COMPOSITIONS AND METHODS FOR TREATMENT OF DISEASES ASSOCIATED

WITH TRINUCLEOTIDE REPEATS IN TRANSCRIPTION FACTOR FOUR

DESCRIPTION

[001] This application relates to compositions and methods for treatment of diseases associated with trinucleotide repeats in the transcription factor four (TNF4) gene, including Fuchs endothelial corneal dystrophy (FECD), posterior polymorphous corneal dystrophy (PPCD), primary sclerosing cholangitis (PSC), and Schizophrenia.

[002] Fuchs endothelial corneal dystrophy (FECD), also known as Fuchs' dystrophy, is a degenerative disease affecting the internal endothelial cell monolayer of the cornea. The role of the corneal endothelium is to ensure corneal clarity by maintaining an endothelial barrier and performing pump functions. In FECD, there is accumulation of focal outgrowths (termed guttae) and abnormal collagen in the corneal endothelium. The presence of guttae interspersed among the corneal endothelial and stromal cells is considered a clinical hallmark of the disease. Advanced FECD is characterized by extensive guttae, endothelial cell loss, and stromal edema.

[003] FECD can result in vision loss, and advanced FECD is only treatable with corneal transplantation. It is estimated that approximately 5% of middle-aged Caucasians in the United States are affected by FECD. Additionally, it is estimated that FECD accounts for more than 14,000 corneal transplantations each year. Risks associated with corneal transplants include acute rejection, chronic rejection, failure of the graft to adhere to host bed, infection, and injury to the host eye. Most transplants leave the recipient with less than 20/20 vision, involve up to a six month recovery period, and require patients to use immunosuppressant drops for two years or more post-operatively.

Extended use of immunosuppressant eye drops can increase the risk for cataracts or glaucoma.

[004] A role for genetic factors in FECD has been reported, including single nucleotide

polymorphisms and trinucleotide repeat (TNR) expansions in the transcription factor 4 (TCF4) gene. A TNR in the third intron of the TCF4 gene accounts for most of the inherited predisposition to disease, with repeat length of greater than 50 repeats being associated with clinical diagnosis of FECD (Wieben et al., PLOS One, 7: 11, e49083 (2012)). Recent studies have suggested that this TNR expansion causes aggregation of the affected TCF4 RNA and sequestration of key RNA splicing factors (Mootha et al, Invest Ophthalmol Vis Sci. 55(l):33-42 (2014); Mootha et al, Invest Ophthalmol Vis Sci. 56(3):2003-l 1(2015); Vasanth, et al., Invest Ophthalmol Vis Sci. 56(8):4531-6 (2015); Soliman et al., JAMA Ophthalmol. 133(12): 1386-91 (2015)). Such sequestration can lead to global changes in gene expression, inducing profound changes in cellular function which ultimately lead to cell death (Du et al, J Biological Chem. 290: 10, 5979-5990 (2015)). TCF4 mutations have also been associated with primary sclerosing cholangitis (PSC) and schizophrenia, see Ellinghas et al., HEPATOLOGY, 58:3, 1074-1083 (2013) and Forrest et al, Trends in Molecular Medicine 20:6 (2014).

[005] In other repeat expansion diseases, RNA toxicity has been proposed. In cases of RNA toxicity, expanded microsatellite DNA sequences can be found in noncoding regions of various genes and the repetitive elements are transcribed into toxic gain-of-function RNAs or toxic protein species {see Mohan et al., Brain Res. 1584, 3-14 (2014)). Recently, RNA toxicity has also been shown in patients with FECD {see Du 2015). Further, it has been proposed that TCF4 TNR transcripts predominantly accumulate in the corneal endothelium and thus lead to the pathogenesis characteristic of FECD. Although the role of RNA toxicity helps to delineate potential disease mechanisms in FECD, treatment is still limited to corneal transplantation.

[006] Other forms of early-onset FECD have been associated with mutations in COL8A2 {see Vedana et al., Clinical Ophthalmology 10 321-330 (2016)). Normally, collagen VIII or COL8 (comprising COL8A1 and COL8A2) is regularly distributed in the Descemet's membrane of the cornea. However, corneas from individuals with mutations in COL8A2 have an irregular mosaic deposition of different amounts of COL8A1 and COL8A2 in a non-coordinated fashion. Three mutations (Gln455Lys, Gln455Val, and Leu450Trp) in COL8A2 result in intracellular accumulation of mutant collagen VIII peptides and can cause early-onset FECD, as well as the related disorder posterior polymorphous corneal dystrophy (PPCD). PPCD is characterized by changes in the Descemet's membrane and endothelial layer of the cornea. The form of PPCD most often associated with mutation in the COL8A2 gene is PPCD2.

