSRDEAINIKYTKNKINYNLYLKQYMRSLTKSLDQ FSEEFINQMIANKTFVLYNPTKNTTRKIKFLRLVNDVKINDIRKNQVINKFNGKNNEPKA FYEN INSLGAIVFKNSANNFKTLS INTQIAIFGDKNWDIEDFKTYNMEKIEKYKEIYGIDKTYNFHSF IFPGTILLDKQNKEFYYI SS IQTVRDI IEIKFLNKIEFKDENKNQDTSKTPKRLMFGIKS IMNN YEQVDI SPFGINKKIFE SEQ ID NO: 359

MGYRIGLDVGITSTGYAVLKTDKNGLPYKILTLDSVIYPRAENPQTGASLAEPRRIKRGL RRRT RRTKFRKQRTQQLFIHSGLLSKPEIEQILATPQAKYSVYELRVAGLDRRLTNSELFRVLY FFIG HRGFKSNRKAELNPENEADKKQMGQLLNS IEEIRKAIAEKGYRTVGELYLKDPKYNDHKRNKGY IDGYLSTPNRQMLVDEIKQILDKQRELGNEKLTDEFYATYLLGDENRAGIFQAQRDFDEG PGAG PYAGDQIKKMVGKDIFEPTEDRAAKATYTFQYFNLLQKMTSLNYQNTTGDTWHTLNGLDR QAII DAVFAKAEKPTKTYKPTDFGELRKLLKLPDDARFNLVNYGSLQTQKEIETVEKKTRFVDF KAYH DLVKVLPEEMWQSRQLLDHIGTALTLYSSDKRRRRYFAEELNLPAELIEKLLPLNFSKFG HLS I KSMQNI IPYLEMGQVYSEATTNTGYDFRKKQI SKDTIREEITNPVVRRAVTKTIKIVEQI IRRY GKPDGI IELARELGRNFKERGDIQKRQDKNRQTNDKIAAELTELGIPVNGQ I IRYKLHKEQN GVDPYTGDQIPFERAFSEGYEVDHI IPYS I SWDDSYTNKVLTSAKCNREKGNRIPMVYLANNEQ RLNALT IAD I IRNSRKRQKLLKQKLSDEELKDWKQR INDTRFITRVLYNYFRQAIEFNPEL EKKQRVLPLNGEVTSKIRSRWGFLKVREDGDLHHAIDATVIAAITPKFIQQVTKYSQHQE VKNN QALWHDAEIKDAEYAAEAQRMDADLFNKIFNGFPLPWPEFLDELLARI SDNPVEMMKSRSWNTY TPIEIAKLKPVFVVRLANHKI SGPAHLDTIRSAKLFDEKGIVLSRVS ITKLKINKKGQVATGDG IYDPENSNNGDKVVYSAIRQALEAHNGSGELAFPDGYLEYVDHGTKKLVRKVRVAKKVSL PVRL KNKAAADNGSMVRIDVFNTGKKFVFVPIYIKDTVEQVLPNKAIARGKSLWYQITESDQFC FSLY PGDMVHIESKTGIKPKYSNKENNTSVVPIKNFYGYFDGADIATAS ILVRAHDSSYTARS IGIAG LLKFEKYQVDYFGRYHKVHEKKRQLFVKRDE

SEQ ID NO: 360

MQKNINTKQNHIYIKQAQKIKEKLGDKPYRIGLDLGVGS IGFAIVSMEENDGNVLLPKEI IMVG SRIFKASAGAADRKLSRGQRNNHRHTRERMRYLWKVLAEQKLALPVPADLDRKENSSEGE TSAK RFLGDVLQKDIYELRVKSLDERLSLQELGYVLYHIAGHRGSSAIRTFENDSEEAQKENTE NKKI AG IKRLMAKKNYRTYGEYLYKEFFENKEKHKREKI SNAANNHKFSPTRDLVIKEAEAILKKQA GKDGFHKELTEEYIEKLTKAIGYESEKLIPESGFCPYLKDEKRLPASHKLNEERRLWETL NNAR YSDPIVDIVTGEITGYYEKQFTKEQKQKLFDYLLTGSELTPAQTKKLLGLKNTNFEDI ILQGRD KKAQKIKGYKLIKLESMPFWARLSEAQQDSFLYDWNSCPDEKLLTEKLSNEYHLTEEEID NAFN EIVLSSSYAPLGKSAMLI ILEKIKNDLSYTEAVEEALKEGKLTKEKQAIKDRLPYYGAVLQEST QKI IAKGFSPQFKDKGYKTPHTNKYELEYGRIANPVVHQTLNELRKLVNEI IDILGKKPCEIGL ETARELKKSAEDRSKLSREQNDNESNRNRIYEIYIRPQQQVI ITRRENPRNYILKFELLEEQKS QCPFCGGQI SPNDI INNQADIEHLFPIAESEDNGRNNLVI SHSACNADKAKRSPWAAFASAAKD SKYDYNRILSNVKE IPHKAWRFNQGAFEKFIENKPMAARFKTDNSYI SKVAHKYLACLFEKPN IICVKGSLTAQLRMAWGLQGLMIPFAKQLITEKESESFNKDVNSNKKIRLDNRHHALDAI VIAY ASRGYGNLLNKMAGKDYKINYSERNWLSKILLPPNNIVWENIDADLESFESSVKTALKNA FISV KHDHSDNGELVKGTMYKIFYSERGYTLTTYKKLSALKLTDPQKKKTPKDFLETALLKFKG RESE MKNEKIKSAIENNKRLFDVIQDNLEKAKKLLEEENEKSKAEGKKEKNINDAS IYQKAI SLSGDK YVQLSKKEPGKFFAI SKPTPTTTGYGYDTGDSLCVDLYYDNKGKLCGEI IRKIDAQQKNPLKYK EQGFTLFERIYGGDILEVDFDIHSDKNSFRNNTGSAPENRVFIKVGTFTEITNNNIQIWF GNI I KSTGGQDDSFTINSMQQYNPRKLILSSCGFIKYRSPILKNKEG

SEQ ID NO: 361

MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDS LAMARRL ARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTP LEWS AVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKE SGHI RNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQ KMLG HCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLT YAQA RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAI SRALEKEGLKDKKSPLNLSPELQDEIGT AFSLFKTDEDITGRLKDRIQPEILEALLKHI SFDKFVQI SLKALRRIVPLMEQGKRYDEACAEI YGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETARE VGKS FKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKE INLG RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK ARVE TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG QITN LLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGE VLHQ KTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPL FVSR APNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARL EAHK DDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKG DKYY LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYF ASCH RGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR

SEQ ID NO: 362

MQTTNLSYILGLDLGIASVGWAVVEINENEDPIGLIDVGVRIFERAEVPKTGESLALSRR LARS TRRLIRRRAHRLLLAKRFLKREGILSTIDLEKGLPNQAWELRVAGLERRLSAIEWGAVLL HLIK HRGYLSKRKNESQTNNKELGALLSGVAQNHQLLQSDDYRTPAELALKKFAKEEGHIRNQR GAYT HTFNRLDLLAELNLLFAQQHQFGNPHCKEHIQQYMTELLMWQKPALSGEAILKMLGKCTH EKNE FKAAKHTYSAERFVWLTKLNNLRILEDGAERALNEEERQLLINHPYEKSKLTYAQVRKLL GLSE QAIFKHLRYSKENAESATFMELKAWHAIRKALENQGLKDTWQDLAKKPDLLDEIGTAFSL YKTD EDIQQYLTNKVPNSVINALLVSLNFDKFIELSLKSLRKILPLMEQGKRYDQACREIYGHH YGEA NQKTSQLLPAIPAQEIRNPVVLRTLSQARKVINAI IRQYGSPARVHIETGRELGKSFKERREIQ KQQEDNRTKRESAVQKFKELFSDFSSEPKSKDILKFRLYEQQHGKCLYSGKEINIHRLNE KGYV EIDHALPFSRTWDDSFNNKVLVLASENQNKGNQTPYEWLQGKINSERWKNFVALVLGSQC SAAK KQRLLTQVIDDNKFIDRNLNDTRYIARFLSNYIQENLLLVGKNKKNVFTPNGQITALLRS RWGL IKARENNNRHHALDAIVVACATPSMQQKITRFIRFKEVHPYKIENRYEMVDQESGEI I SPHFPE PWAYFRQEVNIRVFDNHPDTVLKEMLPDRPQANHQFVQPLFVSRAPTRKMSGQGHMETIK SAKR LAEGI SVLRIPLTQLKPNLLENMVNKEREPALYAGLKARLAEFNQDPAKAFATPFYKQGGQQVK AIRVEQVQKSGVLVRENNGVADNAS IVRTDVFIKNNKFFLVPIYTWQVAKGILPNKAIVAHKNE DEWEEMDEGAKFKFSLFPNDLVELKTKKEYFFGYYIGLDRATG I SLKEHDGEI SKGKDGVYRV GVKLALSFEKYQVDELGKNRQICRPQQRQPVR SEQ ID NO: 363

MGIRFAFDLGTNS IGWAVWRTGPGVFGEDTAASLDGSGVLIFKDGRNPKDGQSLATMRRVPRQS RKRRDRFVLRRRDLLAALRKAGLFPVDVEEGRRLAATDPYHLRAKALDESLTPHEMGRVI FHLN QRRGFRSNRKADRQDREKGKIAEGSKRLAETLAATNCRTLGEFLWSRHRGTPRTRSPTRI RMEG EGAKALYAFYPTREMVRAEFERLWTAQSRFAPDLLTPERHEEIAGILFRQRDLAPPKIGC CTFE PSERRLPRALPSVEARGIYERLAHLRITTGPVSDRGLTRPERDVLASALLAGKSLTFKAV RKTL KILPHALVNFEEAGEKGLDGALTAKLLSKPDHYGAAWHGLSFAEKDTFVGKLLDEADEER LIRR LVTENRLSEDAARRCAS IPLADGYGRLGRTANTEILAALVEETDETGTVVTYAEAVRRAGERTG RNWHHSDERDGVILDRLPYYGEILQRHVVPGSGEPEEKNEAARWGRLANPTVHIGLNQLR KVVN RLIAAHGRPDQIVVELARELKLNREQKERLDRENRKNREENERRTAILAEHGQRDTAENK IRLR LFEEQARANAGIALCPYTGRAIGIAELFTSEVEIDHILPVSLTLDDSLANRVLCRREANR EKRR QTPFQAFGATPAWNDIVARAAKLPPNKRWRFDPAALERFEREGGFLGRQLNETKYLSRLA KIYL GKICDPDRVYVTPGTLTGLLRARWGLNS ILSDSNFKNRSDHRHHAVDAVVIGVLTRGMIQRIAH DAARAEDQDLDRVFRDVPVPFEDFRDHVRERVSTITVAVKPEHGKGGALHEDTSYGLVPD TDPN AALGNLVVRKPIRSLTAGEVDRVRDRALRARLGALAAPFRDESGRVRDAKGLAQALEAFG AENG IRRVRILKPDASVVTIADRRTGVPYRAVAPGENHHVDIVQMRDGSWRGFAASVFEVNRPG WRPE WEVKKLGGKLVMRLHKGDMVELSDKDGQRRVKVVQQIEI SANRVRLSPHNDGGKLQDRHADADD PFRWDLATIPLLKDRGCVAVRVDPIGVVTLRRSNV SEQ ID NO: 364

MMEVFMGRLVLGLDIGITSVGFGI IDLDESEIVDYGVRLFKEGTAAENETRRTKRGGRRLKRRR VTRREDMLHLLKQAGI I STSFHPLNNPYDVRVKGLNERLNGEELATALLHLCKHRGSSVETIED DEAKAKEAGETKKVLSMNDQLLKSGKYVCEIQKERLRTNGHIRGHENNFKTRAYVDEAFQ ILSH QDLSNELKSAI ITI I SRKRMYYDGPGGPLSPTPYGRYTYFGQKEPIDLIEKMRGKCSLFPNEPR APKLAYSAELFNLLNDLNNLS IEGEKLTSEQKAMILKIVHEKGKITPKQLAKEVGVSLEQIRGF RIDTKGSPLLSELTGYKMIREVLEKSNDEHLEDHVFYDEIAEILTKTKDIEGRKKQI SELSSDL NEESVHQLAGLTKFTAYHSLSFKALRLINEEMLKTELNQMQS ITLFGLKQNNELSVKGMK IQA DDTAILSPVAKRAQRETFKVVNRLREIYGEFDS IVVEMAREKNSEEQRKAIRERQKFFEMRNKQ VADI IGDDRKINAKLREKLVLYQEQDGKTAYSLEPIDLKLLIDDPNAYEVDHI IPI S I SLDDS I TNKVLVTHRENQEKGNLTPI SAFVKGRFTKGSLAQYKAYCLKLKEK IKTNKGYRKKVEQYLLN ENDIYKYDIQKEFINRNLVDTSYASRVVLNTLTTYFKQNEIPTKVFTVKGSLTNAFRRKI NLKK DRDEDYGHHAIDALI IASMPKMRLLSTIFSRYKIEDIYDESTGEVFSSGDDSMYYDDRYFAFIA SLKAIKVRKFSHKIDTKPNRSVADETIYSTRVIDGKEKVVKKYKDIYDPKFTALAEDILN NAYQ EKYLMALHDPQTFDQIVKVVNYYFEEMSKSEKYFTKDKKGRIKI SGMNPLSLYRDEHGMLKKYS KKGDGPAITQMKYFDGVLGNHIDI SAHYQVRDKKVVLQQI SPYRTDFYYSKENGYKFVTIRYKD VRWSEKKKKYVIDQQDYAMKKAEKKIDDTYEFQFSMHRDELIGITKAEGEALIYPDETWH NFNF FFHAGETPEILKFTATNNDKSNKIEVKPIHCYCKMRLMPTI SKKIVRIDKYATDVVGNLYKVKK NTLKFEFD

SEQ ID NO: 365

MKKILGVDLGITSFGYAILQETGKDLYRCLDNSVVMRNNPYDEKSGESSQS IRSTQKSMRRLIE KRKKRIRCVAQTMERYGILDYSETMKINDPKNNPIKNRWQLRAVDAWKRPLSPQELFAIF AHMA KHRGYKS IATEDLIYELELELGLNDPEKESEKKADERRQVYNALRHLEELRKKYGGETIAQTIH RAVEAGDLRSYRNHDDYEKMIRREDIEEEIEKVLLRQAELGALGLPEEQVSELIDELKAC ITDQ EMPTIDESLFGKCTFYKDELAAPAYSYLYDLYRLYKKLADLNIDGYEVTQEDREKVIEWV EKKI AQGKNLKKITHKDLRKILGLAPEQKIFGVEDERIVKGKKEPRTFVPFFFLADIAKFKELF ASIQ KHPDALQIFRELAEILQRSKTPQEALDRLRALMAGKGIDTDDRELLELFKNKRSGTRELS HRYI LEALPLFLEGYDEKEVQRILGFDDREDYSRYPKSLRHLHLREGNLFEKEENPINNHAVKS LASW ALGLIADLSWRYGPFDEI ILETTRDALPEKIRKEIDKAMREREKALDKI IGKYKKEFPS IDKRL ARKIQLWERQKGLDLYSGKVINLSQLLDGSADIEHIVPQSLGGLSTDYNTIVTLKSVNAA KGNR LPGDWLAGNPDYRERIGMLSEKGLIDWKKRKNLLAQSLDEIYTENTHSKGIRATSYLEAL VAQV LKRYYPFPDPELRKNGIGVRMIPGKVTSKTRSLLGIKSKSRETNFHHAEDALILSTLTRG WQNR LHRMLRDNYGKSEAELKELWKKYMPHIEGLTLADYIDEAFRRFMSKGEESLFYRDMFDTI RS I S YWVDKKPLSASSHKETVYSSRHEVPTLRKNILEAFDSLNVIKDRHKLTTEEFMKRYDKEI RQKL WLHRIGNTNDESYRAVEERATQIAQILTRYQLMDAQNDKEIDEKFQQALKELITSPIEVT GKLL RKMRFVYDKLNAMQIDRGLVETDKNMLGIHI SKGPNEKLIFRRMDVNNAHELQKERSGILCYLN EMLFIFNKKGLIHYGCLRSYLEKGQGSKYIALFNPRFPANPKAQPSKFTSDSKIKQVGIG SATG I IKAHLDLDGHVRSYEVFGTLPEGS IEWFKEESGYGRVEDDPHH

SEQ ID NO: 366

MRPIEPWILGLDIGTDSLGWAVFSCEEKGPPTAKELLGGGVRLFDSGRDAKDHTSRQAER GAFR RARRQTRTWPWRRDRLIALFQAAGLTPPAAETRQIALALRREAVSRPLAPDALWAALLHL AHHR GFRSNRIDKRERAAAKALAKAKPAKATAKATAPAKEADDEAGFWEGAEAALRQRMAASGA PTVG ALLADDLDRGQPVRMRYNQSDRDGVVAPTRALIAEELAEIVARQSSAYPGLDWPAVTRLV LDQR PLRSKGAGPCAFLPGEDRALRALPTVQDFI IRQTLANLRLPSTSADEPRPLTDEEHAKALALLS TARFVEWPALRRALGLKRGVKFTAETERNGAKQAARGTAGNLTEAILAPLIPGWSGWDLD RKDR VFSDLWAARQDRSALLALIGDPRGPTRVTEDETAEAVADAIQIVLPTGRASLSAKAARAI AQAM APGIGYDEAVTLALGLHHSHRPRQERLARLPYYAAALPDVGLDGDPVGPPPAEDDGAAAE AYYG RIG I SVHIALNETRKIVNALLHRHGPILRLVMVETTRELKAGADERKRMIAEQAERERENAEI DVELRKSDRWMANARERRQRVRLARRQNNLCPYTSTPIGHADLLGDAYDIDHVIPLARGG RDSL DNMVLCQSDANKTKGDKTPWEAFHDKPGWIAQRDDFLARLDPQTAKALAWRFADDAGERV ARKS AEDEDQGFLPRQLTDTGYIARVALRYLSLVTNEPNAVVATNGRLTGLLRLAWDITPGPAP RDLL PTPRDALRDDTAARRFLDGLTPPPLAKAVEGAVQARLAALGRSRVADAGLADALGLTLAS LGGG GKNRADHRHHFIDAAMIAVTTRGLINQINQASGAGRILDLRKWPRTNFEPPYPTFRAEVM KQWD HIHPS IRPAHRDGGSLHAATVFGVRNRPDARVLVQRKPVEKLFLDANAKPLPADKIAEI IDGFA SPRMAKRFKALLARYQAAHPEVPPALAALAVARDPAFGPRGMTANTVIAGRSDGDGEDAG LITP FRANPKAAVRTMGNAVYEVWEIQVKGRPRWTHRVLTRFDRTQPAPPPPPENARLVMRLRR GDLV YWPLESGDRLFLVKKMAVDGRLALWPARLATGKATALYAQLSCPNINLNGDQGYCVQSAE GIRK EKIRTTSCTALGRLRLSKKAT

SEQ ID NO: 367

MKYTLGLDVGIASVGWAVIDKDNNKI IDLGVRCFDKAEESKTGESLATARRIARGMRRRI SRRS QRLRLVKKLFVQYEI IKDSSEFNRIFDTSRDGWKDPWELRYNALSRILKPYELVQVLTHITKRR GFKSNRKEDLSTTKEGVVITS IKNNSEMLRTKNYRTIGEMIFMETPENSNKRNKVDEYIHTIAR EDLLNEIKYIFS IQRKLGSPFVTEKLEHDFLNIWEFQRPFASGDS ILSKVGKCTLLKEELRAPT SCYTSEYFGLLQS INNLVLVEDNNTLTLNNDQRAKI IEYAHFKNEIKYSEIRKLLDIEPEILFK AHNLTHKNPSGNNESKKFYEMKSYHKLKSTLPTDIWGKLHSNKESLDNLFYCLTVYKNDN EIKD YLQANNLDYLIEYIAKLPTFNKFKHLSLVAMKRI IPFMEKGYKYSDACNMAELDFTGSSKLEKC NKLTVEPI IENVTNPVVIRALTQARKVINAI IQKYGLPYMV IELAREAGMTRQDRDNLKKEHE NNRKAREKI SDLIRQNGRVASGLDILKWRLWEDQGGRCAYSGKPIPVCDLLNDSLTQIDHIYPY SRSMDDSYMNKVLVLTDENQNKRSYTPYEVWGSTEKWEDFEARIYSMHLPQSKEKRLLNR NFIT KDLDSFI SRNLNDTRYI SRFLKNYIESYLQFSNDSPKSCVVCVNGQCTAQLRSRWGLNKNREES DLHHALDAAVIACADRKI IKEITNYYNERENHNYKVKYPLPWHSFRQDLMETLAGVFI SRAPRR KITGPAHDETIRSPKHFNKGLTSVKIPLTTVTLEKLETMVKNTKGGI SDKAVYNVLKNRLIEHN NKPLKAFAEKIYKPLKNGTNGAI IRS IRVETPSYTGVFRNEGKGI SDNSLMVRVDVFKKKDKYY LVPIYVAHMIKKELPSKAIVPLKPESQWELIDSTHEFLFSLYQNDYLVIKTKKGITEGYY RSCH RGTGSLSLMPHFANNKNVKIDIGVRTAI S IEKYNVDILGNKS IVKGEPRRGMEKYNSFKSN

SEQ ID NO: 368

MIRTLGIDIGIAS IGWAVIEGEYTDKGLENKEIVASGVRVFTKAENPKNKESLALPRTLARSAR RRNARKKGRIQQVKHYLSKALGLDLECFVQGEKLATLFQTSKDFLSPWELRERALYRVLD KEEL ARVILHIAKRRGYDDITYGVEDNDSGKIKKAIAENSKRIKEEQCKTIGEMMYKLYFQKSL NVRN KKESYNRCVGRSELREELKTIFQIQQELKSPWVNEELIYKLLGNPDAQSKQEREGLIFYQ RPLK GFGDKIGKCSHIKKGENSPYRACKHAPSAEEFVALTKS INFLKNLTNRHGLCFSQEDMCVYLGK ILQEAQKNEKGLTYSKLKLLLDLPSDFEFLGLDYSGKNPEKAVFLSLPSTFKLNKITQDR KTQD KIANILGANKDWEAILKELESLQLSKEQIQTIKDAKLNFSKHINLSLEALYHLLPLMREG KRYD EGVEILQERGIFSKPQPKNRQLLPPLSELAKEESYFDIPNPVLRRALSEFRKVVNALLEK YGGF HYFHIELTRDVCKAKSARMQLEKINKKNKSENDAASQLLEVLGLPNTYNNRLKCKLWKQQ EEYC LYSGEKITIDHLKDQRALQIDHAFPLSRSLDDSQSNKVLCLTSSNQEKSNKTPYEWLGSD EKKW DMYVGRVYSSNFSPSKKRKLTQKNFKERNEEDFLARNLVDTGYIGRVTKEYIKHSLSFLP LPDG KKEHIRI I SGSMTSTMRSFWGVQEKNRDHHLHHAQDAI I IACIEPSMIQKYTTYLKDKETHRLK SHQKAQILREGDHKLSLRWPMSNFKDKIQES IQNI IPSHHVSHKVTGELHQETVRTKEFYYQAF GGEEGVKKALKFGKIREINQGIVDNGAMVRVDIFKSKDKGKFYAVPIYTYDFAIGKLPNK AIVQ GKKNGI IKDWLEMDENYEFCFSLFKNDCIKIQTKEMQEAVLAIYKSTNSAKATIELEHLSKYAL KNEDEEKMFTDTDKEKNKTMTRESCGIQGLKVFQKVKLSVLGEVLEHKPRNRQNIALKTT PKHV SEQ ID NO: 369

MKYS IGLDIGIASVGWSVINKDKERIEDMGVRIFQKAENPKDGSSLASSRREKRGSRRRNRRKK HRLDRIK ILCESGLVKKNEIEKIYKNAYLKSPWELRAKSLEAKI SNKEIAQILLHIAKRRGFK SFRKTDRNADDTGKLLSGIQENKKIMEEKGYLTIGDMVAKDPKFNTHVRNKAGSYLFSFS RKLL EDEVRKIQAKQKELGNTHFTDDVLEKYIEVFNSQRNFDEGPSKPSPYYSEIGQIAKMIGN CTFE SSEKRTAKNTWSGERFVFLQKLNNFRIVGLSGKRPLTEEERDIVEKEVYLKKEVRYEKLR KILY LKEEERFGDLNYSKDEKQDKKTEKTKFISLIGNYTIKKLNLSEKLKSEIEEDKSKLDKI IEILT FNKSDKTIESNLKKLELSREDIEILLSEEFSGTLNLSLKAIKKILPYLEKGLSYNEACEK ADYD YKNNGIKFKRGELLPVVDKDLIANPVVLRAI SQTRKVVNAI IRKYGTPHTIHVEVARDLAKSYD DRQTI IKENKKRELENEKTKKFI SEEFGIKNVKGKLLLKYRLYQEQEGRCAYSRKELSLSEVIL DESMTDIDHI IPYSRSMDDSYSNKVLVLSGENRKKSNLLPKEYFDRQGRDWDTFVLNVKAMKIH PRKKSNLLKEKFTREDNKDWKSRALNDTRYI SRFVANYLENALEYRDDSPKKRVFMIPGQLTAQ LRARWRLNKVRENGDLHHALDAAVVAVTDQKAINNI SNI SRYKELKNCKDVIPS IEYHADEETG EVYFEEVKDTRFPMPWSGFDLELQKRLESENPREEFYNLLSDKRYLGWFNYEEGFIEKLR PVFV SRMPNRGVKGQAHQETIRSSKKI SNQIAVSKKPLNS IKLKDLEKMQGRDTDRKLYEALKNRLEE YDDKPEKAFAEPFYKPTNSGKRGPLVRGIKVEEKQNVGVYVNGGQASNGSMVRIDVFRKN GKFY TVPIYVHQTLLKELPNRAINGKPYKDWDLIDGSFEFLYSFYPNDLIEIEFGKSKS IKNDNKLTK TEIPEVNLSEVLGYYRGMDTSTGAATIDTQDGKIQMRIGIKTVKNIKKYQVDVLGNVYKV KREK RQTF SEQ ID NO: 370

MSKKVSRRYEEQAQEICQRLGSRPYS IGLDLGVGS IGVAVAAYDPIKKQPSDLVFVSSRIFIPS TGAAERRQKRGQRNSLRHRANRLKFLWKLLAERNLMLSYSEQDVPDPARLRFEDAVVRAN PYEL RLKGLNEQLTLSELGYALYHIANHRGSSSVRTFLDEEKSSDDKKLEEQQAMTEQLAKEKG I STF IEVLTAFNTNGLIGYRNSESVKSKGVPVPTRDI I SNEIDVLLQTQKQFYQEILSDEYCDRIVSA ILFENEKIVPEAGCCPYFPDEKKLPRCHFLNEERRLWEAINNARIKMPMQEGAAKRYQSA SFSD EQRHILFHIARSGTDITPKLVQKEFPALKTS I IVLQGKEKAIQKIAGFRFRRLEEKSFWKRLSE EQKDDFFSAWTNTPDDKRLSKYLMKHLLLTENEVVDALKTVSLIGDYGPIGKTATQLLMK HLED GLTYTEALERGMETGEFQELSVWEQQSLLPYYGQILTGSTQALMGKYWHSAFKEKRDSEG FFKP NTNSDEEKYGRIANPVVHQTLNELRKLMNELITILGAKPQEITVELARELKVGAEKREDI IKQQ TKQEKEAVLAYSKYCEPNNLDKRYIERFRLLEDQAFVCPYCLEHI SVADIAAGRADVDHIFPRD DTADNSYGNKVVAHRQCNDIKGKRTPYAAFSNTSAWGPIMHYLDETPGMWRKRRKFETNE EEYA KYLQSKGFVSRFESDNSYIAKAAKEYLRCLFNPNNVTAVGSLKGMETS ILRKAWNLQGIDDLLG SRHWSKDADTSPTMRKNRDDNRHHGLDAIVALYCSRSLVQMINTMSEQGKRAVEIEAMIP IPGY ASEPNLSFEAQRELFRKKILEFMDLHAFVSMKTDNDANGALLKDTVYS ILGADTQGEDLVFVVK KKIKDIGVKIGDYEEVASAIRGRITDKQPKWYPMEMKDKIEQLQSKNEAALQKYKESLVQ AAAV LEESNRKLIESGKKPIQLSEKTI SKKALELVGGYYYLI SNNKRTKTFVVKEPSNEVKGFAFDTG SNLCLDFYHDAQGKLCGEI IRKIQAMNPSYKPAYMKQGYSLYVRLYQGDVCELRASDLTEAESN LAKTTHVRLPNAKPGRTFVI I ITFTEMGSGYQIYFSNLAKSKKGQDTSFTLTTIKNYDVRKVQL SSAGLVRYVSPLLVDKIEKDEVALCGE

SEQ ID NO: 371 MNQKFILGLDIGITSVGYGLIDYETKNI IDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVI DSND DVGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADI IKEI IQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYA YSAD LFNALNDLNNLVIQRDGLSKLEYHEKYHI IENVFKQKKKPTLKQIANEINVNPEDIKGYRITKS GKPQFTEFKLYHDLKSVLFDQS ILENEDVLDQIAEILTIYQDKDS IKSKLTELDILLNEEDKEN IAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMID EFIL SPVVKRTFGQAINLINKI IEKYGVPEDI I IELARENNSKDKQKFINEMQKKNENTRKRINEI IG KYGNQNAKRLVEKIRLHDEQEGKCLYSLES IPLEDLLNNPNHYEVDHI IPRSVSFDNSYHNKVL VKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRI SKKKKEYLLEERDINKFEVQ KEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGY KHHA EDALI IA ADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNYSEMFI IPKQVQDIKDFRN FKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPEKFLM YQHD PRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDV THQF KSSTKKLVKLS IKPYRFDVYLTDKGYKFITI SYLDVLKKDNYYYIPEQKYDKLKLGKAIDKNAK FIASFYKNDLIKLDGEIYKI IGVNSDTRNMIELDLPDIRYKEYCELN IKGEPRIKKTIGKKVN SIEKLTTDVLGNVFTNTQYTKPQLLFKRGN

SEQ ID NO: 372

MIMKLEKWRLGLDLGTNS IGWSVFSLDKDNSVQDLIDMGVRIFSDGRDPKTKEPLAVARRTARS QRKLIYRRKLRRKQVFKFLQEQGLFPKTKEECMTLKSLNPYELRIKALDEKLEPYELGRA LFNL AVRRGFKSNRKDGSREEVSEKKSPDEIKTQADMQTHLEKAIKENGCRTITEFLYKNQGEN GGIR FAPGRMTYYPTRKMYEEEFNLIRSKQEKYYPQVDWDDIYKAIFYQRPLKPQQRGYCIYEN DKER TFKAMPCSQKLRILQDIGNLAYYEGGSKKRVELNDNQDKVLYELLNSKDKVTFDQMRKAL CLAD SNSFNLEENRDFLIGNPTAVKMRSKNRFGKLWDEIPLEEQDLI IETI ITADEDDAVYEVIKKYD LTQEQRDFIVKNTILQSGTSMLCKEVSEKLVKRLEEIADLKYHEAVESLGYKFADQTVEK YDLL PYYGKVLPGSTMEIDLSAPETNPEKHYGKI SNPTVHVALNQTRVVV ALIKEYGKPSQIAIELS RDLKNNVEKKAEIARKQNQRAKENIAINDTI SALYHTAFPGKSFYPNRNDRMKYRLWSELGLGN KCIYCGKGI SGAELFTKEIEIEHILPFSRTLLDAESNLTVAHSSCNAFKAERSPFEAFGTNPSG YSWQEI IQRANQLKNTSKKNKFSPNAMDSFEKDSSFIARQLSDNQYIAKAALRYLKCLVENPSD VWTTNGSMTKLLRDKWEMDS ILCRKFTEKEVALLGLKPEQIGNYKKNRFDHRHHAIDAVVIGLT DRSMVQKLATKNSHKGNRIEIPEFPILRSDLIEKVKNIVVSFKPDHGAEGKLSKETLLGK IKLH GKETFVCRENIVSLSEKNLDDIVDEIKSKVKDYVAKHKGQKIEAVLSDFSKENGIKKVRC VNRV QTPIEITSGKI SRYLSPEDYFAAVIWEIPGEKKTFKAQYIRRNEVEKNSKGLNVVKPAVLENGK PHPAAKQVCLLHKDDYLEFSDKGKMYFCRIAGYAATNNKLDIRPVYAVSYCADWINSTNE TMLT GYWKPTPTQNWVSVNVLFDKQKARLVTVSPIGRVFRK

SEQ ID NO: 373

MSSKAIDSLEQLDLFKPQEYTLGLDLGIKS IGWAILSGERIANAGVYLFETAEELNSTGNKLI S KAAERGRKRRIRRMLDRKARRGRHIRYLLEREGLPTDELEEVVVHQSNRTLWDVRAEAVE RKLT KQELAAVLFHLVRHRGYFPNTKKLPPDDESDSADEEQGKINRATSRLREELKASDCKTIG QFLA QNRDRQRNREGDYSNLMARKLVFEEALQILAFQRKQGHELSKDFEKTYLDVLMGQRSGRS PKLG NCSLIPSELRAPSSAPSTEWFKFLQNLGNLQI SNAYREEWS IDAPRRAQI IDACSQRSTSSYWQ IRRDFQIPDEYRFNLVNYERRDPDVDLQEYLQQQERKTLANFRNWKQLEKI IGTGHPIQTLDEA ARLITLIKDDEKLSDQLADLLPEASDKAITQLCELDFTTAAKI SLEAMYRILPHMNQGMGFFDA CQQESLPEIGVPPAGDRVPPFDEMYNPVVNRVLSQSRKLINAVIDEYGMPAKIRVELARD LGKG RELRERIKLDQLDKSKQNDQRAEDFRAEFQQAPRGDQSLRYRLWKEQNCTCPYSGRMIPV NSVL SEDTQIDHILPI SQSFDNSLSNKVLCFTEENAQKSNRTPFEYLDAADFQRLEAI SGNWPEAKRN KLLHKSFGKVAEEWKSRALNDTRYLTSALADHLRHHLPDSKIQTVNGRITGYLRKQWGLE KDRD KHTHHAVDAIVVACTTPAIVQQVTLYHQDIRRYKKLGEKRPTPWPETFRQDVLDVEEEIF ITRQ PKKVSGGIQTKDTLRKHRSKPDRQRVALTKVKLADLERLVEKDASNRNLYEHLKQCLEES GDQP TKAFKAPFYMPSGPEAKQRPILSKVTLLREKPEPPKQLTELSGGRRYDSMAQGRLDIYRY KPGG KRKDEYRVVLQRMIDLMRGEENVHVFQKGVPYDQGPEIEQNYTFLFSLYFDDLVEFQRSA DSEV IRGYYRTFNIANGQLKI STYLEGRQDFDFFGANRLAHFAKVQVNLLGKVIK

SEQ ID NO: 374

MRSLRYRLALDLGSTSLGWALFRLDACNRPTAVIKAGVRIFSDGRNPKDGSSLAVTR RAARAMR RRRDRLLKRKTRMQAKLVEHGFFPADAGKRKALEQLNPYALRAKGLQEALLPGEFARALF HINQ RRGFKSNRKTDKKDNDSGVLKKAIGQLRQQMAEQGSRTVGEYLWTRLQQGQGVRARYREK PYTT EEGKKRIDKSYDLYIDRAMIEQEFDALWAAQAAFNPTLFHEAARADLKDTLLHQRPLRPV KPGR CTLLPEEERAPLALPSTQRFRIHQEVNHLRLLDENLREVALTLAQRDAVVTALETKAKLS FEQI RKLLKLSGSVQFNLEDAKRTELKGNATSAALARKELFGAAWSGFDEALQDEIVWQLVTEE GEGA LIAWLQTHTGVDEARAQAIVDVSLPEGYGNLSRKALARIVPALRAAVITYDKAVQAAGFD HHSQ LGFEYDASEVEDLVHPETGEIRSVFKQLPYYGKALQRHVAFGSGKPEDPDEKRYGKIANP TVHI GLNQVRMVVNALIRRYGRPTEVVIELARDLKQSREQKVEAQRRQADNQRRNARIRRS IAEVLGI GEERVRGSDIQKWICWEELSFDAADRRCPYSGVQI SAAMLLSDEVEVEHILPFSKTLDDSLNNR TVAMRQANRIKRNRTPWDARAEFEAQGWSYEDILQRAERMPLRKRYRFAPDGYERWLGDD KDFL ARALNDTRYLSRVAAEYLRLVCPGTRVIPGQLTALLRGKFGLNDVLGLDGEKNRNDHRHH AVDA CVIGVTDQGLMQRFATASAQARGDGLTRLVDGMPMPWPTYRDHVERAVRHIWVSHRPDHG FEGA MMEETSYGIRKDGS IKQRRKADGSAGREI SNLIRIHEATQPLRHGVSADGQPLAYKGYVGGSNY CIEITVNDKGKWEGEVI STFRAYGVVRAGGMGRLRNPHEGQNGRKLIMRLVIGDSVRLEVDGAE RTMRIVKI SGSNGQIFMAPIHEANVDARNTDKQDAFTYTSKYAGSLQKAKTRRVTI SPIGEVRD PGFKG

SEQ ID NO: 375

MARPAFRAPRREHVNGWTPDPHRI SKPFFILVSWHLLSRVVIDSSSGCFPGTSRDHTDKFAEWE CAVQPYRLSFDLGTNS IGWGLLNLDRQGKPREIRALGSRIFSDGRDPQDKASLAVARRLARQMR RRRDRYLTRRTRLMGALVRFGLMPADPAARKRLEVAVDPYLARERATRERLEPFEIGRAL FHLN QRRGYKPVRTATKPDEEAGKVKEAVERLEAAIAAAGAPTLGAWFAWRKTRGETLRARLAG KGKE AAYPFYPARRMLEAEFDTLWAEQARHHPDLLTAEAREILRHRIFHQRPLKPPPVGRCTLY PDDG RAPRALPSAQRLRLFQELASLRVIHLDLSERPLTPAERDRIVAFVQGRPPKAGRKPGKVQ KSVP FEKLRGLLELPPGTGFSLESDKRPELLGDETGARIAPAFGPGWTALPLEEQDALVELLLT EAEP ERAIAALTARWALDEATAAKLAGATLPDFHGRYGRRAVAELLPVLERETRGDPDGRVRPI RLDE AVKLLRGGKDHSDFSREGALLDALPYYGAVLERHVAFGTGNPADPEEKRVGRVANPTVHI ALNQ LRHLVNAILARHGRPEEIVIELARDLKRSAEDRRREDKRQADNQKRNEERKRLILSLGER PTPR NLLKLRLWEEQGPVENRRCPYSGETI SMRMLLSEQVDIDHILPFSVSLDDSAANKVVCLREANR IKRNRSPWEAFGHDSERWAGILARAEALPKNKRWRFAPDALEKLEGEGGLRARHLNDTRH LSRL AVEYLRCVCPKVRVSPGRLTALLRRRWGIDAILAEADGPPPEVPAETLDPSPAEKNRADH RHHA LDAVVIGCIDRSMVQRVQLAAASAEREAAAREDNIRRVLEGFKEEPWDGFRAELERRART IVVS HRPEHGIGGALHKETAYGPVDPPEEGFNLVVRKPIDGLSKDEINSVRDPRLRRALIDRLA IRRR DANDPATALAKAAEDLAAQPASRGIRRVRVLKKESNPIRVEHGGNPSGPRSGGPFHKLLL AGEV HHVDVALRADGRRWVGHWVTLFEAHGGRGADGAAAPPRLGDGERFLMRLHKGDCLKLEHK GRVR VMQVVKLEPSSNSVVVVEPHQVKTDRSKHVKI SCDQLRARGARRVTVDPLGRVRVHAPGARVGI GGDAGRTAMEPAEDIS SEQ ID NO: 376

MKRTSLRAYRLGVDLGANSLGWFVVWLDDHGQPEGLGPGGVRIFPDGRNPQSKQSNAAGR RLAR SARRRRDRYLQRRGKLMGLLVKHGLMPADEPARKRLECLDPYGLRAKALDEVLPLHHVGR ALFH LNQRRGLFANRAIEQGDKDASAIKAAAGRLQTSMQACGARTLGEFLNRRHQLRATVRARS PVGG DVQARYEFYPTRAMVDAEFEAIWAAQAPHHPTMTAEAHDTIREAIFSQRAMKRPS IGKCSLDPA TSQDDVDGFRCAWSHPLAQRFRIWQDVRNLAVVETGPTSSRLGKEDQDKVARALLQTDQL SFDE IRGLLGLPSDARFNLESDRRDHLKGDATGAILSARRHFGPAWHDRSLDRQIDIVALLESA LDEA AIIASLGTTHSLDEAAAQRALSALLPDGYCRLGLRAIKRVLPLMEAGRTYAEAASAAGYD HALL PGGKLSPTGYLPYYGQWLQNDVVGSDDERDTNERRWGRLPNPTVHIGIGQLRRVVNELIR WHGP PAEITVELTRDLKLSPRRLAELEREQAENQRKNDKRTSLLRKLGLPASTHNLLKLRLWDE QGDV ASECPYTGEAIGLERLVSDDVDIDHLIPFS I SWDDSAANKVVCMRYANREKGNRTPFEAFGHRQ GRPYDWADIAERAARLPRGKRWRFGPGARAQFEELGDFQARLLNETSWLARVAKQYLAAV THPH RIHVLPGRLTALLRATWELNDLLPGSDDRAAKSRKDHRHHAIDALVAALTDQALLRRMAN AHDD TRRKIEVLLPWPTFRIDLETRLKAMLVSHKPDHGLQARLHEDTAYGTVEHPETEDGANLV YRKT FVDI SEKEIDRIRDRRLRDLVRAHVAGERQQGKTLKAAVLSFAQRRDIAGHPNGIRHVRLTKS I KPDYLVPIRDKAGRIYKSYNAGENAFVDILQAESGRWIARATTVFQANQANESHDAPAAQ PIMR VFKGDMLRIDHAGAEKFVKIVRLSPSNNLLYLVEHHQAGVFQTRHDDPEDSFRWLFASFD KLRE WNAELVRIDTLGQPWRRKRGLETGSEDATRIGWTRPKKWP

SEQ ID NO: 377

MERIFGFDIGTTS IGFSVIDYSSTQSAGNIQRLGVRIFPEARDPDGTPLNQQRRQKRMMRRQLR RRRIRRKALNETLHEAGFLPAYGSADWPVVMADEPYELRRRGLEEGLSAYEFGRAIYHLA QHRH FKGRELEESDTPDPDVDDEKEAANERAATLKALKNEQTTLGAWLARRPPSDRKRGIHAHR NVVA EEFERLWEVQSKFHPALKSEEMRARI SDTIFAQRPVFWRKNTLGECRFMPGEPLCPKGSWLSQQ RRMLEKLNNLAIAGGNARPLDAEERDAILSKLQQQASMSWPGVRSALKALYKQRGEPGAE KSLK FNLELGGESKLLGNALEAKLADMFGPDWPAHPRKQEIRHAVHERLWAADYGETPDKKRVI ILSE KDRKAHREAAANSFVADFGITGEQAAQLQALKLPTGWEPYS IPALNLFLAELEKGERFGALVNG PDWEGWRRTNFPHRNQPTGEILDKLPSPASKEERERI SQLRNPTVVRTQNELRKVVNNLIGLYG KPDRIRIEVGRDVGKSKREREEIQSGIRRNEKQRKKATEDLIKNGIANPSRDDVEKWILW KEGQ ERCPYTGDQIGFNALFREGRYEVEHIWPRSRSFDNSPRNKTLCRKDVNIEKGNRMPFEAF GHDE DRWSAIQIRLQGMVSAKGGTGMSPGKVKRFLAKTMPEDFAARQLNDTRYAAKQILAQLKR LWPD MGPEAPVKVEAVTGQVTAQLRKLWTLNNILADDGEKTRADHRHHAIDALTVACTHPGMTN KLSR YWQLRDDPRAEKPALTPPWDTIRADAEKAVSEIVVSHRVRKKVSGPLHKETTYGDTGTDI KTKS GTYRQFVTRKKIESLSKGELDEIRDPRIKEIVAAHVAGRGGDPKKAFPPYPCVSPGGPEI RKVR LTSKQQLNLMAQTGNGYADLGSNHHIAIYRLPDGKADFEIVSLFDASRRLAQRNPIVQRT RADG ASFVMSLAAGEAIMIPEGSKKGIWIVQGVWASGQVVLERDTDADHSTTTRPMPNPILKDD AKKV SIDPIGRVRPSND SEQ ID NO: 378