[007] Means to directly modulate (CTG)„ TNRs in TCF4 and point mutations in COL8A2 are needed to treat genetic mutations leading to FECD, PPCD, PSC, and Schizophrenia. A recently investigated gene editing/disruption technique is based on the bacterial CRISPR (clustered regularly interspersed short palindromic repeats) system. CRISPR gene editing relies on a single nuclease, such as that embodied by "CRISPR-associated protein 9" (Cas9) and Cpfl, that can induce site- specific breaks in the DNA. Cas endonucleases are guided to a specific DNA sequence by small RNA molecules, termed trRNA and crRNA, along with a protospacer adjacent motif (PAM) adjacent to the target gene. The trRNA and crRNA together form the guide RNA, also known as gRNA. The trRNA and crRNA can be combined into a single guide RNA (sgRNA) to facilitate targeting of the Cas protein, or can be used at the same time but not combined, as a dual guide (dgRNA) system. Cas endonucleases in combination with trRNA and crRNA is termed the Cas ribonucleoprotein (RNP) complex. SUMMARY

[008] We herein describe CRISPR compositions and their methods of use that in some

embodiments are designed to excise some or all of the region within TCF4 containing the TNR expansions. In some embodiments these TNR expansions are found in individuals affected with FECD. Doing so prevents the toxicity associated with the expansion. A reduction or elimination in TNRs within TCF4 will reduce downstream effects of the TNRs, such as RNA toxicity, and improve disease course. Thus, guide RNAs complementary to target sequences flanking the TNRs of intron 3 of TCF4 and other modifications of the nuclease (or Cas RNP) may be a means to treat genetic forms of FECD exhibiting TNRs in TCF4, as well as TNRs in PSC and Schizophrenia. Additionally, guide sequences for use in designing guide RNAs that together with a nuclease knock out or edit COL8A2 in forms of FECD and PPCD displaying mutations in the alpha subunit of collagen VIII are also disclosed.

[009] In accordance with the description, in some embodiments compositions of guide RNAs are described that direct CRISPR/Cas endonucleases to regions 5' and 3' to TNR expansions in the TCF4 gene. The compositions are useful in excising TNR expansions from the TCF4 gene, as well as in treating FECD, PPCD, PSC, and Schizophrenia. In other embodiments compositions of guide RNAs are also described that target to regions of the COL8A2 gene, including guide RNAs that target to mutant alleles that are associated with FECD. These guide RNAs are to be used together with a CRISPR nuclease to excise TNRs, generate indels, or induce gene correction through homologous recombination (HR) or homology-directed repair (HDR) via double-strand breaks, depending on the design of the guide RNAs and methods used in the treatments.

[0010] In one embodiment, the invention comprises a composition comprising at least one guide RNA comprising a guide sequence that directs a nuclease to a target sequence selected from SEQ ID NOs: 1-1084. In some embodiments, the invention comprises a composition comprising at least one guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 1089-1278.

[0011] In some embodiments, a composition comprising at least one guide RNA comprising a guide sequence that is identical to a sequence selected from SEQ ID NOs: 1089-1278 is provided.

[0012] In some embodiments, the guide RNA targets a trinucleotide repeat (TNR) in the

transcription factor four (TCF4) gene, and directs a nuclease to a target sequence selected from SEQ ID NOs: 1-190. In some embodiments, the invention comprises at least one guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence selected from SEQ ID NOs: 1089-1278.

[0013] A composition comprising two guide RNAs selected from the following guide RNA pairings is provided:

a. a first guide RNA that directs a nuclease to SEQ ID NO: 83, and a second guide RNA that directs a nuclease to SEQ ID NO: 109;

b. a first guide RNA that directs a nuclease to SEQ ID NO: 85, and a second guide RNA that directs a nuclease to SEQ ID NO: 109;

c. a first guide RNA that directs a nuclease to SEQ ID NO: 86, and a second guide RNA that directs a nuclease to SEQ ID NO: 112;

d. a first guide RNA that directs a nuclease to SEQ ID NO: 85, and a second guide RNA that directs a nuclease to SEQ ID NO: 112;

e. a first guide RNA that directs a nuclease to SEQ ID NO: 86, and a second guide RNA that directs a nuclease to SEQ ID NO: 109;

f. a first guide RNA that directs a nuclease to SEQ ID NO: 85, and a second guide RNA that directs a nuclease to SEQ ID NO: 107;

g. a first guide RNA that directs a nuclease to SEQ ID NO: 83, and a second guide RNA that directs a nuclease to SEQ ID NO: 125;

h. a first guide RNA that directs a nuclease to SEQ ID NO: 86, and a second guide RNA that directs a nuclease to SEQ ID NO: 125;

i. a first guide RNA that directs a nuclease to SEQ ID NO: 86, and a second guide RNA that directs a nuclease to SEQ ID NO: 107;

j . a first guide RNA that directs a nuclease to SEQ ID NO: 64, and a second guide

RNA that directs a nuclease to SEQ ID NO: 106;

k. a first guide RNA that directs a nuclease to SEQ ID NO: 85, and a second guide

RNA that directs a nuclease to SEQ ID NO: 114;

1. a first guide RNA that directs a nuclease to SEQ ID NO: 86, and a second guide

RNA that directs a nuclease to SEQ ID NO: 114;

m. a first guide RNA that directs a nuclease to SEQ ID NO: 83, and a second guide

RNA that directs a nuclease to SEQ ID NO: 114; n. a first guide RNA that directs a nuclease to SEQ ID NO: 53, and a second guide

RNA that directs a nuclease to SEQ ID NO: 114;

o. a first guide RNA that directs a nuclease to SEQ ID NO: 83, and a second guide

RNA that directs a nuclease to SEQ ID NO: 112; and

p. a first guide RNA that directs a nuclease to SEQ ID NO: 74, and a second guide RNA that directs a nuclease to SEQ ID NO: 114.

[0014] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 83 comprises SEQ ID NO: 1177, and the second guide RNA that directs a nuclease to SEQ ID NO: 109 comprises SEQ ID NO: 1197.

[0015] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 85 comprises SEQ ID NO: 1173, and the second guide RNA that directs a nuclease to SEQ ID NO: 109 comprises SEQ ID NO: 1197.

[0016] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 86 comprises SEQ ID NO: 1174, and the second guide RNA that directs a nuclease to SEQ ID NO: 112 comprises SEQ ID NO: 1200.

[0017] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 85 comprises SEQ ID NO: 1173, and the second guide RNA that directs a nuclease to SEQ ID NO: 112 comprises SEQ ID NO: 1200.

[0018] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 86 comprises SEQ ID NO: 1174, and the second guide RNA that directs a nuclease to SEQ ID NO: 109 comprises SEQ ID NO: 1197.

[0019] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 85 comprises SEQ ID NO: 1173, and the second guide RNA that directs a nuclease to SEQ ID NO: 107 comprises SEQ ID NO: 1195.

[0020] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 83 comprises SEQ ID NO: 1171, and the second guide RNA that directs a nuclease to SEQ ID NO: 125 comprises SEQ ID NO: 1213.

[0021] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 86 comprises SEQ ID NO: 1174, and the second guide RNA that directs a nuclease to SEQ ID NO: 125 comprises SEQ ID NO: 1213. [0022] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 86 comprises SEQ ID NO: 1174, and the second guide RNA that directs a nuclease to SEQ ID NO: 107 comprises SEQ ID NO: 1195.

[0023] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 64 comprises SEQ ID NO: 1152, and the second guide RNA that directs a nuclease to SEQ ID NO: 106 comprises SEQ ID NO: 1194.

[0024] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 85 comprises SEQ ID NO: 1173, and the second guide RNA that directs a nuclease to SEQ ID NO: 114 comprises SEQ ID NO: 1202.

[0025] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 86 comprises SEQ ID NO: 1174, and the second guide RNA that directs a nuclease to SEQ ID NO: 114 comprises SEQ ID NO: 1202.

[0026] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 83 comprises SEQ ID NO: 1171, and the second guide RNA that directs a nuclease to SEQ ID NO: 114 comprises SEQ ID NO: 1202.

[0027] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 53 comprises SEQ ID NO: 1141, and the second guide RNA that directs a nuclease to SEQ ID NO: 114 comprises SEQ ID NO: 1202.

[0028] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 83 comprises SEQ ID NO: 1171, and the second guide RNA that directs a nuclease to SEQ ID NO: 112 comprises SEQ ID NO: 1200.

[0029] In some embodiments comprising two gRNAs, the first guide RNA that directs a nuclease to SEQ ID NO: 74 comprises SEQ ID NO: 1162, and the second guide RNA that directs a nuclease to SEQ ID NO: 114 comprises SEQ ID NO: 1202.

[0030] In some embodiments, the guide RNA targets the alpha 2 subunit of collagen type VIII (Col8A2) gene, and directs a nuclease to a target sequence selected from SEQ ID NOs: 191-1063. In some embodiments, the invention comprises at least one guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence complementary, or identical, to the first 20 nucleotides of a target sequence selected from SEQ ID NOs: 191-1063 (e.g, the target sequence absent the PAM), wherein the thymines in the first 20 nucleotides of SEQ ID NOs: 191-1063 are replaced with uracil. [0031] In some embodiments, the guide RNA targets the Gln455Lys mutation in the Col8A2 gene product and directs a nuclease to a target sequence selected from SEQ ID NOs: 1064-1069. In some embodiments, the invention comprises at least one guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence

complementary, or identical, to the first 20 nucleotides of a target sequence selected from SEQ ID NOs: 1064-1069 (e.g, the target sequence absent the PAM), wherein the thymines in the first 20 nucleotides of SEQ ID NOs: 1064-1069 are replaced with uracil.

[0032] In some embodiments, the guide RNA targets the Gln455Val mutation in the Col8A2 gene product and directs a nuclease to a target sequence selected from SEQ ID NOs: 1070-1075. In some embodiments, the invention comprises at least one guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence

complementary, or identical, to the first 20 nucleotides of a target sequence selected from SEQ ID NOs: 1070-1075 (e.g, the target sequence absent the PAM), wherein the thymines in the first 20 nucleotides of SEQ ID NOs: 1070-1075 are replaced with uracil.