MNKRILGLDTGTNSLGWAVVDWDEHAQSYELIKYGDVIFQEGVKIEKGIESSKAAERSGY KAIR KQYFRRRLRKIQVLKVLVKYHLCPYLSDDDLRQWHLQKQYPKSDELMLWQRTSDEEGKNP YYDR HRCLHEKLDLTVEADRYTLGRALYHLTQRRGFLSNRLDTSADNKEDGVVKSGI SQLSTEMEEAG CEYLGDYFYKLYDAQGNKVRIRQRYTDRNKHYQHEFDAICEKQELSSELIEDLQRAIFFQ LPLK SQRHGVGRCTFERGKPRCADSHPDYEEFRMLCFVNNIQVKGPHDLELRPLTYEEREKIEP LFFR KSKPNFDFEDIAKALAGKKNYAWIHDKEERAYKFNYRMTQGVPGCPTIAQLKS IFGDDWKTGIA ETYTLIQKKNGSKSLQEMVDDVWNVLYSFSSVEKLKEFAHHKLQLDEESAEKFAKIKLSH SFAA LSLKAIRKFLPFLRKGMYYTHASFFANIPTIVGKEIWNKEQNRKYIMENVGELVFNYQPK HREV QGTIEMLIKDFLANNFELPAGATDKLYHPSMIETYPNAQRNEFGILQLGSPRTNAIRNPM AMRS LHILRRVVNQLLKES I IDENTEVHVEYARELNDANKRRAIADRQKEQDKQHKKYGDEIRKLYKE ETGKDIEPTQTDVLKFQLWEEQNHHCLYTGEQIGITDFIGSNPKFDIEHTIPQSVGGDST QMNL TLCDNRFNREVKKAKLPTELANHEEILTRIEPWKNKYEQLVKERDKQRTFAGMDKAVKDI RIQK RHKLQMEIDYWRGKYERFTMTEVPEGFSRRQGTGIGLI SRYAGLYLKSLFHQADSRNKSNVYVV KGVATAEFRKMWGLQSEYEKKCRDNHSHHCMDAITIACIGKREYDLMAEYYRMEETFKQG RGSK PKFSKPWATFTEDVL IYKNLLVVHDTPNNMPKHTKKYVQTS IGKVLAQGDTARGSLHLDTYYG AIERDGEIRYVVRRPLSSFTKPEELENIVDETVKRTIKEAIADKNFKQAIAEPIYMNEEK GILI KKVRCFAKSVKQPINIRQHRDLSKKEYKQQYHVMNENNYLLAIYEGLVKNKVVREFEIVS YIEA AKYYKRSQDRNIFSS IVPTHSTKYGLPLKTKLLMGQLVLMFEENPDEIQVDNTKDLVKRLYKVV GIEKDGRIKFKYHQEARKEGLPIFSTPYKNNDDYAPIFRQS INNINILVDGIDFTIDILGKVTL KE SEQ ID NO: 379

MNYKMGLDIGIASVGWAVINLDLKRIEDLGVRIFDKAEHPQNGESLALPRRIARSARRRL RRRK HRLERIRRLLVSENVLTKEEMNLLFKQKKQIDVWQLRVDALERKLNNDELARVLLHLAKR RGFK SNRKSERNSKESSEFLKNIEENQS ILAQYRSVGEMIVKDSKFAYHKRNKLDSYSNMIARDDLER EIKLIFEKQREFNNPVCTERLEEKYLNIWSSQRPFASKEDIEKKVGFCTFEPKEKRAPKA TYTF QSFIVWEHINKLRLVSPDETRALTEIERNLLYKQAFSKNKMTYYDIRKLLNLSDDIHFKG LLYD PKSSLKQIENIRFLELDSYHKIRKCIENVYGKDGIRMFNETDIDTFGYALTIFKDDEDIV AYLQ NEYITKNGKRVSNLANKVYDKSLIDELLNLSFSKFAHLSMKAIR ILPYMEQGEIYSKACELAG YNFTGPKKKEKALLLPVIPNIANPVVMRALTQSRKVVNAI IKKYGSPVS IHIELARDLSHSFDE RKKIQKDQTENRKKNETAIKQLIEYELTKNPTGLDIVKFKLWSEQQGRCMYSLKPIELER LLEP GYVEVDHILPYSRSLDDSYANKVLVLTKENREKGNHTPVEYLGLGSERWKKFEKFVLANK QFSK KKKQNLLRLRYEETEEKEFKERNLNDTRYI SKFFANFIKEHLKFADGDGGQKVYTINGKITAHL RSRWDFNKNREESDLHHAVDAVIVACATQGMIKKITEFYKAREQNKESAKKKEPIFPQPW PHFA DELKARLSKFPQES IEAFALGNYDRKKLESLRPVFVSRMPKRSVTGAAHQETLRRCVGIDEQSG KIQTAVKTKLSDIKLDKDGHFPMYQKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKK NGEP GPVIRTVKI IDTKNKVVHLDGSKTVAYNS IVRTDVFEKDGKYYCVPVYTMDIMKGTLPNKAIE ANKPYSEWKEMTEEYTFQFSLFPNDLVRIVLPREKTIKTSTNEEI I IKDIFAYYKTIDSATGGL ELI SHDRNFSLRGVGSKTLKRFEKYQVDVLG IHKVKGEKRVGLAAPTNQKKGKTVDSLQSVSD

SEQ ID NO: 380

MRRLGLDLGTNS IGWCLLDLGDDGEPVS IFRTGARIFSDGRDPKSLGSLKATRREARLTRRRRD RFIQRQKNLINALVKYGLMPADEIQRQALAYKDPYPIRKKALDEAIDPYEMGRAIFHINQ RRGF KSNRKSADNEAGVVKQS IADLEMKLGEAGARTIGEFLADRQATNDTVRARRLSGTNALYEFYPD RYMLEQEFDTLWAKQAAFNPSLYIEAARERLKEIVFFQRKLKPQEVGRCIFLSDEDRI SKALPS FQRFRIYQELSNLAWIDHDGVAHRITASLALRDHLFDELEHKKKLTFKAMRAILRKQGVV DYPV GFNLESDNRDHLIGNLTSCIMRDAKKMIGSAWDRLDEEEQDSFILMLQDDQKGDDEVRS ILTQQ YGLSDDVAEDCLDVRLPDGHGSLSKKAIDRILPVLRDQGLIYYDAVKEAGLGEANLYDPY AALS DKLDYYGKALAGHVMGASGKFEDSDEKRYGTI SNPTVHIALNQVRAVVNELIRLHGKPDEVVIE IGRDLPMGADGKRELERFQKEGRAKNERARDELKKLGHIDSRESRQKFQLWEQLAKEPVD RCCP FTGKMMS I SDLFSDKVEIEHLLPFSLTLDDSMANKTVCFRQANRDKGNRAPFDAFGNSPAGYDW QEILGRSQNLPYAKRWRFLPDAMKRFEADGGFLERQLNDTRYI SRYTTEYI STI IPKNKIWVVT GRLTSLLRGFWGLNS ILRGHNTDDGTPAKKSRDDHRHHAIDAIVVGMTSRGLLQKVSKAARRSE DLDLTRLFEGRIDPWDGFRDEVKKHIDAI IVSHRPRKKSQGALHNDTAYGIVEHAENGASTVVH RVPITSLGKQSDIEKVRDPLIKSALLNETAGLSGKSFENAVQKWCADNS IKSLRIVETVS I IPI TDKEGVAYKGYKGDGNAYMDIYQDPTSSKWKGEIVSRFDANQKGFIPSWQSQFPTARLIM RLRI NDLLKLQDGEIEEIYRVQRLSGSKILMAPHTEANVDARDRDKNDTFKLTSKSPGKLQSAS ARKV HISPTGLIREG

SEQ ID NO: 381

MK ILGLDLGLSS IGWSVIRENSEEQELVAMGSRVVSLTAAELSSFTQGNGVS INSQRTQKRTQ RKGYDRYQLRRTLLRNKLDTLGMLPDDSLSYLPKLQLWGLRAKAVTQRIELNELGRVLLH LNQK RGYKS IKSDFSGDKKITDYVKTVKTRYDELKEMRLTIGELFFRRLTENAFFRCKEQVYPRQAYV EEFDCIMNCQRKFYPDILTDETIRCIRDEI IYYQRPLKSCKYLVSRCEFEKRFYLNAAGKKTEA GPKVSPRTSPLFQVCRLWES IN IVVKDRRNEIVFI SAEQRAALFDFLNTHEKLKGSDLLKLLG LSKTYGYRLGEQFKTGIQGNKTRVEIERALGNYPDKKRLLQFNLQEESSSMVNTETGEII PMIS LSFEQEPLYRLWHVLYS IDDREQLQSVLRQKFGIDDDEVLERLSAIDLVKAGFGNKSSKAIRRI LPFLQLGMNYAEACEAAGYNHSNNYTKAENEARALLDRLPAIKKNELRQPVVEKILNQMV NVVN ALMEKYGRFDEIRVELARELKQSKEERSNTYKS INKNQRENEQIAKRIVEYGVPTRSRIQKYKM WEESKHCCIYCGQPVDVGDFLRGFDVEVEHI IPKSLYFDDSFANKVCSCRSCNKEKNNRTAYDY MKSKGEKALSDYVERVNTMYTNNQI SKTKWQNLLTPVDKI S IDFIDRQLRESQYIARKAKEILT SICYNVTATSGSVTSFLRHVWGWDTVLHDLNFDRYKKVGLTEVIEVNHRGSVIRREQIKD WSKR FDHRHHAIDALTIACTKQAYIQRLNNLRAEEGPDFNKMSLERYIQSQPHFSVAQVREAVD RILV SFRAGKRAVTPGKRYIRKNRKRI SVQSVLIPRGALSEESVYGVIHVWEKDEQGHVIQKQRAVMK YPITS INREMLDKEKVVDKRIHRILSGRLAQYNDNPKEAFAKPVYIDKECRIPIRTVRCFAKPA INTLVPLKKDDKGNPVAWVNPGNNHHVAIYRDEDGKYKERTVTFWEAVDRCRVGIPAIVT QPDT IWD ILQRNDI SENVLESLPDVKWQFVLSLQQNEMFILGMNEEDYRYAMDQQDYALLNKYLYRV QKLSKSDYSFRYHTETSVEDKYDGKPNLKLSMQMGKLKRVS IKSLLGLNPHKVHI SVLGEIKEI S

SEQ ID NO: 382

MAEKQHRWGLDIGTNS IGWAVIALIEGRPAGLVATGSRIFSDGRNPKDGSSLAVERRGPRQMRR RRDRYLRRRDRFMQALINVGLMPGDAAARKALVTENPYVLRQRGLDQALTLPEFGRALFH LNQR RGFQSNRKTDRATAKESGKVKNAIAAFRAGMGNARTVGEALARRLEDGRPVRARMVGQGK DEHY ELYIAREWIAQEFDALWASQQRFHAEVLADAARDRLRAILLFQRKLLPVPVGKCFLEPNQ PRVA AALPSAQRFRLMQELNHLRVMTLADKRERPLSFQERNDLLAQLVARPKCGFDMLRKIVFG ANKE AYRFTIESERRKELKGCDTAAKLAKVNALGTRWQALSLDEQDRLVCLLLDGENDAVLADA LREH YGLTDAQIDTLLGLSFEDGHMRLGRSALLRVLDALESGRDEQGLPLSYDKAVVAAGYPAH TADL ENGERDALPYYGELLWRYTQDAPTAKNDAERKFGKIANPTVHIGLNQLRKLVNALIQRYG KPAQ IVVELARNLKAGLEEKERIKKQQTANLERNERIRQKLQDAGVPDNRENRLRMRLFEELGQ GNGL GTPCIYSGRQI SLQRLFSNDVQVDHILPFSKTLDDSFANKVLAQHDANRYKGNRGPFEAFGANR DGYAWDDIRARAAVLPRNKRNRFAETAMQDWLHNETDFLARQLTDTAYLSRVARQYLTAI CSKD DVYVSPGRLTAMLRAKWGLNRVLDGVMEEQGRPAVKNRDDHRHHAIDAVVIGATDRAMLQ QVAT LAARAREQDAERLIGDMPTPWPNFLEDVRAAVARCVVSHKPDHGPEGGLHNDTAYGIVAG PFED GRYRVRHRVSLFDLKPGDLSNVRCDAPLQAELEPIFEQDDARAREVALTALAERYRQRKV WLEE LMSVLPIRPRGEDGKTLPDSAPYKAYKGDSNYCYELFINERGRWDGELI STFRANQAAYRRFRN DPARFRRYTAGGRPLLMRLCINDYIAVGTAAERTIFRVVKMSENKITLAEHFEGGTLKQR DADK DDPFKYLTKSPGALRDLGARRIFVDLIGRVLDPGIKGD

SEQ ID NO: 383 MIERILGVDLGI SSLGWAIVEYDKDDEAANRI IDCGVRLFTAAETPKKKESPNKARREARGIRR VLNRRRVRMNMIKKLFLRAGLIQDVDLDGEGGMFYSKANRADVWELRHDGLYRLLKGDEL ARVL IHIAKHRGYKFIGDDEADEESGKVKKAGVVLRQNFEAAGCRTVGEWLWRERGANGKKRNK HGDY EI S IHRDLLVEEVEAIFVAQQEMRSTIATDALKAAYREIAFFVRPMQRIEKMVGHCTYFPEER R APKSAPTAEKFIAI SKFFSTVI IDNEGWEQKI IERKTLEELLDFAVSREKVEFRHLRKFLDLSD NEIFKGLHYKGKPKTAKKREATLFDPNEPTELEFDKVEAEKKAWI SLRGAAKLREALGNEFYGR FVALGKHADEATKILTYYKDEGQKRRELTKLPLEAEMVERLVKIGFSDFLKLSLKAIRDI LPAM ESGARYDEAVLMLGVPHKEKSAILPPLNKTDIDILNPTVIRAFAQFRKVANALVRKYGAF DRVH FELAREINTKGEIEDIKESQRKNEKERKEAADWIAETSFQVPLTRKNILKKRLYIQQDGR CAYT GDVIELERLFDEGYCEIDHILPRSRSADDSFANKVLCLARANQQKTDRTPYEWFGHDAAR WNAF ETRTSAPSNRVRTGKGKIDRLLKKNFDENSEMAFKDRNLNDTRYMARAIKTYCEQYWVFK NSHT KAPVQVRSGKLTSVLRYQWGLESKDRESHTHHAVDAI I IAFSTQGMVQKLSEYYRFKETHREKE RPKLAVPLANFRDAVEEATRIENTETVKEGVEVKRLLI SRPPRARVTGQAHEQTAKPYPRIKQV KNKKKWRLAPIDEEKFESFKADRVASANQKNFYETSTIPRVDVYHKKGKFHLVPIYLHEM VLNE LPNLSLGTNPEAMDENFFKFS IFKDDLI S IQTQGTPKKPAKI IMGYFKNMHGANMVLSS INNSP CEGFTCTPVSMDKKHKDKCKLCPEENRIAGRCLQGFLDYWSQEGLRPPRKEFECDQGVKF ALDV KKYQIDPLGYYYEVKQEKRLGTIPQMRSAKKLVKK

SEQ ID NO: 384

MNNS IKSKPEVTIGLDLGVGSVGWAIVDNET I IHHLGSRLFSQAKTAEDRRSFRGVRRLIRRR KYKLKRFVNLIWKYNSYFGFKNKEDILNNYQEQQKLHNTVLNLKSEALNAKIDPKALSWI LHDY LKNRGHFYEDNRDFNVYPTKELAKYFDKYGYYKGI IDSKEDNDNKLEEELTKYKFSNKHWLEEV KKVLSNQTGLPEKFKEEYESLFSYVRNYSEGPGS INSVSPYGIYHLDEKEGKVVQKYN IWDKT IGKCNIFPDEYRAPKNSPIAMIFNEINELSTIRSYS IYLTGWFINQEFKKAYLNKLLDLLIKTN GEKPIDARQFKKLREETIAES IGKETLKDVENEEKLEKEDHKWKLKGLKLNTNGKIQYNDLSSL AKFVHKLKQHLKLDFLLEDQYATLDKINFLQSLFVYLGKHLRYSNRVDSANLKEFSDSNK LFER ILQKQKDGLFKLFEQTDKDDEKILAQTHSLSTKAMLLAITRMTNLDNDEDNQKNNDKGWN FEAI KNFDQKFIDITKKNNNLSLKQNKRYLDDRFINDAILSPGVKRILREATKVFNAILKQFSE EYDV TKVVIELARELSEEKELENTKNYKKLIKKNGDKI SEGLKALGI SEDEIKDILKSPTKSYKFLLW LQQDHIDPYSLKEIAFDDIFTKTEKFEIDHI IPYS I SFDDSSSNKLLVLAESNQAKSNQTPYEF ISSGNAGIKWEDYEAYCRKFKDGDSSLLDSTQRSKKFAKMMKTDTSSKYDIGFLARNLND TRYA TIVFRDALEDYANNHLVEDKPMFKVVCINGSVTSFLRKNFDDSSYAKKDRDK IHHAVDAS IIS IFSNETKTLFNQLTQFADYKLFKNTDGSWKKIDPKTGVVTEVTDENWKQIRVRNQVSEIA KVIE KYIQDS IERKARYSRKIENKT I SLFNDTVYSAKKVGYEDQIKRKNLKTLDIHESAKENKNSK VKRQFVYRKLVNVSLLNNDKLADLFAEKEDILMYRANPWVINLAEQIFNEYTENKKIKSQ NVFE KYMLDLTKEFPEKFSEFLVKSMLRNKTAI IYDDKK IVHRIKRLKMLSSELKENKLSNVI IRSK NQSGTKLSYQDTINSLALMIMRS IDPTAKKQYIRVPLNTLNLHLGDHDFDLHNMDAYLKKPKFV KYLKANEIGDEYKPWRVLTSGTLLIHKKDKKLMYISSFQNLNDVIEIKNLIETEYKENDD SDSK KKKKANRFLMTLSTILNDYILLDAKDNFDILGLSKNRIDEILNSKLGLDKIVK

SEQ ID NO: 385

MGGSEVGTVPVTWRLGVDVGERS IGLAAVSYEEDKPKEILAAVSWIHDGGVGDERSGASRLALR GMARRARRLRRFRRARLRDLDMLLSELGWTPLPDKNVSPVDAWLARKRLAEEYVVDETER RRLL GYAVSHMARHRGWRNPWTTIKDLKNLPQPSDSWERTRESLEARYSVSLEPGTVGQWAGYL LQRA PGIRLNPTQQSAGRRAELSNATAFETRLRQEDVLWELRCIADVQGLPEDVVSNVIDAVFC QKRP SVPAERIGRDPLDPSQLRASRACLEFQEYRIVAAVANLRIRDGSGSRPLSLEERNAVIEA LLAQ TERSLTWSDIALEILKLPNESDLTSVPEEDGPSSLAYSQFAPFDETSARIAEFIAKNRRK IPTF AQWWQEQDRTSRSDLVAALADNS IAGEEEQELLVHLPDAELEALEGLALPSGRVAYSRLTLSGL TRVMRDDGVDVHNARKTCFGVDDNWRPPLPALHEATGHPVVDRNLAILRKFLSSATMRWG PPQS IVVELARGASESRERQAEEEAARRAHRKANDRIRAELRASGLSDPSPADLVRARLLELYD CHCM YCGAPI SWENSELDHIVPRTDGGSNRHENLAITCGACNKEKGRRPFASWAETSNRVQLRDVIDR VQKLKYSGNMYWTRDEFSRYKKSVVARLKRRTSDPEVIQS IESTGYAAVALRDRLLSYGEKNGV AQVAVFRGGVTAEARRWLDI S IERLFSRVAIFAQSTSTKRLDRRHHAVDAVVLTTLTPGVAKTL ADARSRRVSAEFWRRPSDVNRHSTEEPQSPAYRQWKESCSGLGDLLI STAARDS IAVAAPLRLR PTGALHEETLRAFSEHTVGAAWKGAELRRIVEPEVYAAFLALTDPGGRFLKVSPSEDVLP ADEN RHIVLSDRVLGPRDRVKLFPDDRGS IRVRGGAAYIASFHHARVFRWGSSHSPSFALLRVSLADL AVAGLLRDGVDVFTAELPPWTPAWRYAS IALVKAVESGDAKQVGWLVPGDELDFGPEGVTTAAG DLSMFLKYFPERHWVVTGFEDDKRINLKPAFLSAEQAEVLRTERSDRPDTLTEAGEILAQ FFPR CWRATVAKVLCHPGLTVIRRTALGQPRWRRGHLPYSWRPWSADPWSGGTP

SEQ ID NO: 386

MHNKKNITIGFDLGIAS IGWAI IDSTTSKILDWGTRTFEERKTANERRAFRSTRRNIRRKAYRN QRFINLILKYKDLFELKNI SDIQRANKKDTENYEKI I SFFTEIYKKCAAKHS ILEVKVKALDS KIEKLDLIWILHDYLENRGFFYDLEEENVADKYEGIEHPS ILLYDFFKKNGFFKSNSS IPKDLG GYSFSNLQWVNEIKKLFEVQEINPEFSEKFLNLFTSVRDYAKGPGSEHSASEYGIFQKDE KGKV FKKYDNIWDKTIGKCSFFVEENRSPVNYPSYEIFNLLNQLINLSTDLKTTNKKIWQLSSN DRNE LLDELLKVKEKAKI I S I SLKKNEIKKI ILKDFGFEKSDIDDQDTIEGRKI IKEEPTTKLEVTKH LLATIYSHSSDSNWININNILEFLPYLDAICI ILDREKSRGQDEVLKKLTEKNIFEVLKIDREK QLDFVKS IFSNTKFNFKKIGNFSLKAIREFLPKMFEQNKNSEYLKWKDEEIRRKWEEQKSKLGK TDKKTKYLNPRIFQDEI I SPGTKNTFEQAVLVLNQI IKKYSKENI IDAI I IESPREKNDKKTIE EIKKRNKKGKGKTLEKLFQILNLENKGYKLSDLETKPAKLLDRLRFYHQQDGIDLYTLDK INID QLINGSQKYEIEHI IPYSMSYDNSQANKILTEKAENLKKGKLIASEYIKRNGDEFYNKYYEKAK ELFINKYKKNKKLDSYVDLDEDSAKNRFRFLTLQDYDEFQVEFLARNLNDTRYSTKLFYH ALVE HFENNEFFTYIDENSSKHKVKI STIKGHVTKYFRAKPVQKNNGPNENLNNNKPEKIEKNRENNE HHAVDAAIVAI IGNKNPQIANLLTLADNKTDKKFLLHDENYKE IETGELVKIPKFEVDKLAKV EDLKKI IQEKYEEAKKHTAIKFSRKTRTILNGGLSDETLYGFKYDEKEDKYFKI IKKKLVTSKN EELKKYFENPFGKKADGKSEYTVLMAQSHLSEFNKLKEIFEKYNGFSNKTGNAFVEYMND LALK EPTLKAEIESAKSVEKLLYYNFKPSDQFTYHDNINNKSFKRFYKNIRI IEYKS IPIKFKILSKH DGGKSFKDTLFSLYSLVYKVYENGKESYKS IPVTSQMRNFGIDEFDFLDENLYNKEKLDIYKSD FAKPIPVNCKPVFVLKKGS ILKKKSLDIDDFKETKETEEGNYYFI STI SKRFNRDTAYGLKPLK LSVVKPVAEPSTNPIFKEYIPIHLDELGNEYPVKIKEHTDDEKLMCTIK

Nucleic Acids Encoding Cas9 Molecules

Nucleic acids encoding the Cas9 molecules or Cas9 polypeptides, e.g., an eaCas9 molecule or eaCas9 polypeptide, are provided herein.