[0033] In some embodiments, the guide RNA targets the Leu450Trp mutation in the Col8A2 gene product, and directs a nuclease to a target sequence selected from SEQ ID NOs: 1076-1084. In some embodiments, the invention comprises at least one guide RNA comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence

complementary, or identical, to the first 20 nucleotides of a target sequence selected from SEQ ID NOs: 1076-1084 (e.g, the target sequence absent the PAM), wherein the thymines in the first 20 nucleotides of SEQ ID NOs: 1070-1075 are replaced with uracil.

[0034] In some embodiments, the guide RNA is a dual guide. In some embodiments, the guide RNA is a single guide. In some embodiments, at least one guide RNA comprises a crRNA, a trRNA, or a crRNA and a trRNA.

[0035] In some embodiments, at least one guide sequence is encoded on a vector. In some embodiments, a first guide sequence and a second guide sequence are encoded on the same vector. In some embodiments, a first guide sequence and a second guide sequence are encoded on different vectors. In some embodiments, the first guide sequence and the second guide sequence are controlled by the same promotor and/or regulatory sequence.

[0036] In some embodiments, the guide sequence is complementary to a target sequence in the positive strand of a target gene. In some embodiments, the guide sequence is complementary to a target sequence in the negative strand of a target gene. In some embodiments, a first guide sequence and second guide sequence are complementary to a first target sequence and a second target sequence in opposite strands of a target gene (i.e., a region of interest such as TNRs in TCF4 in genomic DNA).

[0037] In some embodiments, the guide RNA is chemically modified. In some embodiments, the invention further comprises a nuclease. In some embodiments, the nuclease is a Cas protein or other nuclease that cleaves double or single-stranded DNA. In some embodiments, the Cas protein is from the Type-I, Type-II, or Type-Ill CRISPR/Cas system. In some embodiments, the Cas protein is Cas9 or Cpfl . In some embodiments, the nuclease is a nickase. In some embodiments, the nuclease is modified. In some embodiments, the modified nuclease comprises a nuclear localization signal (NLS).

[0038] In some embodiments, the invention comprises a pharmaceutical formulation of a guide RNA and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation comprises one or more guide RNA and an mRNA encoding a Cas protein. In some embodiments, the pharmaceutical formulation comprises one or more guide RNA and a Cas protein.

[0039] In some embodiments, the invention comprises a method of excising at least a portion of a trinucleotide repeat (TNR) in the transcription factor four (TCF4) gene in a human subject. In some embodiments, two guide RNA are used, wherein the first is complementary to a sequence 5' of the TNR and the second is complementary to a sequence 3' of the TNR. When two guide sequences are used, the DNA sequences between the targeted regions of genomic DNA are excised.

[0040] In some embodiments, the TNR is equal to or greater than about 40 trinucleotide repeats. In some embodiments, the TNR is equal to or greater than about 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5 trinucleotide repeats. In some embodiments, the TNR is equal to or greater than about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 trinucleotide repeats.

[0041] In some embodiments, the composition or pharmaceutical formulation comprises at least two guides that excise at least a portion of the TNR. In some embodiments, the entire TNR is excised.

[0042] In some embodiments, the composition or pharmaceutical formulation is administered via a viral vector. In some embodiments, the composition or pharmaceutical formulation is administered via lipid nanoparticles. Any lipid nanoparticle known to those of skill in the art is suitable for delivering the one or more guide RNA provided herein, optionally together with an mRNA encoding a Cas protein. In some embodiments, the lipid nanoparticles described in PCT US2017/024973, filed 3/30/3017, are utilized. In some embodiments, the lipid nanoparticles comprise one or more guide RNA provided herein and an rnRNA encoding a Cas protein. In some embodiments, the lipid nanoparticles comprise one or more guide RNA provided herein without an rnRNA encoding a Cas protein.

[0043] In some embodiments, the invention further comprises co-administration of eye drops or ointments. In some embodiments, the invention further comprising the use of soft contact lenses.

[0044] In some embodiments, the human subject has schizophrenia.

[0045] In some embodiments, the human subject has primary sclerosing cholangitis (PSC).

[0046] In some embodiments, the invention comprises a method of decreasing expression of a mutant allele of the COL8A2 gene, such as Gln455Lys, Gln455Val, or Leu450Trp, or altering the nucleotide sequence to correct said mutant allele in a human subject.

[0047] In some embodiments, the human subject has Fuchs endothelial corneal dystrophy (FECD) or posterior polymorphous corneal dystrophy (PPCD). In some embodiments, the human subject has FECD. In some embodiments, the subject has a family history of FECD.

[0048] In some embodiment, the subject has an improvement, stabilization, or slowing of decline in visual acuity as a result of administration. In some embodiments, the subject has an improvement, stabilization, or slowing of change as measured by corneal pachymetry as a result of administration. In some embodiments, the subject has an improvement, stabilization, or slowing of change based on specular microscopy as a result of administration. In some embodiments, the subject has a delay in the time until a corneal transplant is needed as a result of administration.

[0049] In some embodiments, the invention comprises use of a composition or a pharmaceutical for the preparation of a medicament for treating a human subject having a TNR expansion in the TCF4 gene, or having mutation in the COL8A2 gene leading to a Gln455Lys, Gln455Val, or a Leu450Trp mutation in the gene product.

[0050] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0051] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0052] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 provides a schematic of excision of the TNR expansion region in intron 3 of TCF4 using a pair of gRNAs, with one gRNA having a guide sequence that targets to a region of intron 3 that is 5' of the TNRs and the other gRNA having a guide sequence that targets to a region of intron 3 that is 3' of the TNRs. While the drawing shows the excision occurring at the exact boundaries of the TNR, in practice the excision can be larger or smaller, and include upstream and/or downstream regions of the intron.

[0054] Figure 2 provides a schematic showing the predicted sizes of excised fragments for the 93 pairs of gRNAs that were tested for excision. The numbers correspond to the SEQ ID NOs of each target sequence for the guides tested. The pairs are rank ordered by excision percent (the top pair of the list having the highest excision rate). The "0" marks the center of the TNR region.

DESCRIPTION OF THE SEQUENCES

[0055] Table 1 provides a listing of certain sequences referenced herein.

Presented in Table 3 Target 191- sequence 1063 s for

wild

type

COL8A2

Presented in Table 4 Target 1064- sequence 1069 s for

COL8A2

Gln455Ly s

Mutation

Presented in Table 5 Target 1070- sequence 1075 s for

COL8A2

Gln455Va

1

Mutation

Presented in Table 6 Target 1076- sequence 1084 s for

COL8A2

Leu450Tr

P

Mutation

GTTTGTGTGA TTTTGCTAAA ATGCATCACC AACAGCGAAT TCF4 1085 GGCTGCCTTA GGGACGGACA AAGAGCTGAG TGATTTACTG intron 3

GATTTCAGTG CGgtaagaaa gaacggtgga aactaacaac sequence agctgtgaaa aaaacaaaac aaaaacccaa acacttcagc with

tagaaaccag taggaatcta aaggacagta ataattttta flanking attggctgaa tccttggtaa atatgaaggt ctttttgaca exons ,

agtttttaac tataattttg tggtgtgatg gaagattcag reverse gctttttttt ttttttgagt tttattactg gccttcaatt strand

ccctacccac tgattacccc aaataatgga atctcacccc (GRCh37/ agtggaaagc aaaaatagac acccctaaaa ctaaaccacc hgl9) .

cctaaaactt ggccatgtct gaacactgag actactaata While

ctttgcacac tactcttcgt tttatttatt gtttttggaa commonly atggaaaata gaaaatagga gacccagttg tctctttaaa referred gttttaagct aatgatgctt tggattggta ggacctgttc to as

cttacatctt acctcctagt tacatctttt cctaggattc intron

ttaaaactag tatggatatg ctgagcatac attctttaga 3, many accttttgga ctgttttggt aaatttcgta gtcgtaggat alternat cagcacaaag cggaacttga cacacttgtg gagttttacg ely gctgtacttg gtccttctcc atccctttgc ttccttttcc spliced taaaccaagt cccagacatg tcaggagaat gaattcattt isoforms ttaatgccag atgagtttgg tgtaagatgc atttgtaaag of the caaaataaaa agaatccaca aaacacacaa ataaaatcca gene aaccgccttc caagtggggc tctttcatgc tgctgctgct exist, gctgctgctg ctgctgctgc tgctgctgct gctgctgctg such ctgctgctgc tgctgctgct gctcctcctc ctcctcctcc that ttctcctcct cctcctcctc ttctagacct tcttttggag this aaatggcttt cggaagtttt gccaggaaac gtagccctag intron gcaggcagct ttgcagcccc ctttctgctt gttgcacttt may not ctccattcgt tcctttgctt tttgcaggct ctgactcagg fall gaaggtgtgc attatccact agatacgtcg aagaagaggg between aaaccaatta gggtcgaaat aaatgctgga gagagaggga the 3 ^rd gtgaaagaga gagtgagagt gagagagaga gagagtcttg and 4 ^th cttcaaattg ctctcctgtt agagacgaaa tgagaattta exons of gtgcaggtgg cacttttatt tttatttggg ttcacatatg every acaggcaaat cctatacgag atggaaatgg acattgccac transcri gtttatggcc aaggttttca atataaaaca aaacaacttt pt .

tttcttctcc ttggtgaaac tagtgttttt ctagagaggc

tgctggcctc caacctgaat cttgataaca ttatggggac

tgtgtttgtt ccaaatgtag cagtagtact gcttggccat

Bold ctaatgaacc tgaggaaaaa gaaagaacag agtgataatg

font ggggctgggg tgggatctgt aatgttgttt ctcttttagt

indicate tttaagttgg atggtgatgt attttactaa ataaaccctt

s ctg agcataaact ctaagctgtt tggtaacagt atgaaagatc

repeats tttgaggagc tctgaaggca caagtgtctt cttttcaact

(TNRs) . gtaatatttc tttgtttctt ttagATGTTT TCACCTCCTG

This TGAGCAGTGG GAAAAATGGA CCAACTTCTT TGGCAAGTGG

region ACATTTTACT GGCTCAA

is variable in size.