Exemplary nucleic acids encoding Cas9 molecules or Cas9 polypeptides are described in

Cong et al., SCIENCE 2013, 399(6121):819-823; Wang et al., CELL 2013, 153(4):910-918; Mali et al., SCIENCE 2013, 399(6121):823-826; Jinek et al., SCIENCE 2012, 337(6096):816-821. Another exemplary nucleic acid encoding a Cas9 molecule or Cas9 polypeptide is shown in black in Fig. 8.

In an embodiment, a nucleic acid encoding a Cas9 molecule or Cas9 polypeptide can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section VIII. In an embodiment, the Cas9 mRNA has one or more (e.g., all of the following properties: it is capped, polyadenylated, substituted with 5- methylcytidine and/or pseudouridine.

In addition, or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.

In addition, or alternatively, a nucleic acid encoding a Cas9 molecule or Cas9

polypeptide may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. pyogenes.

ATGGATAAAA AGTACAGCAT CGGGCTGGAC ATCGGTACAA ACTCAGTGGG GTGGGCCGTG AT TACGGACG AGTACAAGGT ACCCTCCAAA AAAT T TAAAG TGCTGGGTAA CACGGACAGA CACTCTATAA AGAAAAATCT TAT TGGAGCC T TGCTGT TCG ACTCAGGCGA GACAGCCGAA GCCACAAGGT TGAAGCGGAC CGCCAGGAGG CGGTATACCA GGAGAAAGAA CCGCATATGC TACCTGCAAG AAATCT TCAG TAACGAGATG GCAAAGGT TG ACGATAGCT T T T TCCATCGC CTGGAAGAAT CCT T TCT TGT TGAGGAAGAC AAGAAGCACG AACGGCACCC CATCT T TGGC AATAT TGTCG ACGAAGTGGC ATATCACGAA AAGTACCCGA CTATCTACCA CCTCAGGAAG AAGCTGGTGG ACTCTACCGA TAAGGCGGAC CTCAGACT TA T T TAT T TGGC ACTCGCCCAC AT GAT T AAA T T TAGAGGACA T T TCT TGATC GAGGGCGACC TGAACCCGGA CAACAGTGAC GTCGATAAGC TGT TCATCCA ACT TGTGCAG ACCTACAATC AACTGT TCGA AGAAAACCCT ATAAATGCT T CAGGAGTCGA CGCTAAAGCA ATCCTGTCCG CGCGCCTCTC AAAATCTAGA AGACT TGAGA ATCTGAT TGC TCAGT TGCCC GGGGAAAAGA AAAATGGAT T GT T TGGCAAC CTGATCGCCC TCAGTCTCGG ACTGACCCCA AAT T TCAAAA GTAACT TCGA CCTGGCCGAA GACGCTAAGC TCCAGCTGTC CAAGGACACA TACGATGACG ACCTCGACAA TCTGCTGGCC CAGAT TGGGG ATCAGTACGC CGATCTCT T T T TGGCAGCAA AGAACCTGTC CGACGCCATC CTGT TGAGCG ATATCT TGAG AGTGAACACC GAAAT TACTA AAGCACCCCT TAGCGCATCT ATGATCAAGC GGTACGACGA GCATCATCAG GATCTGACCC TGCTGAAGGC TCTTGTGAGG CAACAGCTCC CCGAAAAATA CAAGGAAATC TTCTTTGACC AGAGCAAAAA CGGCTACGCT GGCTATATAG ATGGTGGGGC CAGTCAGGAG GAATTCTATA AATTCATCAA GCCCATTCTC GAGAAAATGG ACGGCACAGA GGAGTTGCTG GTCAAACTTA ACAGGGAGGA CCTGCTGCGG AAGCAGCGGA CCTTTGACAA CGGGTCTATC CCCCACCAGA TTCATCTGGG CGAACTGCAC GCAATCCTGA GGAGGCAGGA GGATTTTTAT CCTTTTCTTA AAGATAACCG CGAGAAAATA GAAAAGATTC TTACATTCAG GATCCCGTAC TACGTGGGAC CTCTCGCCCG GGGCAATTCA CGGTTTGCCT GGATGACAAG GAAGTCAGAG GAGACTATTA CACCTTGGAA CTTCGAAGAA GTGGTGGACA AGGGTGCATC TGCCCAGTCT TTCATCGAGC GGATGACAAA TTTTGACAAG AACCTCCCTA ATGAGAAGGT GCTGCCCAAA CATTCTCTGC TCTACGAGTA CTTTACCGTC TACAATGAAC TGACTAAAGT CAAGTACGTC ACCGAGGGAA TGAGGAAGCC GGCATTCCTT AGTGGAGAAC AGAAGAAGGC GATTGTAGAC CTGTTGTTCA AGACCAACAG GAAGGTGACT GTGAAGCAAC TTAAAGAAGA CTACTTTAAG AAGATCGAAT GTTTTGACAG TGTGGAAATT TCAGGGGTTG AAGACCGCTT CAATGCGTCA TTGGGGACTT ACCATGATCT TCTCAAGATC ATAAAGGACA AAGACTTCCT GGACAACGAA GAAAATGAGG ATATTCTCGA AGACATCGTC CTCACCCTGA CCCTGTTCGA AGACAGGGAA ATGATAGAAG AGCGCTTGAA AACCTATGCC CACCTCTTCG ACGATAAAGT TATGAAGCAG CTGAAGCGCA GGAGATACAC AGGATGGGGA AGATTGTCAA GGAAGCTGAT CAATGGAATT AGGGATAAAC AGAGTGGCAA GACCATACTG GATTTCCTCA AATCTGATGG CTTCGCCAAT AGGAACTTCA TGCAACTGAT TCACGATGAC TCTCTTACCT TCAAGGAGGA CATTCAAAAG GCTCAGGTGA GCGGGCAGGG AGACTCCCTT CATGAACACA TCGCGAATTT GGCAGGTTCC CCCGCTATTA AAAAGGGCAT CCTTCAAACT GTCAAGGTGG TGGATGAATT GGTCAAGGTA ATGGGCAGAC ATAAGCCAGA AAATATTGTG ATCGAGATGG CCCGCGAAAA CCAGACCACA CAGAAGGGCC AGAAAAATAG TAGAGAGCGG ATGAAGAGGA TCGAGGAGGG CATCAAAGAG CTGGGATCTC AGATTCTCAA AGAACACCCC GTAGAAAACA CACAGCTGCA GAACGAAAAA TTGTACTTGT ACTATCTGCA GAACGGCAGA GACATGTACG TCGACCAAGA ACTTGATATT AATAGACTGT CCGACTATGA CGTAGACCAT ATCGTGCCCC AGTCCTTCCT GAAGGACGAC TCCATTGATA ACAAAGTCTT GACAAGAAGC GACAAGAACA GGGGTAAAAG TGATAATGTG CCTAGCGAGG AGGTGGTGAA AAAAATGAAG AACTACTGGC GACAGCTGCT TAATGCAAAG CTCATTACAC AACGGAAGTT CGATAATCTG ACGAAAGCAG AGAGAGGTGG CTTGTCTGAG TTGGACAAGG CAGGGTTTAT TAAGCGGCAG CTGGTGGAAA CTAGGCAGAT CACAAAGCAC GTGGCGCAGA TTTTGGACAG CCGGATGAAC ACAAAATACG ACGAAAATGA TAAACTGATA CGAGAGGTCA AAGTTATCAC GCTGAAAAGC AAGCTGGTGT CCGATTTTCG GAAAGACTTC CAGTTCTACA AAGTTCGCGA GATTAATAAC TACCATCATG CTCACGATGC GTACCTGAAC GCTGTTGTCG GGACCGCCTT GATAAAGAAG TACCCAAAGC TGGAATCCGA GTTCGTATAC GGGGATTACA AAGTGTACGA TGTGAGGAAA ATGATAGCCA AGTCCGAGCA GGAGATTGGA AAGGCCACAG CTAAGTACTT CTTTTATTCT AACATCATGA ATTTTTTTAA GACGGAAATT ACCCTGGCCA ACGGAGAGAT CAGAAAGCGG CCCCTTATAG AGACAAATGG TGAAACAGGT GAAATCGTCT GGGATAAGGG CAGGGATTTC GCTACTGTGA GGAAGGTGCT GAGTATGCCA CAGGTAAATA TCGTGAAAAA AACCGAAGTA CAGACCGGAG GATTTTCCAA GGAAAGCATT TTGCCTAAAA GAAACTCAGA CAAGCTCATC GCCCGCAAGA AAGATTGGGA CCCTAAGAAA TACGGGGGAT TTGACTCACC CACCGTAGCC TATTCTGTGC TGGTGGTAGC TAAGGTGGAA AAAGGAAAGT CTAAGAAGCT GAAGTCCGTG AAGGAACTCT TGGGAATCAC TATCATGGAA AGATCATCCT TTGAAAAGAA CCCTATCGAT TTCCTGGAGG CTAAGGGTTA CAAGGAGGTC AAGAAAGACC TCATCATTAA ACTGCCAAAA TACTCTCTCT TCGAGCTGGA AAATGGCAGG AAGAGAATGT TGGCCAGCGC CGGAGAGCTG CAAAAGGGAA ACGAGCTTGC TCTGCCCTCC AAATATGTTA ATTTTCTCTA TCTCGCTTCC CACTATGAAA AGCTGAAAGG GTCTCCCGAA GATAACGAGC AGAAGCAGCT GTTCGTCGAA CAGCACAAGC ACTATCTGGA TGAAATAATC GAACAAATAA GCGAGTTCAG CAAAAGGGTT ATCCTGGCGG ATGCTAATTT GGACAAAGTA CTGTCTGCTT ATAACAAGCA CCGGGATAAG CCTATTAGGG AACAAGCCGA GAATATAATT CACCTCTTTA CACTCACGAA TCTCGGAGCC CCCGCCGCCT TCAAATACTT TGATACGACT ATCGACCGGA AACGGTATAC CAGTACCAAA GAGGTCCTCG ATGCCACCCT CATCCACCAG TCAATTACTG GCCTGTACGA AACACGGATC GACCTCTCTC AACTGGGCGG CGACTAG

(SEQ ID NO: 22)

Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.

MDKKYS IGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFDSGETAEATRL KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAY HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF KSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP LSAS MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPIL EKMD GTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVL PKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI ECFD SVEI SGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD SLTF KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINR LSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK MIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLS MPQV IVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQI SEFSKRV ILADANLDKVLSAYNKHRDKPIREQAE I IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQS ITGLYETRIDLSQLGGD*

(SEQ ID NO: 23) Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitidis.

ATGGCCGCCT TCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACATCGGCATCGCCAGCG TGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGAACCCCATCTGCCTGATCGACCTGG GTGT GCGCGTGT TCGAGCGCGCTGAGGTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCT T GCTCGCTCTGT TCGGCGCCT TACTCGCCGGCGCGCTCACCGCCT TCTGCGCGCTCGCCGCCTGC TGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACT TCGACGAGAACGGCCTGATCAAGAGCCTGCC CAACACTCCT TGGCAGCTGCGCGCTGCCGCTCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGC GCCGTGCTGCTGCACCTGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGC GAGA CCGCCGACAAGGAGCTGGGTGCTCTGCTGAAGGGCGTGGCCGACAACGCCCACGCCCTGC AGAC TGGTGACT TCCGCACTCCTGCTGAGCTGGCCCTGAACAAGT TCGAGAAGGAGAGCGGCCACATC CGCAACCAGCGCGGCGACTACAGCCACACCT TCAGCCGCAAGGACCTGCAGGCCGAGCTGATCC TGCTGT TCGAGAAGCAGAAGGAGT TCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGGGCAT CGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCCGTGCAGAAGATGCT GGGC CACTGCACCT TCGAGCCAGCCGAGCCCAAGGCCGCCAAGAACACCTACACCGCCGAGCGCT TCA TCTGGCTGACCAAGCTGAACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGA CCGA CACCGAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTACGCCCA GGCC CGCAAGCTGCTGGGTCTGGAGGACACCGCCT TCT TCAAGGGCCTGCGCTACGGCAAGGACAACG CCGAGGCCAGCACCCTGATGGAGATGAAGGCCTACCACGCCATCAGCCGCGCCCTGGAGA AGGA GGGCCTGAAGGACAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGG CACC GCCT TCAGCCTGT TCAAGACCGACGAGGACATCACCGGCCGCCTGAAGGACCGCATCCAGCCCG AGATCCTGGAGGCCCTGCTGAAGCACATCAGCT TCGACAAGT TCGTGCAGATCAGCCTGAAGGC CCTGCGCCGCATCGTGCCCCTGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGA GATC TACGGCGACCACTACGGCAAGAAGAACACCGAGGAGAAGATCTACCTGCCTCCTATCCCC GCCG ACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGGCCCGCAAGGTGATCAACG GCGT GGTGCGCCGCTACGGCAGCCCCGCCCGCATCCACATCGAGACCGCCCGCGAGGTGGGCAA GAGC T TCAAGGACCGCAAGGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGG CCG CCGCCAAGT TCCGCGAGTACT TCCCCAACT TCGTGGGCGAGCCCAAGAGCAAGGACATCCTGAA GCTGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCTGTACAGCGGCAAGGAGATCAACCT GGGC CGCCTGAACGAGAAGGGCTACGTGGAGATCGACCACGCCCTGCCCT TCAGCCGCACCTGGGACG ACAGCT TCAACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCAGACCCC CTACGAGTACT TCAACGGCAAGGACAACAGCCGCGAGTGGCAGGAGT TCAAGGCCCGCGTGGAG ACCAGCCGCT TCCCCCGCAGCAAGAAGCAGCGCATCCTGCTGCAGAAGT TCGACGAGGACGGCT TCAAGGAGCGCAACCTGAACGACACCCGCTACGTGAACCGCT TCCTGTGCCAGT TCGTGGCCGA CCGCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGT TCGCCAGCAACGGCCAGATCACCAAC CTGCTGCGCGGCT TCTGGGGCCTGCGCAAGGTGCGCGCCGAGAACGACCGCCACCACGCCCTGG ACGCCGTGGTGGTGGCCTGCAGCACCGTGGCCATGCAGCAGAAGATCACCCGCT TCGTGCGCTA CAAGGAGATGAACGCCT TCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCACCAG AAGACCCACT TCCCCCAGCCCTGGGAGT TCT TCGCCCAGGAGGTGATGATCCGCGTGT TCGGCA AGCCCGACGGCAAGCCCGAGT TCGAGGAGGCCGACACCCCCGAGAAGCTGCGCACCCTGCTGGC CGAGAAGCTGAGCAGCCGCCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGT TCGTGAGCCGC GCCCCCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCCAAGCGC CTGG ACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCTGAAGCTGAAGGACCTGGAGA AGAT GGTGAACCGCGAGCGCGAGCCCAAGCTGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCA CAAG GACGACCCCGCCAAGGCCT TCGCCGAGCCCT TCTACAAGTACGACAAGGCCGGCAACCGCACCC AGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGTGCGCAACC ACAA CGGCATCGCCGACAACGCCACCATGGTGCGCGTGGACGTGTTCGAGAAGGGCGACAAGTA CTAC CTGGTGCCCATCTACAGCTGGCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTG CAGG GCAAGGACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGCCTGC ACCC CAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTTCGGCTACTTCGCCAGCTG CCAC CGCGGCACCGGCAACATCAACATCCGCATCCACGACCTGGACCACAAGATCGGCAAGAAC GGCA TCCTGGAGGGCATCGGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGC TGGG CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA

(SEQ ID NO: 24)

Provided below is the corresponding amino acid sequence of a N. meningitidis Cas9 molecule.

MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDSLAM ARRL ARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTP LEWS AVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKE SGHI RNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQ KMLG HCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLT YAQA RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAI SRALEKEGLKDKKSPLNLSPELQDEIGT AFSLFKTDEDITGRLKDRIQPEILEALLKHI SFDKFVQI SLKALRRIVPLMEQGKRYDEACAEI YGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETARE VGKS FKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKE INLG RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK ARVE TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG QITN LLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGE VLHQ KTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPL FVSR APNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARL EAHK DDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKG DKYY LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYF ASCH RGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR* (SEQ ID NO: 25)

Provided below is an amino acid sequence of a S. aureus Cas9 molecule.

MKRNYILGLDIGITSVGYGI IDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEV EEDT GNELSTKEQI SRNSKALEEKYVAELQLERLKKDGEVRGS INRFKTSDYVKEAKQLLKVQKAYHQ LDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYN ADLY NALNDLNNLVITRDENEKLEYYEKFQI IENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK PEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQIS NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDF ILSP VVKRSFIQS IKVINAI IKKYGLPNDI I IELAREKNSKDAQKMINEMQKRNRQTNERIEEI IRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVK QEENSKKGNRTPFQYLSSSDSKI SYETFKKHILNLAKGKGRI SKTKKEYLLEERDINRFSVQKD FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFTSFLRRKWKFKKERNKGYKHHAED ALI IANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFK D YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLL MYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLD ITDD YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKI SNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKT QS IKKYSTDILGNLYEVKSKKHPQI IKKG*

(SEQ ID NO: 26) Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. aureus Cas9.

ATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGGGTATGGGATT ATTG ACTATGAAACAAGGGACGTGATCGACGCAGGCGTCAGACTGTTCAAGGAGGCCAACGTGG AAAA CAATGAGGGACGGAGAAGCAAGAGGGGAGCCAGGCGCCTGAAACGACGGAGAAGGCACAG AATC CAGAGGGTGAAGAAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTGAGCTGAGT GGAA TTAATCCTTATGAAGCCAGGGTGAAAGGCCTGAGTCAGAAGCTGTCAGAGGAAGAGTTTT CCGC AGCTCTGCTGCACCTGGCTAAGCGCCGAGGAGTGCATAACGTCAATGAGGTGGAAGAGGA CACC GGCAACGAGCTGTCTACAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAG TATG TCGCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTCAATTAATA GGTT CAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGCTGAAAGTGCAGAAGGCTTACCA CCAG CTGGATCAGAGCTTCATCGATACTTATATCGACCTGCTGGAGACTCGGAGAACCTACTAT GAGG GACCAGGAGAAGGGAGCCCCTTCGGATGGAAAGACATCAAGGAATGGTACGAGATGCTGA TGGG ACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACGCTTATAACGCAGATCT GTAC AACGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAAAACGAGAAACTGGAA TACT ATGAGAAGTTCCAGATCATCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTACACTGAAAC AGAT TGCTAAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAGCACTGG AAAA CCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGGACATCACAGCACGGAAAGAA ATCA TTGAGAACGCCGAACTGCTGGATCAGATTGCTAAGATCCTGACTATCTACCAGAGCTCCG AGGA CATCCAGGAAGAGCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGAT TAGT AATCTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATCAATCTGATTCTG GATG AGCTGTGGCATACAAACGACAATCAGATTGCAATCTTTAACCGGCTGAAGCTGGTCCCAA AAAA GGTGGACCTGAGTCAGCAGAAAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTC ACCC GTGGTCAAGCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTAC GGCC TGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACAGCAAGGACGCACAGAAGA TGAT CAATGAGATGCAGAAACGAAACCGGCAGACCAATGAACGCATTGAAGAGATTATCCGAAC TACC GGGAAAGAGAACGCAAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGCAGGAGGGA AAGT GTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCATTCAACTACG AGGT CGATCATATTATCCCCAGAAGCGTGTCCTTCGACAATTCCTTTAACAACAAGGTGCTGGT CAAG CAGGAAGAGAACTCTAAAAAGGGCAATAGGACTCCTTTCCAGTACCTGTCTAGTTCAGAT TCCA AGATCTCTTACGAAACCTTTAAAAAGCACATTCTGAATCTGGCCAAAGGAAAGGGCCGCA TCAG CAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACAGATTCTCCGTCCAGAA GGAT TTTATTAACCGGAATCTGGTGGACACAAGATACGCTACTCGCGGCCTGATGAATCTGCTG CGAT CCTATTTCCGGGTGAACAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTCACAT CTTT TCTGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCACCATGCCGA AGAT GCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGAGTGGAAAAAGCTGGACAAAGCC AAGA AAGTGATGGAGAACCAGATGTTCGAAGAGAAGCAGGCCGAATCTATGCCCGAAATCGAGA CAGA ACAGGAGTACAAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAA GGAC TACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGATCAATGACACCCTG TATA GTACAAGAAAAGACGATAAGGGGAATACCCTGATTGTGAACAATCTGAACGGACTGTACG ACAA AGATAATGACAAGCTGAAAAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCA CCAT GATCCTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACGAGAAGAAC CCAC TGTATAAGTACTATGAAGAGACTGGGAACTACCTGACCAAGTATAGCAAAAAGGATAATG GCCC CGTGATCAAGAAGATCAAGTACTATGGGAACAAGCTGAATGCCCATCTGGACATCACAGA CGAT TACCCTAACAGTCGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGATTCGATGTC TATC TGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCATCAAAAAGGAGA ACTA CTATGAAGTGAATAGCAAGTGCTACGAAGAGGCTAAAAAGCTGAAAAAGATTAGCAACCA GGCA GAGTTCATCGCCTCCTTTTACAACAACGACCTGATTAAGATCAATGGCGAACTGTATAGG GTCA TCGGGGTGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACATCACTTACC GAGA GTATCTGGAAAACATGAATGATAAGCGCCCCCCTCGAATTATCAAAACAATTGCCTCTAA GACT CAGAGTATCAAAAAGTACTCAACCGACATTCTGGGAAACCTGTATGAGGTGAAGAGCAAA AAGC ACCCTCAGATTATCAAAAAGGGC

(SEQ ID NO: 39)

If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C- terminus, it is understood that the stop codon will be removed.