Capital letters indicate sequence s of adj acent 5' and 3' exons . mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAG sgRNA

AmGmCmUmAmGmAmAmAmUmAmGmCAAGOT modified 1086 AUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAm sequence

Aii AmAii AmGmUmGmGmCmAmCmCmGmAmGmUmC

mGmGmUmGmCmU*mU*mU*mU "N" may

be any

natural

or non- natural

nucleoti

de .

* = PS

linkage ;

Ί' =

2 ' -O-Me

nucleoti

de

crRNA

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUG sequence 1087

"N" may

be any

natural

or non- natural

nucleoti

de .

trRNA

AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG sequence 1088 AAAAAGUGGCACCGAGUCGGUGCUUUUUUU

DESCRIPTION OF THE EMBODIMENTS

Definitions

[0056] The term "treatment," as used herein, covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of FECD may comprise alleviating symptoms of FECD, as well as reducing the number of TNRs in the TCF4 gene resulting in an amelioration of symptoms of FECD, a slowing of disease progression, or cure/prevention of reoccurrence of symptoms of the disease. [0057] As used herein, "FECD" refers to Fuchs endothelial corneal dystrophy, also known as Fuchs' dystrophy. FECD would also include individuals without symptoms but with a genetic disorder, such as a TNR expansion in intron 3 of TCF4, linked to increased occurrence of FECD. FECD would also include individuals without symptoms, but having a known family history of FECD and a TNR expansion in intron 3 of TCF4.

[0058] As used herein, "TNRs" refers to trinucleotide repeats. "Microsatellite repeats" refers to short sequence of DNA consisting of multiple repetitions of a set of two to nine base pairs. The term microsatellite repeats encompasses TNRs. "TNR expansion" refers to a higher than normal number of trinucleotide repeats. For intron 3 of TCF4, for example, a TNR expansion can be characterized by about 50 or more TNRs. The range of TNR expansion associated with disease is usually between 50 and 1000, though some patients with > 1000 repeats have been identified. Patients with < 50 TNRs in intron 3 of TCF4 are generally not considered to be at increased risk of disease through a TNR expansion mechanism, though they may still benefit from a reduced number of TNRs.

[0059] Diseases caused by TNRs and/or characterized by the presence of TNRs may be referred to as "trinucleotide repeat disorders," "trinucleotide repeat expansion disorders," "triplet repeat expansion disorders," or "codon reiteration disorders."

[0060] A "guide RNA" and "gRNA" are used interchangeably herein. The gRNA comprises or consists a CRISPR RNA (crRNA) and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated on one RNA molecule (single guide RNA (sgRNA)), or may be disassociated on separate RNA molecules (dual guide RNA (dgRNA)).

[0061] As used in this application, "the guide sequence" refers to an about 20-base pair sequence within the crRNA or trRNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by a nuclease. Slightly shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-base pairs in length. In some embodiments, the length of the guide sequence corresponds to the length of the target sequence, e.g., as described herein.

[0062] As used herein, a "target sequence" refers to a sequence of nucleic acid to which the guide RNA directs a nuclease for cleavage. The target sequence is within the genomic DNA of a subject. In some embodiments, a Cas protein may be directed by a guide RNA to a target sequence, where the guide RNA hybridizes with and the nuclease cleaves the target sequence. Target sequences include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for a Cas protein is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be "complementary to a target sequence", it is to be understood that the guide sequence may direct a guide RNA (e.g., in a RNP) to bind to the reverse complement of a target sequence provided herein. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to the first 20 nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0063] As used herein, a "PAM" or "protospacer adjacent motif refers to a sequence that must be adjacent to the target sequence. The PAM needed varies depending on the specific CRISPR system. In the CRISPR/Cas system derived from Streptococcus pyogenes, the target DNA must immediately precede a 5'-NGG PAM (where "N" is any nucleobase followed by two guanine nucleobases) for optimal cutting, while other Cas9 orthologs have different PAM requirements. While Streptococcus pyogenes Cas9 can also recognize the 5 '-NAG PAM, it appears to cut less efficiently at these PAM sites. The target sequences of Table 2 comprise a PAM.

[0064] In some embodiments, the guide RNA and the Cas protein may form a "ribonucleoprotein" (RNP). In some embodiments, the guide RNA guides the nuclease such as Cas9 to a target sequence, and the guide RNA hybridizes with and the nuclease cleaves the target sequence.