Other Cas Molecules and Cas Polypeptides

Various types of Cas molecules or Cas polypeptides can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of Type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et al, PLoS COMPUTATIONAL BIOLOGY 2005, 1(6): e60 and Makarova et al, NATURE REVIEW MICROBIOLOGY 2011, 9:467-477, the contents of both references are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table 16.

Table 16: Cas Systems

Gene System type Name from Structure of Families (and Representatives name* or subtype Haft ei a/. ^§ encoded protein superfamily) of

(PDB encoded

accessions) ¹ protein

cas4 • Subtype I-A cas4 and csal NA COG1468 APE1239 and BH0340

• Subtype I-B

• Subtype I-C

• Subtype I-D

• Subtype II- B

cas5 • Subtype I-A cas5a, cas5d, 3KG4 COG1688 APE1234, BH0337,

• Subtype I-B cas5e, cas5h, (RAMP) devS and ygcl

• Subtype I-C cas5p, cas5t

• Subtype I-E and cmx5

cas6 • Subtype I-A cas6 and cmx6 3I4H COG1583 and PF1131 and slr7014

• Subtype I-B COG5551

• Subtype I-D (RAMP)

• Subtype III- Α· Subtype

III-B

cas6e • Subtype I-E cse3 1WJ9 (RAMP) ygcH

cas6f • Subtype I-F csy4 2XLJ (RAMP) yl727

cas7 • Subtype I-A csal, csd2, NA COG 1857 and devR and ygcJ

• Subtype I-B cse4, csh2, COG3649

• Subtype I-C cspl and cst2 (RAMP)

• Subtype I-E

cas8al • Subtype I- cmxl, cstl, NA BH0338-like LA3191 and

A** csx8, csx 13 PG2018

and CXXC- CXXC

cas8a2 • Subtype I- csa4 and csx9 NA PH0918 AF0070, AF1873,

A** MJ0385, PF0637,

PH0918 and SSO1401 cas8b • Subtype I- cshl and NA BH0338-like MTH1090 and

B« TM1802 TM1802 cas8c • Subtype I- csdl and cspl NA BH0338-like BH0338

C**

cas9 • Type II** csnl and csxl2 NA COG3513 FTN_0757 and

SPyl046 cas 10 • Type III** cmr2, csml NA COG1353 MTH326, Rv2823c and csxll and TM1794 caslOd • Subtype I- csc3 NA COG1353 slr7011

D**

csyl • Subtype I- csyl NA yl724-like yl724

F**

csy2 • Subtype I-F csy2 NA (RAMP) y!725 Table 16: Cas Systems

Gene System type Name from Structure of Families (and Representatives name* or subtype Hatt et al encoded protein superfamily) of

(PDB encoded

accessions) ¹ protein

csy3 • Subtype I-F csy3 NA (RAMP) yl726

csel • Subtype I- csel NA YgcL-like ygcL

E«

cse2 • Subtype I-E cse2 2ZCA YgcK-like ygcK

cscl • Subtype I-D cscl NA alrl563-like alrl563

(RAMP)

csc2 • Subtype I-D cscl and csc2 NA COG1337 slr7012

(RAMP)

csa5 • Subtype I-A csa5 NA AF1870 AF1870, MJ0380,

PF0643 and SS01398 csn2 • Subtype II- csn2 NA SPyl049-like SPyl049

A

csm.2 • Subtype III- csm.2 NA COG1421 MTH1081 and

A« SERP2460 csm.3 • Subtype III- csc2 and csm.3 NA COG1337 MTH1080 and

A (RAMP) SERP2459 csm.4 • Subtype III- csm4 NA COG 1567 MTH1079 and

A (RAMP) SERP2458 csm5 • Subtype III- csm5 NA COG1332 MTH1078 and

A (RAMP) SERP2457 csm.6 • Subtype III- APE2256 and 2WTE COG1517 APE2256 and

A csm6 SS01445 cmrl • Subtype Hi- cmrl NA COG 1367 PF1130

fi (RAMP)

cmr3 • Subtype Hi- cmr3 NA COG 1769 PF1128

fi (RAMP)

cmr4 • Subtype Hi- cmr4 NA COG1336 PF1126

fi (RAMP)

cmr5 • Subtype III- cmr5 2ZOP and 20EB COG3337 MTH324 and PF1125

B«

cmr6 • Subtype Hi- cmr6 NA COG 1604 PF1124

fi (RAMP)

csbl • Subtype I-U GSU0053 NA (RAMP) Balac_1306 and

GSU0053 csb2 • Subtype I- NA NA (RAMP) Balac_1305 and

U GSU0054 csb3 • Subtype I-U NA NA (RAMP) Balac_1303 csx 17 • Subtype I-U NA NA NA Btus_2683 csx 14 • Subtype I-U NA NA NA GSU0052 Table 16: Cas Systems

Gene System type Name from Structure of Families (and Representatives name* or subtype Hatt et al encoded protein superfamily) of

(PDB encoded

accessions) ¹ protein

csx 10 • Subtype I-U csx 10 NA (RAMP) Caur_2274 csx 16 • Subtype III- VVA1548 NA NA VVA1548

U

csaX • Subtype III- csaX NA NA SS01438

U

csx3 • Subtype III- csx3 NA NA AF1864

U

csxl • Subtype III- csa3, csxl, lXMX and 2171 COG1517 and MJ1666, NE0113,

U csx2, DXTHG, COG4006 PF1127 and TM1812

NE0113 and

TIGR02710

csx 15 • Unknown NA NA TTE2665 TTE2665 csfl • Type U csfl NA NA AFE_1038 csfl • Type U csfl NA (RAMP) AFE_1039 csf3 • Type U csf3 NA (RAMP) AFE_1040 csf4 • Type U csf4 NA NA AFE_1037

IV. Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are described, e.g., in Jinek et al, SCIENCE 2012, 337(6096):816-821.

Binding and Cleavage Assay: Testing the endonuclease activity of Cas9 molecule

The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro- transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95 °C and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (~8 nM)) is incubated for 60 min at 37°C with purified Cas9 protein molecule (50-500 nM) and gRNA (50-500 nM, 1: 1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KC1, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl ₂ . The reactions are stopped with 5X DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.

Alternatively, the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotide kinase and -3-6 pmol (-20-40 mCi) [γ-32Ρ]-ΑΤΡ in IX T4 polynucleotide kinase reaction buffer at 37°C for 30 min, in a 50 μΐ _^ reaction. After heat inactivation (65°C for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates (100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95 °C for 3 min, followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95°C for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) is pre- incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM

HEPES pH 7.5, 100 mM KC1, 5 mM MgC12, 1 mM DTT, 5% glycerol) in a total volume of 9 μΐ. Reactions are initiated by the addition of 1 μΐ target DNA (10 nM) and incubated for 1 h at 37°C. Reactions are quenched by the addition of 20 μΐ of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95°C for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging. The resulting cleavage products indicate that whether the complementary strand, the non- complementary strand, or both, are cleaved.

One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule or candidate Cas9 molecule. Binding Assay: Testing the binding of Cas9 molecule to target DNA

Exemplary methods for evaluating the binding of Cas9 molecule to target DNA are described, e.g., in Jinek et al, SCIENCE 2012; 337(6096):816-821.

For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand (10 nmol) in deionized water, heating to 95 °C for 3 min and slow cooling to room temperature. All DNAs are purified on 8% native gels containing IX TBE. DNA bands are visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H ₂ 0. Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H ₂ 0. DNA samples are 5' end labeled with [γ-32Ρ]-ΑΤΡ using T4 polynucleotide kinase for 30 min at 37°C. Polynucleotide kinase is heat denatured at 65°C for 20 min, and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KC1, 5 mM MgCl ₂ , 1 mM DTT and 10% glycerol in a total volume of 10 μΐ. Cas9 protein molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 pM to 1 μΜ. Radiolabeled DNA is added to a final concentration of 20 pM. Samples are incubated for 1 h at 37°C and resolved at 4°C on an 8% native polyacrylamide gel containing IX TBE and 5 mM MgCl ₂ . Gels are dried and DNA visualized by

phosphorimaging.

Differential Scanning Flourimetry (DSF)

The thermostability of Cas9-gRNA ribonucleoprotein (RNP) complexes can be measured via DSF. This technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g., a gRNA.

The assay is performed using two different protocols, one to test the best stoichiometric ratio of gRNA:Cas9 protein and another to determine the best solution conditions for RNP formation.

To determine the best solution to form RNP complexes, a 2uM solution of Cas9 in water+10x SYPRO Orange® (Life Techonologies cat#S-6650) and dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for lO'and brief centrifugation to remove any bubbles,a Bio-Rad CFX384™ Real-Time System CI 000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1° increase in temperature every 10 seconds.

The second assay consists of mixing various concentrations of gRNA with 2uM Cas9 in optimal buffer from assay 1 above and incubating at RT for 10' in a 384 well plate. An equal volume of optimal buffer + lOx SYPRO Orange® (Life Techonologies cat#S-6650) is added and the plate sealed with Microseal® B adhesive (MSB-1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System CIOOO Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20°C to 90°C with a 1° increase in temperature every 10 seconds.

V. Genome Editing Approaches

While not wishing to be bound by theory, altering the LCA10 target position may be achieved using one of the approaches discussed herein.

V.l NHEJ Approaches for Gene Targeting

As described herein, nuclease-induced non-homologous end-joining (NHEJ) can be used to introduce indels at a target position. Nuclease-induced NHEJ can also be used to remove (e.g., delete) genomic sequence including the mutation at a target position in a gene of interest.

While not wishing to be bound by theory, it is believed that, in an embodiment, the genomic alterations associated with the methods described herein rely on nuclease-induced NHEJ and the error-prone nature of the NHEJ repair pathway. NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair.

The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most commonly in the 1-50 bp range, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it can also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. If a double- strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences; however, the error-prone nature of NHEJ may still produce indel mutations at the site of deletion.

Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate break- induced indels.

Double strand break

In an embodiment, double strand cleavage is effected by a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-terminal RuvC-like domain, e.g., a wild type Cas9. Such embodiments require only a single gRNA.

Single strand break

In other embodiments, two single strand breaks are effected by a Cas9 molecule having nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments require two gRNAs, one for placement of each single strand break. In an embodiment, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is

complementary to the strand to which the gRNA hybridizes. In an embodiment, the Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes.

In an embodiment, the nickase has HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation. D10A inactivates RuvC therefore the Cas9 nickase has (only) HNH activity and will cut on the strand to which the gRNA hybridizes (the complementary strand, which does not have the NGG PAM on it). In other embodiments, a Cas9 molecule having an H840, e.g., an H840A, mutation can be used as a nickase. H840A inactivates HNH therefore the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (the strand that has the NGG PAM and whose sequence is identical to the gRNA). In other embodiments, a Cas9 molecule having an H863, e.g., an H863A, mutation can be used as a nickase. H863A inactivates HNH therefore the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (the strand that has the NGG PAM and whose sequence is identical to the gRNA).

In an embodiment, in which a nickase and two gRNAs are used to position two single strand breaks, one nick is on the + strand and one nick is on the - strand of the target nucleic acid. The PAMs can be outwardly facing. The gRNAs can be selected such that the gRNAs are separated by, from 0-50, 0-100, or 0-200 nucleotides. In an embodiment, there is no overlap between the target sequences that are complementary to the targeting domains of the two gRNAs. In an embodiment, the gRNAs do not overlap and are separated by as much as 50, 100, or 200 nucleotides. In an embodiment, the use of two gRNAs can increase specificity, e.g., by decreasing off-target binding (Ran et al., Cell 2013; 154(6): 1380-1389).

Placement of double strand or single strand breaks relative to the target position

In an embodiment, in which a gRNA and Cas9 nuclease generate a double strand break for the purpose of inducing break- induced indels, a gRNA, e.g., a unimolecular (or chimeric) or modular gRNA molecule, is configured to position one double-strand break in close proximity to a nucleotide of the target position. In an embodiment, the cleavage site is between 0-40 bp away from the target position (e.g., less than 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position).

In an embodiment, in which two gRNAs complexing with a Cas9 nickase induce two single strand breaks for the purpose of introducing break-induced indels, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position two single-strand breaks to provide for NHEJ-mediated alteration of a nucleotide of the target position. In an embodiment, the gRNAs are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, essentially mimicking a double strand break. In an embodiment, the two nicks are between 0-40 bp away from the target position (e.g., less than 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position) respectively, and the two single strand breaks are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). In an embodiment, the gRNAs are configured to place a single strand break on either side of the target position. In an embodiment, the gRNAs are configured to place a single strand break on the same side (either 5' or 3') of the target position.

Regardless of whether a break is a double strand or a single strand break, the gRNA should be configured to avoid unwanted target chromosome elements, such as repeated elements, e.g., an Alu repeat, in the target domain. In addition, a break, whether a double strand or a single strand break, should be sufficiently distant from any sequence that should not be altered. For example, cleavage sites positioned within introns should be sufficiently distant from any intron/exon border, or naturally occurring splice signal, to avoid alteration of the exonic sequence or unwanted splicing events.

V.2 Single- Strand Annealing

Single strand annealing (SSA) is another DNA repair process that repairs a double-strand break between two repeat sequences present in a target nucleic acid. Repeat sequences utilized by the SSA pathway are generally greater than 30 nucleotides in length. Resection at the break ends occurs to reveal repeat sequences on both strands of the target nucleic acid. After resection, single strand overhangs containing the repeat sequences are coated with RPA protein to prevent the repeats sequences from inappropriate annealing, e.g., to themselves. RAD52 binds to and each of the repeat sequences on the overhangs and aligns the sequences to enable the annealing of the complementary repeat sequences. After annealing, the single-strand flaps of the overhangs are cleaved. New DNA synthesis fills in any gaps, and ligation restores the DNA duplex. As a result of the processing, the DNA sequence between the two repeats is deleted. The length of the deletion can depend on many factors including the location of the two repeats utilized, and the pathway or processivity of the resection.

In contrast to HDR pathways, SSA does not require a template nucleic acid to alter or correct a target nucleic acid sequence. Instead, the complementary repeat sequence is utilized.

V. 3 Other DNA Repair Pathways

SSBR (single strand break repair)

Single- stranded breaks (SSB) in the genome are repaired by the SSBR pathway, which is a distinct mechanism from the DSB repair mechanisms discussed above. The SSBR pathway has four major stages: SSB detection, DNA end processing, DNA gap filling, and DNA ligation. A more detailed explanation is given in Caldecott, Nature Reviews Genetics 9, 619-631 (August

2008), and a summary is given here.

In the first stage, when a SSB forms, PARP1 and/or PARP2 recognize the break and recruit repair machinery. The binding and activity of PARP1 at DNA breaks is transient and it seems to accelerate SSBr by promoting the focal accumulation or stability of SSBr protein complexes at the lesion. Arguably the most important of these SSBr proteins is XRCCl, which functions as a molecular scaffold that interacts with, stabilizes, and stimulates multiple enzymatic components of the SSBr process including the protein responsible for cleaning the

DNA 3' and 5' ends. For instance, XRCCl interacts with several proteins (DNA polymerase beta, PNK, and three nucleases, APE1, APTX, and APLF) that promote end processing. APE1 has endonuclease activity. APLF exhibits endonuclease and 3' to 5' exonuclease activities.

APTX has endonuclease and 3' to 5' exonuclease activity.

This end processing is an important stage of SSBR since the 3'- and/or 5 '-termini of most, if not all, SSBs are 'damaged'. End processing generally involves restoring a damaged 3'- end to a hydroxylated state and and/or a damaged 5' end to a phosphate moiety, so that the ends become ligation-competent. Enzymes that can process damaged 3' termini include PNKP,

APE1, and TDP1. Enzymes that can process damaged 5' termini include PNKP, DNA polymerase beta, and APTX. LIG3 (DNA ligase III) can also participate in end processing.

Once the ends are cleaned, gap filling can occur.

At the DNA gap filling stage, the proteins typically present are PARP1, DNA polymerase beta, XRCCl, FEN1 (flap endonculease 1), DNA polymerase delta/epsilon, PCNA, and LIG1.

There are two ways of gap filling, the short patch repair and the long patch repair. Short patch repair involves the insertion of a single nucleotide that is missing. At some SSBs, "gap filling" might continue displacing two or more nucleotides (displacement of up to 12 bases have been reported). FEN1 is an endonuclease that removes the displaced 5'-residues. Multiple DNA polymerases, including Pol β , are involved in the repair of SSBs, with the choice of DNA polymerase influenced by the source and type of SSB.

In the fourth stage, a DNA ligase such as LIG1 (Ligase I) or LIG3 (Ligase III) catalyzes joining of the ends. Short patch repair uses Ligase III and long patch repair uses Ligase I.

Sometimes, SSBR is replication-coupled. This pathway can involve one or more of CtIP,

MRN, ERCC1, and FEN1. Additional factors that may promote SSBR include: aPARP, PARP1, PARP2, PARG, XRCC1, DNA polymerase b, DNA polymerase d, DNA polymerase e, PCNA, LIGl, PNK, PNKP, APEl, APTX, APLF, TDPl, LIG3, FENl, CtIP, MRN, and ERCCl.

MMR (mismatch repair)

Cells contain three excision repair pathways: MMR, BER, and NER. The excision repair pathways hace a common feature in that they typically recognize a lesion on one strand of the DNA, then exo/endonucleaseases remove the lesion and leave a 1-30 nucleotide gap that is sub- sequentially filled in by DNA polymerase and finally sealed with ligase. A more complete picture is given in Li, Cell Research (2008) 18:85-98, and a summary is provided here.

Mismatch repair (MMR) operates on mispaired DNA bases.

The MSH2/6 or MSH2/3 complexes both have ATPases activity that plays an important role in mismatch recognition and the initiation of repair. MSH2/6 preferentially recognizes base- base mismatches and identifies mispairs of 1 or 2 nucleotides, while MSH2/3 preferentially recognizes larger ID mispairs.

hMLHl heterodimerizes with hPMS2 to form hMutL a which possesses an ATPase activity and is important for multiple steps of MMR. It possesses a PCNA/replication factor C (RFC)-dependent endonuclease activity which plays an important role in 3 ' nick-directed MMR involving EXOl. (EXOl is a participant in both HR and MMR.) It regulates termination of mismatch-provoked excision. Ligase I is the relevant ligase for this pathway. Additional factors that may promote MMR include: EXOl, MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, DNA Pol d, RPA, HMGB1, RFC, and DNA ligase I.

Base excision repair (BER)

The base excision repair (BER) pathway is active throughout the cell cycle; it is responsible primarily for removing small, non-helix-distorting base lesions from the genome. In contrast, the related Nucleotide Excision Repair pathway (discussed in the next section) repairs bulky helix-distorting lesions. A more detailed explanation is given in Caldecott, Nature

Reviews Genetics 9, 619-631 (August 2008), and a summary is given here.

Upon DNA base damage, base excision repair (BER) is initiated and the process can be simplified into five major steps: (a) removal of the damaged DNA base; (b) incision of the subsequent a basic site; (c) clean-up of the DNA ends; (d) insertion of the correct nucleotide into the repair gap; and (e) ligation of the remaining nick in the DNA backbone. These last steps are similar to the SSBR.

In the first step, a damage- specific DNA glycosylase excises the damaged base through cleavage of the N-glycosidic bond linking the base to the sugar phosphate backbone. Then AP endonuclease-1 (APE1) or bifunctional DNA glycosylases with an associated lyase activity incised the phosphodiester backbone to create a DNA single strand break (SSB). The third step of BER involves cleaning-up of the DNA ends. The fourth step in BER is conducted by Pol β that adds a new complementary nucleotide into the repair gap and in the final step

XRCCl/Ligase III seals the remaining nick in the DNA backbone. This completes the short- patch BER pathway in which the majority (-80%) of damaged DNA bases are repaired.

However, if the 5 ' -ends in step 3 are resistant to end processing activity, following one nucleotide insertion by Pol β there is then a polymerase switch to the replicative DNA polymerases, Pol δ/ε, which then add -2-8 more nucleotides into the DNA repair gap. This creates a 5 ' -flap structure, which is recognized and excised by flap endonuclease-1 (FEN-1) in association with the processivity factor proliferating cell nuclear antigen (PCNA). DNA ligase I then seals the remaining nick in the DNA backbone and completes long-patch BER. Additional factors that may promote the BER pathway include: DNA glycosylase, APE1, Polb, Pold, Pole, XRCC1, Ligase III, FEN-1, PCNA, RECQL4, WRN, MYH, PNKP, and APTX. Nucleotide excision repair (NER)

Nucleotide excision repair (NER) is an important excision mechanism that removes bulky helix-distorting lesions from DNA. Additional details about NER are given in Marteijn et al., Nature Reviews Molecular Cell Biology 15, 465-481 (2014), and a summary is given here.

NER a broad pathway encompassing two smaller pathways: global genomic NER (GG-NER) and transcription coupled repair NER (TC-NER). GG-NER and TC-NER use different factors for recognizing DNA damage. However, they utilize the same machinery for lesion incision, repair, and ligation.

Once damage is recognized, the cell removes a short single- stranded DNA segment that contains the lesion. Endonucleases XPF/ERCC1 and XPG (encoded by ERCC5) remove the lesion by cutting the damaged strand on either side of the lesion, resulting in a single-strand gap of 22-30 nucleotides. Next, the cell performs DNA gap filling synthesis and ligation. Involved in this process are: PCNA, RFC, DNA Pol δ, DNA Pol ε or DNA Pol κ, and DNA ligase I or XRCCl/Ligase III. Replicating cells tend to use DNA pol ε and DNA ligase I, while non- replicating cells tend to use DNA Pol δ, DNA Pol κ, and the XRCC1/ Ligase III complex to perform the ligation step.

NER can involve the following factors: XPA-G, POLH, XPF, ERCCl, XPA-G, and

LIGl. Transcription-coupled NER (TC-NER) can involve the following factors: CSA, CSB, XPB, XPD, XPG, ERCCl, and TTDA. Additional factors that may promote the NER repair pathway include XPA-G, POLH, XPF, ERCCl, XPA-G, LIGl, CSA, CSB, XPA, XPB, XPC, XPD, XPF, XPG, TTDA, UVSSA, USP7, CETN2, RAD23B, UV-DDB, CAK subcomplex, RPA, and PCNA.

Interstrand Crosslink (ICL)

A dedicated pathway called the ICL repair pathway repairs interstrand crosslinks.

Interstrand crosslinks, or covalent crosslinks between bases in different DNA strand, can occur during replication or transcription. ICL repair involves the coordination of multiple repair processes, in particular, nucleolytic activity, translesion synthesis (TLS), and HDR. Nucleases are recruited to excise the ICL on either side of the crosslinked bases, while TLS and HDR are coordinated to repair the cut strands. ICL repair can involve the following factors:

endonucleases, e.g., XPF and RAD51C, endonucleases such as RAD51, translesion polymerases, e.g., DNA polymerase zeta and Revl), and the Fanconi anemia (FA) proteins, e.g., FancJ.

Other pathways

Several other DNA repair pathways exist in mammals.

Translesion synthesis (TLS) is a pathway for repairing a single stranded break left after a defective replication event and involves translesion polymerases, e.g., DNA ροΐζ and Revl..

Error-free postreplication repair (PRR) is another pathway for repairing a single stranded break left after a defective replication event. V.4 Examples of gRNAs in Genome Editing Methods

gRNA molecules as described herein can be used with Cas9 molecules that cleave both or a single strand to alter the sequence of a target nucleic acid, e.g., of a target position or target genetic signature. gRNA molecules useful in these method are described below.

In an embodiment, the gRNA, e.g., a chimeric gRNA, molecule is configured such that it comprises one or more of the following properties;

a) it can position, e.g., when targeting a Cas9 molecule that makes double strand breaks, a double strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;

b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17,

(ii) 18, or (iii) 20 nucleotides; and

c)

(i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;

(ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S.

pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;

(iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;

iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or

(v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the

corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.

In an embodiment, the gRNA molecule is configured such that it comprises properties: a(i); and b(i).

In an embodiment, the gRNA molecule is configured such that it comprises properties: a(i); and b(ii).

In an embodiment, the gRNA molecule is configured such that it comprises properties: a(i); and b(iii).

In an embodiment, the gRNA molecule is configured such that it comprises properties: a(ii); and b(i).

In an embodiment, the gRNA molecule is configured such that it comprises properties: a(ii); and b(ii).

In an embodiment, the gRNA molecule is configured such that it comprises properties: a(ii); and b(iii).

In an embodiment, the gRNA molecule is configured such that it comprises properties: b(i); and c(i).

In an embodiment, the gRNA molecule is configured such that it comprises properties: b(i); and c(ii).

In an embodiment, the gRNA molecule is configured such that it comprises properties: b(ii); and c(i).

In an embodiment, the gRNA molecule is configured such that it comprises properties: b(ii); and c(ii).

In an embodiment, the gRNA molecule is configured such that it comprises properties: b(iii); and c(i).

In an embodiment, the gRNA molecule is configured such that it comprises properties: b(iii); and c(ii). In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.

In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H863, e.g., a H863A.

In an embodiment, a pair of gRNA molecules, e.g., a pair of chimeric gRNA molecules, comprising a first and a second gRNA molecule, is configured such that they comprises one or more of the following properties:

a) the first and second gRNA molecules position, e.g., when targeting a Cas9 molecule that makes single strand or double strand breaks:

(i) as positioned by a first and second gRNA molecule described herein; or

(ii) sufficiently close that the target position is altered when the break is repaired; b) one or both, independently, has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and

c) one or both, independently, has a the tail domain is (i) at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length or (ii) the tail domain comprises, 15, 20, 25, 30, 35, 40, or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S.

pyogenes, S. aureus, or S. thermophilus tail domain.

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: a(i); and b(i).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: a(i); and b(ii).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: a(i); and b(iii).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: a(ii); and b(i). embodiment, one or both of the gRNA molecules is configured such that it comprises properties: a(ii); and b(ii).

embodiment, one or both of the gRNA molecules is configured such that it comprises properties: a(ii); and b(iii).

embodiment, one or both of the gRNA molecules is configured such that it comprises properties: b(i); and c(i).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: b(i); and c(ii).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: b(ii); and c(i).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: b(ii); and c(ii).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: b(iii); and c(i).

In an embodiment, one or both of the gRNA molecules is configured such that it comprises properties: b(iii); and c(ii).

In an embodiment the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.

In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.

In an embodiment the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H863, e.g., a H863A.

VI. Targets: Cells

Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell, e.g., to edit a target nucleic acid, in a wide variety of cells. In some embodiments, a cell is manipulated by altering one or more target genes, e.g., as described herein. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vivo.

In an embodiment, the target cell is a retinal cell, e.g., a cell of the retinal pigment epithelium cell or a photoreceptor cell. In another embodiment, the target cell is a horizontal cell, a bipolar cell, an amacrine cell, or a ganglion cell. In an embodiment, the target cell is a cone photoreceptor cell or cone cell, a rod photoreceptor cell or rod cell, or a macular cone photoreceptor cell. In an exemplary embodiment, cone photoreceptors in the macula are targeted, i.e., cone photoreceptors in the macula are the target cells.

In an embodiment, the target cell is removed from the subject, the gene altered ex vivo, and the cell returned to the subject. In an embodiment, a photoreceptor cell is removed from the subject, the gene altered ex vivo, and the photoreceptor cell returned to the subject. In an embodiment, a cone photoreceptor cell is removed from the subject, the gene altered ex vivo, and the cone photoreceptor cell returned to the subject.

In an embodiment, the cells are induced pluripotent stem cells (iPS) cells or cells derived from iPS cells, e.g., iPS cells from the subject, modified to alter the gene and differentiated into retinal progenitor cells or retinal cells, e.g., retinal photoreceptors, and injected into the eye of the subject, e.g., subretinally, e.g., in the submacular region of the retina.

In an embodiment, the cells are targeted in vivo, e.g., by delivery of the components, e.g., a Cas9 molecule and a gRNA molecule, to the target cells. In an embodiment, the target cells are retinal pigment epithelium, photoreceptor cells, or a combination thereof. In an embodiment, AAV is used to deliver the components, e.g., a Cas9 molecule and a gRNA molecule, e.g., by transducing the target cells. VII. Delivery, Formulations and Routes of Administration

The components, e.g., a Cas9 molecule and gRNA molecule can be delivered, formulated, or administered in a variety of forms, see, e.g., Table 17. In an embodiment, one Cas9 molecule and two or more (e.g., 2, 3, 4, or more) different gRNA molecules are delivered, e.g., by an AAV vector. In an embodiment, the sequence encoding the Cas9 molecule and the sequence(s) encoding the two or more (e.g., 2, 3, 4, or more) different gRNA molecules are present on the same nucleic acid molecule, e.g., an AAV vector. When a Cas9 or gRNA component is delivered encoded in DNA the DNA will typically include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 molecule sequences include CMV, EFS, EF-la, MSCV, PGK, CAG, hGRKl, hCRX, hNRL, and hRCVRN control promoters. In an embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is a tissue specific promoter. Exemplary promoter sequences are disclosed in

Table 19. Useful promoters for gRNAs include HI, 7SK, and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment, the sequence encoding a Cas9 molecule comprises at least two nuclear localization signals. In an embodiment a promoter for a Cas9 molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific. To detect the expression of a Cas9, an affinity tag can be used. Useful affinity tag sequences include, but are not limted to, 3xFlag tag, single Flag tag, HA tage, Myc tag or HIS tage. Exemplary affinity tage sequences are disclosed in Table 25. To regulate Cas9 expression, e.g., in mammalian cells, polyadenylation signals (poly(A) signals) can be used. Exemplary polyadenylation signals are disclosed in Table 26.

Table 17 provides examples of how the components can be formulated, delivered, or administered.

Table 17

an eaCas9 molecule, is transcribed from DNA. mRNA RNA In this embodiment a Cas9 molecule, typically

an eaCas9 molecule, is transcribed from DNA.

Protein DNA In this embodiment a Cas9 molecule, typically

an eaCas9 molecule, is provided as a protein. A

gRNA is transcribed from DNA.

Protein RNA In this embodiment an eaCas9 molecule is

provided as a protein.

Table 18 summarizes various delivery methods for the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, as described herein.

Table 18

Transient

Herpes Simplex YES Stable NO DNA Virus

Non-Viral Cationic YES Transient Depends on Nucleic Acids

Liposomes what is and Proteins

delivered

Polymeric YES Transient Depends on Nucleic Acids Nanoparticles what is and Proteins

delivered

Biological Attenuated YES Transient NO Nucleic Acids Non-Viral Bacteria

Delivery Engineered YES Transient NO Nucleic Acids Vehicles Bacteriophages

Mammalian YES Transient NO Nucleic Acids

Virus-like

Particles

Biological YES Transient NO Nucleic Acids liposomes:

Erythrocyte

Ghosts and

Exosomes

Table 19 describes exemplary promoter sequences that can be used in AAV vectors, e.g., for Cas9 expression.

Table 19

TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT

GACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTA

TTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT

ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT

TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTT

TTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAA

CAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGT

ACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAA

CCGTCAGATCCGCTAGAGATCCGC (SEQ ID NO: 401)

EFS 252 TCGAGTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA

TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGC

AATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC

TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG

AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCG

TGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA

GGTGTCGTGACCGCGG (SEQ ID NO: 402)

Human 292 GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGG GRK1 AAAAGTGAGGCGGCCCCTTGGAGGAAGGGGCCGGGCAG (rhodopsin AATGATCTAATCGGATTCCAAGCAGCTCAGGGGATTGTCT kinase) TTTTCTAGCACCTTCTTGCCACTCCTAAGCGTCCTCCGTGA

CCCCGGCTGGGATTTCGCCTGGTGCTGTGTCAGCCCCGGT

CTCCCAGGGGCTTCCCAGTGGTCCCCAGGAACCCTCGAC

AGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGACGGG

CCACAGGCCAAGGGC (SEQ ID NO: 403)

Human 113 GCCTGTAGCCTTAATCTCTCCTAGCAGGGGGTTTGGGGGA CRX (cone GGGAGGAGGAGAAAGAAAGGGCCCCTTATGGCTGAGAC rod ACAATGACCCAGCCACAAGGAGGGATTACCGGGCG (SEQ homeobox ID NO: 404)

transcriptio

n factor)

Human 281 AGGTAGGAAGTGGCCTTTAACTCCATAGACCCTATTTAAA

NRL CAGCTTCGGACAGGTTTAAACATCTCCTTGGATAATTCCT

(neural AGTATCCCTGTTCCCACTCCTACTCAGGGATGATAGCTCT retina AAGAGGTGTTAGGGGATTAGGCTGAAAATGTAGGTCACC leucine CCTCAGCCATCTGGGAACTAGAATGAGTGAGAGAGGAGA zipper GAGGGGCAGAGACACACACATTCGCATATTAAGGTGACG transcriptio CGTGTGGCCTCGAACACCGAGCGACCCTGCAGCGACCCG n factor CTTAA (SEQ ID NO: 405)

enhance

upstream of

the human

TK terminal

promoter) Human 235 ATTTTAATCTCACTAGGGTTCTGGGAGCACCCCCCCCCAC

RCVRN CGCTCCCGCCCTCCACAAAGCTCCTGGGCCCCTCCTCCCT

(recoverin) TCAAGGATTGCGAAGAGCTGGTCGCAAATCCTCCTAAGC

CACCAGCATCTCGGTCTTCAGCTCACACCAGCCTTGAGCC

CAGCCTGCGGCCAGGGGACCACGCACGTCCCACCCACCC

AGCGACTCCCCAGCCGCTGCCCACTCTTCCTCACTCA

(SEQ ID NO: 406)

Table 25 describes exemplary affinity tag sequences that can be used in AAV vectors, ., for Cas9 expression.

Table 26 describes exemplary polyA sequences that can be used in AAV vectors, e.g., for Cas9 expression.

Table 24 describes exemplary Inverted Terminal Repeat (ITR) sequences that used in AAV vectors.

Table 24. Sequences of ITRs from exemplary AAV Serotypes

NO: 411) NO: 440)

AAV6 ATACCCCTAGTGATGGAGTTGCCCACT TTGCCCACTCCCTCTATGCGCGCTCGCT

CCCTCTATGCGCGCTCGCTCGCTCGGT CGCTCGGTGGGGCCTGCGGACCAAAGG

GGGGCCGGCAGAGCAGAGCTCTGCCGT TCCGCAGACGGCAGAGCTCTGCTCTGC

CTGCGGACCTTTGGTCCGCAGGCCCCA CGGCCCCACCGAGCGAGCGAGCGCGC

CCGAGCGAGCGAGCGCGCATAGAGGG ATAGAGGGAGTGGGCAACTCCATCACT

AGTGGGCAA (SEQ ID NO: 412) AGGGGTAT (SEQ ID NO: 441)

AAV7 TTGGCCACTCCCTCTATGCGCGCTCGCT CGGTACCCCTAGTGATGGAGTTGGCCA

CGCTCGGTGGGGCCTGCGGACCAAAGG CTCCCTCTATGCGCGCTCGCTCGCTCGG

TCCGCAGACGGCAGAGCTCTGCTCTGC TGGGGCCGGCAGAGCAGAGCTCTGCCG

CGGCCCCACCGAGCGAGCGAGCGCGC TCTGCGGACCTTTGGTCCGCAGGCCCC

ATAGAGGGAGTGGCCAACTCCATCACT ACCGAGCGAGCGAGCGCGCATAGAGG

AGGGGTACCG (SEQ ID NO: 413) GAGTGGCCAA (SEQ ID NO: 442)

AAV8 CAGAGAGGGAGTGGCCAACTCCATCAC GGTGTCGCAAAATGCCGCAAAAGCACT

TAGGGGTAGCGCGAAGCGCCTCCCACG CACGTGACAGCTAATACAGGACCACTC

CTGCCGCGTCAGCGCTGACGTAAATTA CCCTATGACGTAATTTACGTCAGCGCT

CGTCATAGGGGAGTGGTCCTGTATTAG GACGCGGCAGCGTGGGAGGCGCTTCGC

CTGTCACGTGAGTGCTTTTGCGGCATTT GCTACCCCTAGTGATGGAGTTGGCCAC

TGCGACACC (SEQ ID NO: 414) TCCCTCTCTG (SEQ ID NO: 443)

AAV9 CAGAGAGGGAGTGGCCAACTCCATCAC GTGTCGCAAAATGTCGCAAAAGCACTC

TAGGGGTAATCGCGAAGCGCCTCCCAC ACGTGACAGCTAATACAGGACCACTCC

GCTGCCGCGTCAGCGCTGACGTAGATT CCTATGACGTAATCTACGTCAGCGCTG

ACGTCATAGGGGAGTGGTCCTGTATTA ACGCGGCAGCGTGGGAGGCGCTTCGCG

GCTGTCACGTGAGTGCTTTTGCGACAT ATTACCCCTAGTGATGGAGTTGGCCAC

TTTGCGACAC (SEQ ID NO: 415) TCCCTCTCTG (SEQ ID NO: 444)

Additional exemplary sequences for the recombinant AAV genome components described herein are provided below.

Exemplary Left and right ITR sequences are provided in Table 24 (SEQ ID NOS: 407- 415 and 436-444).

Exemplary spacer 1 sequence: CAGATCTGAATTCGGTACC (SEQ ID NO: 416). Exemplary U6 promoter sequence:

AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGA TACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTA GTAC AAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGC AGTTTTAAAA TTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCT TGGCTTTATATATCTTGTGGAAAGGACGAAACACC (SEQ ID NO: 417)

Exemplary gRNA targeting domain sequences are described herein, e.g., in Tables 1A- 1D, Tables 2A-2C, Tables 3A-3D, Tables 4A-4D, Tables 5A-5B, Tables 6A-6D, Tables 7A- 7D, Tables 8A-8E, Tables 9A-9B, or Table 10.

Exemplary gRNA scaffold domain sequence:

GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCT CGTCAACTTGTTGGCGAGATTTTTT (SEQ ID NO: 418).

Exemplary spacer 2 domain sequence:

GGTACCGCTAGCGCTTAAGTCGCGATGTACGGGCCAGATATACGCGTTGA (SEQ ID NO: 419).

Exemplary Polymerase II promoter sequences are provided in Table 19.

Exemplary N-ter NLS nucleotide sequence:

CCGAAGAAAAAGCGCAAGGTCGAAGCGTCC (SEQ ID NO: 420)

Exemplary N-ter NLS amino acid sequence: PKKKRKV (SEQ ID NO: 434)

Exemplary Cas9 nucleotide sequence:

ATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGGGTATGG GATTATTGACTATGAAACAAGGGACGTGATCGACGCAGGCGTCAGACTGTTCAAGG AGGCCAACGTGGAAAACAATGAGGGACGGAGAAGCAAGAGGGGAGCCAGGCGCCT GAAACGACGGAGAAGGCACAGAATCCAGAGGGTGAAGAAACTGCTGTTCGATTACA ACCTGCTGACCGACCATTCTGAGCTGAGTGGAATTAATCCTTATGAAGCCAGGGTGA AAGGCCTGAGTCAGAAGCTGTCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTG GCTAAGCGCCGAGGAGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACG AGCTGTCTACAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTAT GTCGCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTCAA TTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGCTGAAAGTG CAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACTTATATCGACCTGCTG GAGACTCGGAGAACCTACTATGAGGGACCAGGAGAAGGGAGCCCCTTCGGATGGAA AGACATCAAGGAATGGTACGAGATGCTGATGGGACATTGCACCTATTTTCCAGAAG AGCTGAGAAGCGTCAAGTACGCTTATAACGCAGATCTGTACAACGCCCTGAATGAC CTGAACAACCTGGTCATCACCAGGGATGAAAACGAGAAACTGGAATACTATGAGAA GTTCCAGATCATCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTACACTGAAACAGA TTGCTAAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAGC ACTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGGACATCAC AGCACGGAAAGAAATCATTGAGAACGCCGAACTGCTGGATCAGATTGCTAAGATCC TGACTATCTACCAGAGCTCCGAGGACATCCAGGAAGAGCTGACTAACCTGAACAGC GAGCTGACCCAGGAAGAGATCGAACAGATTAGTAATCTGAAGGGGTACACCGGAAC ACACAACCTGTCCCTGAAAGCTATCAATCTGATTCTGGATGAGCTGTGGCATACAAA CGACAATCAGATTGCAATCTTTAACCGGCTGAAGCTGGTCCCAAAAAAGGTGGACC TGAGTCAGCAGAAAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTCACCCG TGGTCAAGCGGAGCTTCATCC AGAGCATCAAAGTGATCAACGCC ATC ATC AAGAAG TACGGCCTGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACAGCAAGGA CGCACAGAAGATGATCAATGAGATGCAGAAACGAAACCGGCAGACCAATGAACGC ATTGAAGAGATTATCCGAACTACCGGGAAAGAGAACGCAAAGTACCTGATTGAAAA AATCAAGCTGCACGATATGCAGGAGGGAAAGTGTCTGTATTCTCTGGAGGCCATCCC CCTGGAGGACCTGCTGAACAATCC ATTCA ACTACGAGGTCGATC ATATTATCCCC AG AAGCGTGTCCTTCGACAATTCCTTTAACAACAAGGTGCTGGTCAAGCAGGAAGAGA ACTCTAAAAAGGGCAATAGGACTCCTTTCCAGTACCTGTCTAGTTCAGATTCCAAGA TCTCTTACGAAACCTTTAAAAAGCACATTCTGAATCTGGCCAAAGGAAAGGGCCGC ATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACAGATTCTC CGTCCAGAAGGATTTTATTAACCGGAATCTGGTGGACACAAGATACGCTACTCGCGG CCTGATGAATCTGCTGCGATCCTATTTCCGGGTGAACAATCTGGATGTGAAAGTCAA GTCCATCAACGGCGGGTTCACATCTTTTCTGAGGCGCAAATGGAAGTTTAAAAAGGA GCGCAACAAAGGGTACAAGCACCATGCCGAAGATGCTCTGATTATCGCAAATGCCG ACTTCATCTTTAAGGAGTGGAAAAAGCTGGACAAAGCCAAGAAAGTGATGGAGAAC CAGATGTTCGAAGAGAAGCAGGCCGAATCTATGCCCGAAATCGAGACAGAACAGGA GTACAAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAAGGA CTACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGATCAATGACA CCCTGTATAGTACAAGAAAAGACGATAAGGGGAATACCCTGATTGTGAACAATCTG AACGGACTGTACGACAAAGATAATGACAAGCTGAAAAAGCTGATCAACAAAAGTCC CGAGAAGCTGCTGATGTACCACCATGATCCTCAGACATATCAGAAACTGAAGCTGA TTATGGAGCAGTACGGCGACGAGAAGAACCCACTGTATAAGTACTATGAAGAGACT GGGAACTACCTGACCAAGTATAGCAAAAAGGATAATGGCCCCGTGATCAAGAAGAT CAAGTACTATGGGAACAAGCTGAATGCCCATCTGGACATCACAGACGATTACCCTA ACAGTCGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGATTCGATGTCTATC TGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCATCAAAAAG GAGAACTACTATGAAGTGAATAGCAAGTGCTACGAAGAGGCTAAAAAGCTGAAAA AGATTAGCAACCAGGCAGAGTTCATCGCCTCCTTTTACAACAACGACCTGATTAAGA TCAATGGCGAACTGTATAGGGTCATCGGGGTGAACAATGATCTGCTGAACCGCATTG AAGTGAATATGATTGACATCACTTACCGAGAGTATCTGGAAAACATGAATGATAAG CGCCCCCCTCGAATTATCAAAACAATTGCCTCTAAGACTCAGAGTATCAAAAAGTAC TC AACCGACATTCTGGGAAACCTGTATGAGGTGAAG AGCAAAA AGCACCCTCAGAT TATCAAAAAGGGC (SEQ ID NO: 421)

Exemplary Cas9 amino acid sequence:

MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLK RRR RHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVH NVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVK EAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGH CTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPT LKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKIL TIY QSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIF NR LKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR EKN SKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPL EDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYET F KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYF RVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLD KAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNR ELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQK LKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDY PNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLK KISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPP R IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO: 435) Exemplary C-ter NLS sequence: CCCAAGAAGAAGAGGAAAGTC (SEQ ID NO:

422).