[0065] As used herein, "indels" refer to insertion/deletion mutations consisting of a number of nucleotides that are either inserted or deleted at the site of double-stranded breaks (DSBs) in the nucleic acid.

[0066] As used herein, "excision fragment(s)" refers to deletions of a consecutive number of nucleotides that may occur when two or more guide RNAs are used together with a Cas mRNA or protein.

Compositions

[0067] Compositions useful in the treatment of FECD are described. In some aspects, the compositions comprise a guide RNA that directs a nuclease to a TNR in the TCF4 gene thereby cleaving the TNR thereby treating diseases having TNRs in the TCF4 gene, including FECD, PPCD, PSC, and Schizophrenia. In some embodiments, the composition comprises two guide RNAs that direct nuclease to a first and second location in intron 3 of TCF4, wherein the nuclease cleaves the intron 3 of TCF4 at the first and second locations and excises a fragment of nucleic acid between the first and the second cleavage, thereby excising some or all of the TNRs contained within intron 3 of TCF4 and treating diseases having TNRs in the TCF4 gene, including FECD, PPCD, PSC, and Schizophrenia. In other aspects, the compositions comprise a guide RNA that directs a nuclease to the COL8A2 gene via a target sequence in the DNA thereby mediating NHEJ for the purpose of cleaving the sequence and leading to introduction of indels or mediating HR or HDR wherein a mutation in the DNA can be corrected by use of a template and treating FECD or PPCD.

Embodiments of the compositions are described below.

Guide RNA

[0068] In some embodiments, the compositions of the invention comprise guide RNA (gRNA) comprising a guide sequence(s) that directs a nuclease such as Cas9 to a target DNA sequence. The gRNA comprises a crRNA and a trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated on one RNA (sgRNA), or may be disassociated on separate RNAs (dgRNA).

[0069] In each of the composition and method embodiments described herein, the guide RNA may comprise two RNA molecules as a "dual guide RNA" or "dgRNA". The dgRNA comprises a first RNA molecule comprising a crRNA, and a second RNA molecule comprising a trRNA. The first and second RNA molecules are not covalently linked, but may form a RNA duplex via the base pairing between the flagpole regions on the crRNA and the trRNA.

[0070] In each of the composition and method embodiments described herein, the guide RNA may comprise a single RNA molecule as a "single guide RNA" or "sgRNA". The sgRNA comprises a crRNA covalently linked to a trRNA. In some embodiments, the crRNA and the trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via the base pairing between the flagpole regions on the crRNA and the trRNA.

[0071] In some embodiments, the trRNA may comprise all or a portion of a wild type trRNA sequence from a naturally -occurring CRISPR/Cas system. In some embodiments, the trRNA comprises a truncated or modified wild type trRNA. The length of the trRNA depends on the CRISPR/Cas system used. In some embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In certain embodiments, the trRNA is at least 26 nucleotides in length. In additional embodiments, the trRNA is at least 40 nucleotides in length. In some embodiments, the trRNA may comprise certain secondary structures, such as, e.g., one or more hairpins or stem-loop structures, or one or more bulge structures.

[0072] In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a "modified" gRNA or "chemically modified" gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called "modified." 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 (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with "dephospho" linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose- phosphate backbone (an exemplary backbone modification); (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, cap or linker (such 3 Or 5' cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).

[0073] The modifications listed above can be combined to provide modified gRNAs comprising nucleosides and nucleotides (collectively "residues") that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In some embodiments, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA.

[0074] In some embodiments, the gRNA comprises one, two, three or more modified residues. 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 gRNA are modified nucleosides or nucleotides. [0075] 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 gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases. In some embodiments, the modified gRNA molecules 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, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0076] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0077] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. 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. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone 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.

[0078] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged 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, carboxy methyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. [0079] 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. Such modifications may comprise backbone and sugar modifications. 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.

[0080] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. 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.

[0081] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); poly ethylenegly cols (PEG), 0(CH2CH20)nCH2CH20R 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 2' hydroxyl group modification can be 2'-0-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include "locked" nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a Ci-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., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can included "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).

[0082] "Deoxy" 2' modifications can include hydrogen {i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo {e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) _n CH2CH2- 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.

[0083] The sugar modification can comprise a sugar group which may 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 modified nucleic acids can also include abasic sugars. 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.

[0084] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a 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 residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally -occurring and synthetic derivatives of a base.

[0085] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification. In certain embodiments, one or more or all of the nucleotides in single stranded overhang of a guide RNA molecule are deoxynucleotides.

[0086] In some embodiments, the guide RNAs disclosed herein comprise one of the modification patterns disclosed in US 62/431,756, filed December 8, 2016, titled "Chemically Modified Guide RNAs," the contents of which are hereby incorporated by reference in their entirety.