Exemplary C-ter NLS amino acid sequence: PKKKRKV (SEQ ID NO: 434)

Exemplary poly(A) signal sequence:

TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGC G CG (SEQ ID NO: 424).

Exemplary Spacer 3 sequence:

TCCAAGCTTCGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCGTTAACTC TAGATTTAAATGCATGCTGGGGAGAGATCT (SEQ ID NO: 425)

Exemplary 3xFLAG nucleotide sequence:

GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGA CGATGACAAG (SEQ ID NO: 423).

Exemplary 3xFLAG amino acid sequence:

DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 435)

Exemplary Spacer 4 sequence: CGACTTAGTTCGATCGAAGG (SEQ ID NO: 427).

Exemplary recombinant AAV genome sequences are provided in Figs. 19A-24F (SEQ IDNOS: 428-433 and 445-450). Exemplary sequences of the recombinant AAV genome components (e.g., one or more of the components described above) are also shown in Figs. 19A- 24F (SEQ IDNOS: 428-433 and 445-450).

DNA-based Delivery of a Cas9 molecule and or a gRNA molecule

Nucleic acids encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNA molecules, can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.

DNA encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNA molecules can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., the target cells described herein). Donor template molecules can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., the target cells described herein). In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).

A vector can comprise a sequence that encodes a Cas9 molecule and/or a gRNA molecule. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, a vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.

One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter). In other embodiments, the promoter is recognized by RNA polymerase III (e.g., a U6 promoter). In some embodiments, the promoter is a regulated promoter (e.g., inducible promoter). In other embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a viral promoter. In other embodiments, the promoter is a non-viral promoter.

In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA virus). In other embodiments, the virus is an RNA virus (e.g., an ssRNA virus).

Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno- associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.

In some embodiments, the virus infects dividing cells. In other embodiments, the virus infects non-dividing cells. In some embodiments, the virus infects both dividing and non- dividing cells. In some embodiments, the virus can integrate into the host genome. In some embodiments, the virus is engineered to have reduced immunity, e.g., in human. In some embodiments, the virus is replication-competent. In other embodiments, the virus is replication- defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In some

embodiments, the virus causes transient expression of the Cas9 molecule and/or the gRNA molecule. In other embodiments, the virus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRNA molecule. The packaging capacity of the viruses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

In an embodiment, the viral vector recognizes a specific cell type or tissue. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., genetic modification(s) of one or more viral envelope glycoproteins to incorporate a targeting ligand such as a peptide ligand, a single chain antibody, or a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., a ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant retrovirus. In some embodiments, the retrovirus (e.g., Moloney murine leukemia virus) comprises a reverse transcriptase, e.g., that allows integration into the host genome. In some embodiments, the retrovirus is replication-competent. In other embodiments, the retrovirus is replication-defective, e.g., having one of more coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted. In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant lentivirus. For example, the lentivirus is replication-defective, e.g., does not comprise one or more genes required for viral replication.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant adenovirus. In some embodiments, the adenovirus is engineered to have reduced immunity in human.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant AAV. In some embodiments, the AAV does not incorporate its geneome into that of a host cell, e.g., a target cell as describe herein. In some embodiments, the AAV can incorporate at least part of its genome into that of a host cell, e.g., a target cell as described herein. In some embodiments, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods, include AAV1,

AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y731F and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8, AAV 8.2, AAV9, AAV rhlO, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods. In an embodiment, an AAV capsid that can be used in the methods described herein is a capsid sequence from serotype AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rhlO, AAV.rh32/33, AAV.rh43, AAV.rh64Rl, or AAV7m8. Exemplary AAV serotypes and ITR sequences are disclosed in Table 24.

In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered in a re- engineered AAV capsid, e.g., with 50% or greater, e.g., 60% or greater, 70% or greater, 80% or greater, 90% or greater, or 95% or greater, sequence homology with a capsid sequence from serotypes AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rhl0, AAV.rh32/33, AAV.rh43, or AAV.rh64Rl.

In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a chimeric AAV capsid. Exemplary chimeric AAV capsids include, but are not limited to, AAV9il, AAV2i8, AAV-DJ, AAV2G9, AAV2i8G9, or AAV8G9.

In an embodiment, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a hybrid virus, e.g., a hybrid of one or more of the viruses described herein. In an embodiment, the hybrid virus is hybrid of an AAV (e.g., of any AAV serotype), with a Bocavirus, B19 virus, porcine AAV, goose AAV, feline AAV, canine AAV, or MVM.

A packaging cell is used to form a virus particle that is capable of infecting a target cell. Such a cell includes a 293 cell, which can package adenovirus, and a ψ2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into a host or target cell (if applicable), with other viral sequences being replaced by an expression cassette encoding the protein to be expressed, e.g., Cas9. For example, an AAV vector used in gene therapy typically only possesses inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and gene expression in the host or target cell. The missing viral functions can be supplied in trans by the packaging cell line and/or plasmid containing E2A, E4, and VA genes from adenovirus, and plasmid encoding Rep and Cap genes from AAV, as described in "Triple Transfection Protocol." Henceforth, the viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. In embodiment, the viral DNA is packaged in a producer cell line, which contains El A and/or EIB genes from adenovirus. The cell line is also infected with adenovirus as a helper. The helper virus (e.g., adenovirus or HSV) or helper plasmid promotes replication of the AAV vector and expression of AAV genes from the plasmid with rrP S. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., genetic modification of the viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibody, a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).

In an embodiment, the viral vector achieves cell type specific expression. For example, a tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cell. The specificity of the vector can also be mediated by microRNA- dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of the viral vector and a target cell membrane. For example, a fusion protein such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, a virus that requires the breakdown of the cell wall (during cell division) and therefore will not infect a non-diving cell can be altered to incorporate a nuclear localization peptide in the matrix protein of the virus thereby enabling the transduction of non-proliferating cells.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a non-vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a combination of a vector and a non-vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer, e.g., in a respiratory epithelial cell than either a viral or a liposomal method alone.

In an embodiment, the delivery vehicle is a non- viral vector. In an embodiment, the non- viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe ₃ Mn0 ₂ ) and silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.

Exemplary lipids for gene transfer are shown below in Table 20.

Table 20: Lipids Used for Gene Transfer

l-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium 20c Cationic

2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimeth yl- DOSPA Cationic 1-propanaminium trifluoroacetate

l,2-Dioleyl-3-trimethylammonium-propane DOPA Cationic

N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- MDRIE Cationic propanaminium bromide

Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide DMRI Cationic

3P-[N-(N',N'-Dimethylaminoethane)-carbamoyl]cholesterol DC-Choi Cationic

Bis-guanidium-tren-cholesterol BGTC Cationic l,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide DOSPER Cationic

Dimethyloctadecylammonium bromide DDAB Cationic

Dioctadecylamidoglicylspermidin DSL Cationic rac- [ (2, 3 -Dioctadecyloxypropyl) (2-hydroxyethyl) ] - CLIP-1 Cationic dimethylammonium chloride

rac-[2(2,3-Dihexadecyloxypropyl- CLIP-6 Cationic oxymethyloxy)ethyl] trimethylammonium bromide

Ethyldimyristoylphosphatidylcholine EDMPC Cationic l,2-Distearyloxy-N,N-dimethyl-3-aminopropane DSDMA Cationic

1 ,2-Dimyristoyl-trimethylammonium propane DMTAP Cationic

0, 0 '-Dimyristyl-N-lysyl aspartate DMKE Cationic l,2-Distearoyl-sn-glycero-3-ethylphosphocholine DSEPC Cationic

N-Palmitoyl D-erythro-sphingosyl carbamoyl- spermine CCS Cationic

N-i-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine diC14-amidine Cationic

Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] DOTIM Cationic imidazolinium chloride

M-Cholesteryloxycarbonyl-3,7-diazanonane-l,9-diamine CD AN Cationic

2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N- RPR209120 Cationic ditetradecylcarbamoylme-ethyl-acetamide

1 ,2-dilinoleyloxy-3- dimethylaminopropane DLinDMA Cationic

2,2-dilinoleyl-4-dimethylaminoethyl- [1,3]- dioxolane DLin-KC2- Cationic DMA

dilinoleyl- methyl-4-dimethylaminobutyrate DLin-MC3- Cationic

DMA

Exemplary polymers for gene transfer are shown below in Table 21.

Table 21: Polymers Used for Gene Transfer

Polymer Abbreviation

Poly(ethylene)glycol PEG

Polyethylenimine PEI

Dithiobis(succinimidylpropionate) DSP

Dimethyl-3,3'-dithiobispropionimidate DTBP

Poly(ethylene imine)biscarbamate PEIC

Poly(L-lysine) PLL

Histidine modified PLL

Poly(N-vinylpyrrolidone) PVP

Poly(propylenimine) PPI

Poly(amidoamine) PAMAM

Poly(amidoethylenimine) SS-PAEI

Triethylenetetramine TETA

Poly ( β- aminoester)

Poly(4-hydroxy-L-proline ester) PHP

Poly(allylamine)

Poly(a-[4-aminobutyl]-L-glycolic acid) PAGA

Poly(D,L-lactic-co-glycolic acid) PLGA

Poly(N-ethyl-4-vinylpyridinium bromide)

Poly (pho sphazene) s PPZ

Poly (pho sphoester) s PPE

Poly (pho sphoramidate) s PPA

Poly(N-2-hydroxypropylmethacrylamide) pHPMA

Poly (2-(dimethylamino)ethyl methacrylate) pDMAEMA Poly(2-aminoethyl propylene phosphate) PPE-EA

Chitosan

Galactosylated chitosan

N-Dodacylated chitosan

Histone

Collagen

Dextran- spermine D-SPM

In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacteriumlongum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the "empty" particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes - subject (i.e., patient) derived membrane-bound nanovescicle (30 -100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).

In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein.

Delivery of RNA encoding a Cas9 molecule

RNA encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Cas9-encoding and/or gRNA-encoding RNA can be conjugated to molecules (e.g., GalNAc) promoting uptake by the target cells (e.g., target cells described herein). Delivery Cas9 molecule protein

Cas9 molecules (e.g., eaCas9 molecules) can be delivered into cells by art-known methods or as described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA. Cas9-encoding and/or gRNA-encoding RNA can be conjugated to molecules (e.g., GalNAc) promoting uptake by the target cells (e.g., target cells described herein).

Route of administration

Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to the eye.

Local modes of administration include, by way of example, intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intravitreally) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.

In an embodiment, components described herein are delivered subretinally, e.g., by subretinal injection. Subretinal injections may be made directly into the macular, e.g., submacular injection.

In an embodiment, components described herein are delivered by intravitreal injection. Intravitreal injection has a relatively low risk of retinal detachment. In an embodiment, nanoparticle or viral, e.g., AAV vector, is delivered intravitreally.

Methods for administration of agents to the eye are known in the medical arts and can be used to administer components described herein. Exemplary methods include intraocular injection (e.g., retrobulbar, subretinal, submacular, intravitreal and intrachoridal), iontophoresis, eye drops, and intraocular implantation (e.g., intravitreal, sub-Tenons and sub-conjunctival).

Administration may be provided as a periodic bolus (for example, subretinally, intravenously or intravitreally) or as continuous infusion from an internal reservoir (for example, from an implant disposed at an intra- or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device immobilized to an inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT/USOO/00207, PCT/US02/14279, Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41: 1181- 1185, and Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41: 1186-1191). A variety of devices suitable for administering components locally to the inside of the eye are known in the art. See, for example, U.S. Pat. Nos. 6,251,090, 6,299,895, 6,416,777, 6,413,540, and PCT/USOO/28187.

In addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.

Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters;

polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non- degradable polymers include, for example: polyethers such as poly(ethylene oxide),

poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.

Poly(lactide-co-glycolide) microsphere can also be used for intraocular injection.

Typically the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein.

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.

In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery that confer different pharmacodynamic or

pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, template nucleic acid, or payload. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.

Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno associated virus or lentivirus, delivery.

By way of example, the components, e.g., a Cas9 molecule and a gRNA molecule, can be delivered by modes that differ in terms of resulting half-life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA molecule can be delivered by such modes. The Cas9 molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.

More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.

In an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.

In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.

In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.

In an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.

In an embodiment, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a Cas9 molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 molecule/gRNA molecule complex is only present and active for a short period of time. Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.

Use of differential delivery modes can enhance performance, safety and efficacy. E.g., the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part delivery system can alleviate these drawbacks.

Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.

When the Cas9 molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.

Ex vivo delivery

In some embodiments, components described in Table 17 are introduced into cells which are then introduced into the subject. Methods of introducing the components can include, e.g., any of the delivery methods described in Table 18. VIII. Modified Nucleosides, Nucleotides, and Nucleic Acids

Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRNA, but also other forms of RNA, e.g., mRNA, RNAi, or siRNA. As described herein, "nucleoside" is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, "nucleotide" is defined as a nucleoside further comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;

(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;

(iii) wholesale replacement of the phosphate moiety with "dephospho" linkers;

(iv) modification or replacement of a naturally occurring nucleobase;

(v) replacement or modification of the ribose-phosphate backbone;

(vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal,

modification or replacement of a terminal phosphate group or conjugation of a moiety; and

(vii) modification of the sugar.

The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an

embodiment, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule are replaced with

phosphorothioate groups.

In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a "modified nucleic acid." In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.

In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In some embodiments, the modified

nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.

Definitions of Chemical Groups

As used herein, "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl,

phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.

As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.

As used herein, "arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

As used herein, "cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non- aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.

As used herein, "heterocyclyl" refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl,

tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.

As used herein, "heteroaryl" refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

Phosphate Backbone Modifications

The Phosphate Group

In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with

unsymmetrical charge distribution.

Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR ₃ (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR ₂ (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).

The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo,

methylenedimethylhydrazo and methyleneoxymethylimino. Replacement of the Ribophosphate Backbone

Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

The modified nucleosides and modified nucleotides can include one or more

modifications to the sugar group. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.

Examples of "oxy"-2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar);

polyethyleneglycols (PEG), 0(CH ₂ CH ₂ 0) _n CH ₂ CH ₂ OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2' hydroxyl group modification can include

"locked" nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C _1-6 alkylene or Ci-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH ₂ ) _n -amino, (wherein amino can be, e.g., NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2' hydroxyl group modification can include the methoxyethyl group (MOE),

(OCH ₂ CH ₂ OCH ₃ , e.g., a PEG derivative).

"Deoxy" modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH ₂ CH ₂ NH) _n CH ₂ CH ₂ - amino (wherein amino can be, e.g., as described herein), -NHC(0)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;

thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C- . These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.

Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3'→2')).

Modifications on the Nucleobase

The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil

(U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

Uracil

In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio- uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5 -iodo -uridine or 5-bromo-uridine), 3-

3 5 5

methyl-uridine (m U), 5-methoxy-uridine (mo U), uridine 5-oxyacetic acid (cmo U), uridine 5- oxyacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uridine (cm ⁵ U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s2U), 5-aminomethyl-2-thio-uridine (nm ⁵ s2U), 5- methylaminomethyl-uridine (mnm ⁵ U), 5-methylaminomethyl-2-thio-uridine (mnm ⁵ s2U), 5-

5 2 5 methylaminomethyl-2-seleno-uridine (mnm se U), 5-carbamoylmethyl-uridine (ncm U), 5- carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (xcm ⁵ U), 1-taurinomethyl -pseudouridine, 5-taurinomethyl-2-thio-uridine(xm ⁵ s2U), l-taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m ⁵ U, i.e., having the nucleobase deoxythymine), 1- methyl-pseudouridine (πι'ψ), 5-methyl-2-thio-uridine (m ⁵ s2U), l-methyl-4-thio-pseudouridine 2-thio-l-methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D),

2- thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio- uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine,

3- (3-amino-3-carboxypropyl)uridine (acp U), l-methyl-3-(3-amino-3-

3 5

carboxypropyl)pseudouridine (acp ψ), 5-(isopentenylaminomethyl)uridine (inm U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s2U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-0-dimethyl-uridine (m ⁵ Um), 2'-0-methyl-pseudouridine (\|/m), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl -uridine (mem ⁵ Um), 5-carbamoylmethyl-2'-0- methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm ⁵ Um),

3,2'-0-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.

Cytosine

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m C), N4-acetyl-cytidine (act), 5- formyl-cytidine (f ⁵ C), N4-methyl-cytidine (m ⁴ C), 5-methyl-cytidine (m ⁵ C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm ⁵ C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy- 5 -methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine (k C), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m ⁵ Cm), N4-acetyl-2'-0- methyl-cytidine (ac ⁴ Cm), N4,2'-0-dimethyl-cytidine (m ⁴ Cm), 5-formyl-2'-0-methyl-cytidine (f ⁵ Cm), N4,N4,2'-0-trimethyl-cytidine (m ⁴ ₂ Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

Adenine

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino- purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza- 8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyl- adenosine (i A), 2-methyl-adenine (m ² A), N6-methyl-adenosine (m ⁶ A), 2-methylthio-N6-methyl-adenosine (ms2m ⁶ A), N6- isopentenyl-adenosine (i ⁶ A), 2-methylthio-N6-isopentenyl-adenosine (ms ² i ⁶ A), N6-(cis- hydroxyisopentenyl)adenosine (io ⁶ A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io ⁶ A), N6-glycinylcarbamoyl-adenosine (g ⁶ A), N6-threonylcarbamoyl-adenosine (t ⁶ A), N6- methyl-N6-threonylcarbamoyl-adenosine (m ⁶ t ⁶ A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms ² g ⁶ A), N6,N6-dimethyl-adenosine (m ⁶ ₂ A), N6-hydroxynorvalylcarbamoyl- adenosine (hn ⁶ A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn ⁶ A), N6- acetyl-adenosine (ac ⁶ A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2'-0-methyl-adenosine (Am), N ⁶ ,2'-0-dimethyl-adenosine (m ⁶ Am), N ⁶ -Methyl-2'- deoxyadenosine, N6,N6,2'-0-trimethyl-adenosine (m ⁶ ₂ Am), l,2'-0-dimethyl-adenosine (i Am), 2'-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8- azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)- adenosine.

Guanine

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1- methyl-inosine (iVl), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o ₂ yW), hydroxy wybuto sine (OHyW), undermodified hydroxy wybuto sine (OHyW*), 7-deaza-guanosine, queuosine (Q),

epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQO, archaeosine (G ⁺ ), 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,

1 -methyl-guanosine (m'G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 ₂ G),

N2,7-dimethyl-guanosine (m 2 ,7G), N2, N2,7-dimethyl-guanosine (m 2 ,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2- dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methyl-2'-0- methyl-guanosine (m 2"Gm), N2,N2-dimethyl-2'-0-methyl- guano sine (m 2 ₂ Gm), l-methyl-2'-0- methyl-guanosine (m'Gm), N2,7-dimethyl-2'-0-methyl-guanosine (m",7Gm), 2'-0-methyl- inosine (Im), l,2'-0-dimethyl-inosine (m'lm), 0 ⁶ -phenyl-2'-deoxyinosine, 2'-0-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, 0 ⁶ -methyl- guanosine, 0 ⁶ -Methyl-2'-deoxy guanosine, Z - F-ara-guanosine, and 2'-F-guanosine. Modified gRNAs

In some embodiments, the modified nucleic acids can be modified gRNAs. In some embodiments, gRNAs can be modified at the 3' end. In this embodiment, the gRNAs can be modified at the 3' terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as sown below:

wherein "U" can be an unmodified or modified uridine.

In another embodiment, the 3' terminal U can be modified with a 2' 3' cyclic phosphate as shown below:

wherein "U" can be an unmodified or modified uridine.

In some embodiments, the gRNA molecules may contain 3' nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7- deaza-adenosine, can be incorporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2' OH- group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH ₂ ; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2'-sugar modified, such as, 2-F 2'-0-methyl, thymidine (T), 2'-0- methoxyethyl-5-methyluridine (Teo), 2'-0-methoxyethyladenosine (Aeo ), 2'-0-methoxyethyl- 5-methylcytidine (m5Ceo ), and any combinations thereof.

In an embodiment, one or more or all of the nucleotides in single stranded RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.

miRNA binding sites

microRNAs (or miRNAs) are naturally occurring cellular 19-25 nucleotide long noncoding RNAs. They bind to nucleic acid molecules having an appropriate miRNA binding site, e.g., in the 3' UTR of an mRNA, and down-regulate gene expression. While not wishing to be bound by theory, in an embodiment, it is believed that the down regulation is either by reducing nucleic acid molecule stability or by inhibiting translation. An RNA species disclosed herein, e.g., an mRNA encoding Cas9 can comprise an miRNA binding site, e.g., in its 3'UTR. The miRNA binding site can be selected to promote down regulation of expression is a selected cell type. By way of example, the incorporation of a binding site for miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest in the liver.

Governing gRNA molecules and the use thereof to limit the activity of a Cas9 system

Methods and compositions that use, or include, a nucleic acid, e.g., DNA, that encodes a Cas9 molecule or a gRNA molecule, can, in addition, use or include a "governing gRNA molecule." The governing gRNA can limit the activity of the other CRISPR/Cas components introduced into a cell or subject. In an embodiment, a gRNA molecule comprises a targeting domain that is complementary to a target domain on a nucleic acid that comprises a sequence that encodes a component of the CRISPR/Cas system that is introduced into a cell or subject. In an embodiment, a governing gRNA molecule comprises a targeting domain that is

complementary with a target sequence on: (a) a nucleic acid that encodes a Cas9 molecule; (b) a nucleic acid that encodes a gRNA which comprises a targeting domain that targets the CEP290 gene (a target gene gRNA); or on more than one nucleic acid that encodes a CRISPR/Cas component, e.g., both (a) and (b). The governing gRNA molecule can complex with the Cas9 molecule to inactivate a component of the system. In an embodiment, a Cas9

molecule/governing gRNA molecule complex inactivates a nucleic acid that comprises the sequence encoding the Cas9 molecule. In an embodiment, a Cas9 molecule/governing gRNA molecule complex inactivates the nucleic acid that comprises the sequence encoding a target gene gRNA molecule. In an embodiment, a Cas9 molecule/governing gRNA molecule complex places temporal, level of expression, or other limits, on activity of the Cas9 molecule/target gene gRNA molecule complex. In an embodiment, a Cas9 molecule/governing gRNA molecule complex reduces off-target or other unwanted activity. In an embodiment, a governing gRNA molecule targets the coding sequence, or a control region, e.g., a promoter, for the CRISPR/Cas system component to be negatively regulated. For example, a governing gRNA can target the coding sequence for a Cas9 molecule, or a control region, e.g., a promoter, that regulates the expression of the Cas9 molecule coding sequence, or a sequence disposed between the two. In an embodiment, a governing gRNA molecule targets the coding sequence, or a control region, e.g., a promoter, for a target gene gRNA. In an embodiment, a governing gRNA, e.g., a Cas9- targeting or target gene gRNA-targeting, governing gRNA molecule, or a nucleic acid that encodes it, is introduced separately, e.g., later, than is the Cas9 molecule or a nucleic acid that encodes it. For example, a first vector, e.g., a viral vector, e.g., an AAV vector, can introduce nucleic acid encoding a Cas9 molecule and one or more target gene gRNA molecules, and a second vector, e.g., a viral vector, e.g., an AAV vector, can introduce nucleic acid encoding a governing gRNA molecule, e.g., a Cas9-targeting or target gene gRNA targeting, gRNA molecule. In an embodiment, the second vector can be introduced after the first. In other embodiments, a governing gRNA molecule, e.g., a Cas9-targeting or target gene gRNA targeting, governing gRNA molecule, or a nucleic acid that encodes it, can be introduced together, e.g., at the same time or in the same vector, with the Cas9 molecule or a nucleic acid that encodes it, but, e.g., under transcriptional control elements, e.g., a promoter or an enhancer, that are activated at a later time, e.g., such that after a period of time the transcription of Cas9 is reduced. In an embodiment, the transcriptional control element is activated intrinsically. In an embodiment, the transcriptional element is activated via the introduction of an external trigger.

Typically a nucleic acid sequence encoding a governing gRNA molecule, e.g., a Cas9- targeting gRNA molecule, is under the control of a different control region, e.g., promoter, than is the component it negatively modulates, e.g., a nucleic acid encoding a Cas9 molecule. In an embodiment, "different control region" refers to simply not being under the control of one control region, e.g., promoter, that is functionally coupled to both controlled sequences. In an embodiment, different refers to "different control region" in kind or type of control region. For example, the sequence encoding a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule, is under the control of a control region, e.g., a promoter, that has a lower level of expression, or is expressed later than the sequence which encodes is the component it negatively modulates, e.g., a nucleic acid encoding a Cas9 molecule.

By way of example, a sequence that encodes a governing gRNA molecule, e.g., a Cas9- targeting governing gRNA molecule, can be under the control of a control region (e.g., a promoter) described herein, e.g., human U6 small nuclear promoter, or human HI promoter. In an embodiment, a sequence that encodes the component it negatively regulates, e.g., a nucleic acid encoding a Cas9 molecule, can be under the control of a control region (e.g., a promoter) described herein, e.g., CMV, EF-la, MSCV, PGK, CAG control promoters.

Examples

The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

Example 1 : Cloning and Initial Screening of gRNAs

The suitability of candidate gRNAs can be evaluated as described in this example.

Although described for a chimeric gRNA, the approach can also be used to evaluate modular gRNAs.

Cloning gRNAs into plasmid vector

For each gRNA, a pair of overlapping oligonucleotides is designed and obtained.

Oligonucleotides are annealed and ligated into a digested vector backbone containing an upstream U6 promoter and the remaining sequence of a long chimeric gRNA. Plasmid is sequence-verified and prepped to generate sufficient amounts of transfection-quality DNA. Alternate promoters maybe used to drive in vivo transcription (e.g. HI promoter) or for in vitro transcription (eg. T7 promoter). Cloning gRNAs in linear dsDNA molecule (STITCHR)

For each gRNA, a single oligonucleotide is designed and obtained. The U6 promoter and the gRNA scaffold (e.g. including everything except the targeting domain, e.g., including sequences derived from the crRNA and tracrRNA, e.g., including a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain) are separately PCR amplified and purified as dsDNA molecules. The gRNA- specific oligonucleotide is used in a PCR reaction to stitch together the U6 and the gRNA scaffold, linked by the targeting domain specified in the oligonucleotide. Resulting dsDNA molecule (STITCHR product) is purified for transfection. Alternate promoters may be used to drive in vivo transcription (e.g., HI promoter) or for in vitro transcription (e.g., T7 promoter). Any gRNA scaffold may be used to create gRNAs compatible with Cas9s from any bacterial species.

Initial gRNA Screen

Each gRNA to be tested is transfected, along with a plasmid expressing Cas9 and a small amount of a GFP-expressing plasmid into human cells. In preliminary experiments, these cells can be immortalized human cell lines such as 293T, K562 or U20S. Alternatively, primary human cells may be used. In this case, cells may be relevant to the eventual therapeutic cell target (for example, photoreceptor cells). The use of primary cells similar to the potential therapeutic target cell population may provide important information on gene targeting rates in the context of endogenous chromatin and gene expression.

Transfection may be performed using lipid transfection (such as Lipofectamine or

Fugene) or by electroporation. Following transfection, GFP expression can be determined either by fluorescence microscopy or by flow cytometry to confirm consistent and high levels of transfection. These preliminary transfections can comprise different gRNAs and different targeting approaches (17-mers, 20-mers, nuclease, dual-nickase, etc.) to determine which gRNAs/combinations of gRNAs give the greatest activity.

Efficiency of cleavage with each gRNA may be assessed by measuring NHEJ-induced indel formation at the target locus by a T7El-type assay or by sequencing. Alternatively, other mismatch- sensitive enzymes, such as Cell/Surveyor nuclease, may also be used.

For the T7E1 assay, PCR amplicons are approximately 500-700bp with the intended cut site placed asymmetrically in the amplicon. Following amplification, purification and size- verification of PCR products, DNA is denatured and re-hybridized by heating to 95 °C and then slowly cooling. Hybridized PCR products are then digested with T7 Endonuclease I (or other mismatch- sensitive enzyme) which recognizes and cleaves non-perfectly matched DNA. If indels are present in the original template DNA, when the amplicons are denatured and re- annealed, this results in the hybridization of DNA strands harboring different indels and therefore lead to double- stranded DNA that is not perfectly matched. Digestion products may be visualized by gel electrophoresis or by capillary electrophoresis. The fraction of DNA that is cleaved (density of cleavage products divided by the density of cleaved and uncleaved) may be used to estimate a percent NHEJ using the following equation: %NHEJ = (1-(1 -fraction cleaved)' ⁷² ). The T7E1 assay is sensitive down to about 2-5% NHEJ.

Sequencing may be used instead of, or in addition to, the T7E1 assay. For Sanger sequencing, purified PCR amplicons are cloned into a plasmid backbone, transformed, miniprepped and sequenced with a single primer. For large sequencing numbers, Sanger sequencing may be used for determining the exact nature of indels after determining the NHEJ rate by T7E1.

Sequencing may also be performed using next generation sequencing techniques. When using next generation sequencing, amplicons may be 300-500bp with the intended cut site placed asymmetrically. Following PCR, next generation sequencing adapters and barcodes (for example Illumina multiplex adapters and indexes) may be added to the ends of the amplicon, e.g., for use in high throughput sequencing (for example on an Illumina MiSeq). This method allows for detection of very low NHEJ rates.

Example 2: Assessment of Gene Targeting by NHEJ

The gRNAs that induce the greatest levels of NHEJ in initial tests can be selected for further evaluation of gene targeting efficiency. For example, cells may be derived from disease subjects, relevant cell lines, and/or animal models and, therefore, harbor the relevant mutation.

Following transfection (usually 2-3 days post-transfection,) genomic DNA may be isolated from a bulk population of transfected cells and PCR may be used to amplify the target region. Following PCR, gene targeting efficiency to generate the desired mutations (either knockout of a target gene or removal of a target sequence motif) may be determined by sequencing. For Sanger sequencing, PCR amplicons may be 500-700 bp long. For next generation sequencing, PCR amplicons may be 300-500 bp long. If the goal is to knockout gene function, sequencing may be used to assess what percent of alleles have undergone NHEJ- induced indels that result in a frameshift or large deletion or insertion that would be expected to destroy gene function. If the goal is to remove a specific sequence motif, sequencing may be used to assess what percent of alleles have undergone NHEJ-induced deletions that span this sequence.

Example 3: Assessment of Activity of Individual gRNAs Targeting CEP290

Guide RNA were identified using a custom guide RNA design software based on the public tool cas-offinder (Bae et al. Bioinformatics. 2014; 30(10): 1473-1475). Each gRNA to be tested was generated as a STITCHR product and co-transfected with a plasmid expressing either S. aureus Cas9 (pAF003) or S. pyogenes Cas9 (pJDS246) into either HEK293 cells or primary fibroblasts derived from and LCA10 patient harboring homozygous IVS26 C.2991+1655A to G mutations (hereafter referred to as IVS26 fibroblasts). The pAF003 plasmid encodes the S. aureus Cas9, with N-terminal and C-terminal nuclear localization signals (NLS) and a C- terminal triple flag tag, driven by a CMV promoter. The pJDS246 plasmid encodes the S.

pyogenes Cas9, with a C-terminal nuclear localization signal (NLS) and a C-terminal triple flag tag, driven by a CMV promoter. gRNA and Cas9-encoding DNA was introduced into cells by either Minis TransIT-293 transfection reagent (for 293 cells) or by Amaxa nucleofection (for IVS26 fibroblasts). Nucleofection was optimized for transfection of IVS26 fibroblasts using solution P2 and various pulse codes and assaying for highest levels of gene editing and cell viability. Transfection efficiency in both cell types was assessed by transfecting with GFP and assaying expression by fluorescent microscopy. Three to seven days post-transfection, genomic DNA was isolated from bulk populations of transfected cells and the region of the CEP290 locus surrounding the target site was PCR amplified. PCR amplicons were then cloned into a plasmid backbone using the Zero-Blunt TOPO cloning kit (Lifetechnologies) and transformed into chemically competent ToplO cells. Bacterial colonies were then cultured and plasmid DNA was isolated and sequenced. Sequencing of PCR products allowed for the detection and

quantification of targeted insertion and deletion (indel) events at the target site. Fig. 11A and 11B show the rates of indels induced by various gRNAs at the CEP290 locus. Fig. 11A shows gene editing ( indels) as assessed by sequencing for S. pyogenes and S. aureus gRNAs when co-expressed with Cas9 in patient-derived IVS26 primary fibroblasts. Fig. 11B shows gene editing (% indels) as assessed by sequencing for S. aureus gRNAs when co-expressed with Cas9 in HEK293 cells.

Example 4: Detection of gRNA Pair- Induced Deletions by PCR

To assess the ability of a pair of gRNAs to induce a genomic deletion (in which the sequence between the two cut sites is removed), PCR was performed across the predicted deletion. Pairs of gRNAs (encoded as STITCHR products) were co-transfected with pAF003 into rVS26 fibroblasts. Genomic DNA was isolated from transfected cells and PCR was performed to amplify a segment of the CEP290 locus spanning the two predicted cut sites. PCR was run on a QIAxcel capillary electrophoresis machine. The predicted amplicon on a wildtype allele is 1816 bps. Assuming that cleavage occurs within the gRNA target region, amplicon sizes for alleles having undergone the deletion event were calculated and the presence of this smaller band indicates that the desired genomic deletion event has occurred (Table 22).

Table 22

23 CEP290-368 CEP290-132 1079 737 no

24 CEP290-368 CEP290-139 1528 288 no

25 CEP290-323 CEP290-11 990 826 yes

26 CEP290-323 CEP290-252 1173 643 no

27 CEP290-323 CEP290-64 1176 640 yes

28 CEP290-323 CEP290-230 1609 207 yes

29 Cas9 only wt amplicon = 1816 no

30 GFP only wt amplicon = 1816 no

31 no DNA PCR neg Ctrl

Example 5: Gene Expression Analysis of CEP290

Targeted deletion of a region containing the IVS26 splice mutation is predicted to correct the splicing defect and restore expression of the normal wild-type CEP290 allele. To quantify expression of the wild-type and mutant (containing additional cryptic splice mutation) alleles, TaqMan assays were designed. Multiple assays were tested for each RNA species and a single wt and single mutant assay were selected. The assay for the wild-type allele contains a forward primer that anneals in exon 26, a reverse primer that anneals in exon 27 and a TaqMan probe that spans the exon26-exon-27 junction. The assay for the mutant allele contains a forward primer that anneals in exon 26, a reverse primer that anneals in the cryptic exon and a TaqMan probe that spans the exon26-cryptic exon junction. A TaqMan assay designed to beta-actin was used as a control. Total RNA was isolated from IVS26 cells transfected with pairs of gRNAs and Cas9- expressing plasmid by either Trizol RNA purification (Ambion), Agencourt RNAdvance (Beckman Coulter) or direct cells-to-Ct lysis (Lifetechnologies). Reverse transcription to generate cDNA was performed and cDNA was used as a template for qRT-PCR using selected taqman assays on a BioRad real time PCR machine. Relative gene expression was calculated by AACt, relative to beta-actin control and GFP-only sample. Increases in expression of wt allele and decreases in expression of mutant allele relative to GFP-only control indicate corrected splicing due to gene targeting. Figs. 12A-12B show initial qRT-PCR data for pairs of gRNAs that had shown activity as either individual gRNAs (measured as described in Example 3) or as pairs (measured as described in Example 4). Pairs of gRNAs that showed the desired gene expression changes were repeated in replicate experiments and the cumulative qRT-PCR data is shown in Fig. 13 (error bars represent standard error of the mean calculated from 2 to 6 biological replicates per sample). Example 6: Quantification of Genomic Deletions by ddPCR

Droplet digital PCR (ddPCR) is a method for performing digital PCR in which a single PCR reaction is fractionated into 20,000 droplets in a water-oil emulsion and PCR amplification occurs separately in individual droplets. PCR conditions are optimized for a concentration of DNA template such that each droplet contains either one or no template molecules. Assays were designed to perform amplification using BioRad EvaGreen Supermix PCR system with all amplicons ranging in size from 250-350 bp. Control assays were designed to amplify segments of the CEP290 gene at least 5 kb away from the IVS26 C.2991+1655A to G mutation. Assays to detect targeted genomic deletion were designed such that amplification of an allele that has undergone deletion will yield a PCR product in the size range of 250-350 bp and amplification will not occur on a wild-type allele due to the increased distance between forward and reverse primers. PCR conditions were optimized on genomic DNA isolated from 293 cells that had been transfected with pairs of gRNAs and Cas9-expressing plasmid. Deletion assays were verified to generate no positive signal on genomic DNA isolated from unmodified IVS26 fibroblasts.

Assays were further tested and optimized on genomic DNA isolated from IVS26 fibroblasts that had been transfected with pairs of gRNAs and Cas9-encoding plasmid. Of the three assays tested for each of two deletions (CEP290-323 and CEP290-11; and CEP290-323 and CEP290- 64) and the 4 control assays tested, a single assay was selected for each deletion and a control based on quality data and replicability in the ddPCR assay. Fig. 14 shows deletion rates on three biological replicates calculated by taking the number of positive droplets for the deletion assay and dividing by the number of positive droplets for the control assay.

Example 7: Cloning AAV Expression Vectors

Cloning saCas9 into an AAV expression vector

The pAF003 plasmid encodes the CMV-driven S. aureus Cas9 (saCas9), with N-terminal and C-terminal nuclear localization signals (NLS) and a C-terminal triple flag tag, followed by a bovine growth hormone poly(A) tail (bGH polyA). BGH polyA tail was substituted with a 60-bp minimal polyA tail to obtain pAF003-minimal-pA. The CMV-driven NLS-saCas9-NLS-3xFlag with the minimal polyA tail was amplified with PCR and subcloned into pTR-UFl 1 plasmid (ATCC #MBA-331) with Kpnl and Sphl sites to obtain the pSS3 (pTR-CMV-saCas9-minimal- pA) vector. The CMV promoter sequence can be substituted with EFS promoter (pSSlO vector), or tissue-specific promoters (Table 19, e.g. photo -receptor- specific promoters, e.g. Human GRK1, CRX, NRL, RCVRN promoters, etc.) using Spel and Notl sites.

Constructing the All-in-One AAV expression vector with one gRNA sequence

For each individual gRNA sequence, a STITCHR product with a U6 promoter, gRNA, and the gRNA scaffold was obtained by PCR with an oligonucleotide encoding the gRNA sequence. The STITCHR product with one dsDNA molecule of U6-driven gRNA and scaffold was subcloned into pSS3 or pSSlO vectors using Kpnl sites flanking the STITCHR product and downstream of the left Inverted Terminal Repeat (ITR) in the AAV vectors. The orientation of the U6-gRNA- scaffold insertion into pSS3 or pSSlO was determined by Sanger sequencing. Alternate promoters may be used to drive gRNA expression (e.g. HI promoter, 7SK promoter). Any gRNA scaffold sequences compatible with Cas variants from other bacterial species could be incorporated into STITCHR products and the AAV expression vector therein.

Cloning two gRNA into an AAV expression vector

For each pair of gRNA sequences, two ssDNA oligonucleotides were designed and obtained as the STITCHR primers, i.e. the left STrfCHR primer and the right STITCHR primer. Two STITCHR PCR reactions (i.e. the left STrfCHR PCR and the right STITCHR PCR) amplified the U6 promoter and the gRNA scaffold with the corresponding STITCHR primer separately. The pSS3 or pSSlO backbone was linearized with Kpnl restriction digest. Two dsDNA STITCHR products were purified and subcloned into pSS3 or pSSlO backbone with Gibson Assembly. Due to the unique overlapping sequences upstream and downstream of the STITCHR products, the assembly is unidirectional. The sequences of the constructs were confirmed by Sanger Sequencing. Table 23 lists the names and compositions of AAV expression vectors constructed, including the names of gRNAs targeting human CEP290, the promoter to drive Cas9 expression, and the length of the AAV vector including the Inverted Terminal Repeats (ITRs) from wild type AAV2 geome. Alternative promoters (e.g., HI promoter or 7SK promoter) or gRNA scaffold sequences compatible with any Cas variants could be adapted into this cloning strategy to obtain the corresponding All-in-One AAV expression vectors with two gRNA sequences. Table 23. Components of AAV expression vectors

Example 8: Assessment of the Functions of All-in-One AAV Expression Vectors Each individual AAV expression vectors were transfected into 293T cells with TransIT- 293 (Minis, Inc.) to test their function before being packaged into AAV viral vectors. 293T cells were transfected with the same amount of plasmid and harvested at the same time points.

SaCas9 protein expression was assessed by western blotting with primary antibody probing for the triple Flag tag at the C-terminus of saCas9, while loading control was demonstrated by aTubulin expression. Deletion events at IVS26 mutation could be determined by PCR amplification followed by Sanger sequencing or ddPCR. The results are shown in Fig. 15.

Example 9: Production, purification and titering of recombinant AAV2 vectors

Prior to packaging into AAV viral vectors, all AAV expression vector (plasmids) underwent primer walk with Sanger sequencing and function analysis. In recombinant AAV (rAAV), two ITRs flanking the transgene cassettes are the only cis-acting elements from the wild-type AAV. They are critical for packaging intact rAAVs and genome -release for rAAV vectors during transduction. All AAV expression vectors were restriction digested with Smal or Xmal to ensure the presence of two intact ITRs.

rAAV2 vectors were produced with "Triple Transfection Protocol": (1) pSS vectors with ITRs and transgene cassetts; (2) pHelper plasmid with E2A, E4, VA genes from Adenovirus; (3) pAAV-RC2 plasmid with Rep and Cap genes from AAV2. These three plasmids were mixed at a mass ratio of 3:6:5 and transfected into HEK293 with polymer or lipid-based transfection reagent (e.g. PEI, PEI max, Lipofectamine, TransIT-293, etc.). 60-72 hours post-transfection, HEK293 cells were harvested and sonicated to release viral vectors. Cell lysates underwent CsCl ultracentrifuge to purify and concentrate the viral vectors. Additional purification procedures were performed to obtain higher purity for biophysical assays, including another round of CsCl ultracentrifuge, or sucrose gradient ultracentrifuge, or affinity chromatography. Viral vectors were dialyzed with lxDPBS twice before being aliquoted for storage in -80°C. Viral preps can be tittered with Dot-Blot protocol or/and quantitative PCR with probes annealing to sequences on the transgenes. PCR primer sequences are: AACATGCTACGCAGAGAGGGAGTGG (SEQ ID NO: 482) (ITR-Titer-fwd) and CATGAGACAAGGAACCCCTAGTGATGGAG (SEQ ID NO: 483) (ITR-Titer-rev). Reference AAV preps were obtained from the Vector Core at University of North Carolina-Chapel Hill as standards. To confirm the presence of three nonstructural viral proteins composing the AAV capsid, viral preps were denatured and probed with anti-AAV VP1/VP2/VP3 monoclonal antibody B 1 (American Research Products, Inc. Cat #03- 65158) on western blots. The results are shown in Fig. 16.

Example 10: rAAV-mediated CEP290 modification in vitro

293T were transduced with rAAV2 vectors expressing saCas9 with or without gRNA sequences to demonstrate the deletion events near the IVS26 splicing mutant. 293T cells were transduced with rAAV2 viral vectors at an MOI of 1,000 viral genome (vg)/cell or 10,000 vg/cell and harvested at three to seven days post transduction. Western blotting with the primary antibody for Flag (anti-Flag, M2, Sigma- Aldrich) showed that the presence of U6-gRNA- scaffold does not interfere with saCas9 expression. Genomic DNA from 293T was isolated with the Agencourt DNAdvance Kit (Beckman Coulter). Regions including the deletions were PCR amplified from genomic DNA isolated, and analyzed on the QIAxcel capillary electrophoresis machine. Amplicons smaller than the full-length predicted PCR products represent the deletion events in 293T cells. The PCR results are shown in Fig. 17. To further understand the nature of these deletion events, PCR products were cloned into Zero-Blunt TOPO Cloning Kit (Life Technologies) and transformed into chemically competent Top 10 cells. Bacterial colonies were then cultured and sequenced using Sanger sequencing. Sequence results were aligned with the wt CEP290 locus for analysis.

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.