[0087] In some embodiments, the invention comprises a gRNA comprising one or more

modifications. In some embodiments, the modification comprises a 2'-0-methyl (2'-0-Me) modified nucleotide. In some embodiments, the modification comprises a phosphorothioate (PS) bond between nucleotides. [0088] The terms "mA," "mC," "mU," or "mG" may be used to denote a nucleotide that has been modified with 2'-0-Me.

[0089] Modification of 2'-0-methyl can be depicted as follows:

[0090] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2'-fluoro (2'-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.

[0091] In this application, the terms "fA," "fC," "fU," or "fG" may be used to denote a nucleotide that has been substituted with 2'-F.

[0092] Substitution of 2'-F can be depicted as follows:

Natural composition of R A 2'F substitution

[0093] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[0094] A "*" may be used to depict a PS modification. In this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide with a PS bond. [0095] In this application, the terms "mA*," "mC*," "mU*," or "mG*" may be used to denote a nucleotide that has been substituted with 2'-0-Me and that is linked to the next (e.g., 3') nucleotide with a PS bond.

[0096] The diagram below shows the substitution of S- into a nonbridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

Natural phosphodiester Modified phosphorothioate

linkage of WA (PS} bond

[0097] Abasic nucleotides refer to those which lack nitrogenous bases. The figure below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base:

[0098] Inverted bases refer to those with linkages that are inverted from the normal 5' to 3' linkage (i.e., either a 5' to 5' linkage or a 3' to 3' linkage). For example:

Normal oligonucleotide inverted oligonucieottde

linkage itnkage

[0099] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5' nucleotide via a 5' to 5' linkage, or an abasic nucleotide may be attached to the terminal 3' nucleotide via a 3' to 3' linkage. An inverted abasic nucleotide at either the terminal 5' or 3' nucleotide may also be called an inverted abasic end cap.

[00100] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus of the guide RNA are modified. In some embodiments, the modification is a 2'-0-Me, 2'-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance.

[00101] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.

[00102] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-0-methyl (2'-0-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise an inverted abasic nucleotide.

[00103] In some embodiments, the guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in SEQ ID NO: 1086, where N is any natural or non-natural nucleotide, and where the totality of the N's comprise a guide sequence as described herein that directs a nuclease to a TC4 target sequence. Guide RNAs for TCF4

[00104] In some embodiments, the composition comprises at least one guide RNA (gRNA) comprising or consisting of a guide sequence complementary to any one of the nucleic acids of SEQ ID NOs: 1-190. In some embodiments, the composition comprises at least one guide RNA (gRNA) comprising or consisting of a guide sequence that directs a nuclease to any one of the nucleic acids of SEQ ID NOs: 1-190. In one aspect, the composition comprises at least one gRNA comprising or consisting of a guide sequence complementary to a target sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 1- 190. In one aspect, the composition comprises at least one gRNA comprising or consisting of a guide sequence that directs a nuclease to a target sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 1-190.

[00105] In some aspects, the composition comprises at least one gRNA comprising or consisting of a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 1089-1278. In some aspects, the composition comprises at least one gRNA comprising or consisting of a guide sequence identical to any of the nucleic acids of SEQ ID NOs: 1089-1278.

[00106] In other embodiments, the composition comprises at least two gRNA's comprising or consisting of at least two guide sequences complementary to any one of the target sequences selected from any two or more of the nucleic acids of SEQ ID NOs: 1-190. In some embodiments, the composition comprises at least two gRNA's comprising or consisting of at least two guide sequences complementary to any one of the target sequences selected from any two or more of the nucleic acids that are at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 1-190.

[00107] In some embodiments, a gRNA that targets to a sequence 5' of the TNRs of TCF4 is used together with a gRNA that targets to a sequence 3 ' of the TNRs of TCF4 for the purpose of excising the TNRs of TCF4. In some embodiments, a guide sequence complementary to a target sequence of SEQ ID NOs: 1-93 is used together with a guide sequence complementary to a target sequence of SEQ ID NOs: 94-190.

[00108] In some embodiments, use of a gRNA that targets to a sequence 5' of the TNRs of

TCF4 together with a gRNA that targets to a sequence 3 ' of the TNRs of TCF4 excises the full sequence of TNRs in intron 3 of TCF4 in patients with extended TNR sequences. For example, in some embodiments the combination of gRNAs targeting sequences 5' and 3' to the TNR expansion excises a TNR having at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 or more repeats. In some embodiments, this approach is used to excise TNR expansions greater than 40 in number. In some embodiments, use of a gRNA that targets to a sequence 5' of the TNRs of TCF4 together with a gRNA that targets within the TNR repeats, or use of a gRNA that targets within the TNR repeats together with a gRNA that targets to a sequence 3' of the TNRs of TCF4, excises a portion of the extended TNRs in intron of TCF4 in patients with extended TNR sequences, thereby shortening the length of the TNRs. In some embodiments, the one guide RNA targets a sequence that is 5' of the TNRs of TCF4, and the other guide RNA targets a sequence that is 3' of the TNRs of TCF4, thereby excising all of the TNRs.

Combinations of Two or More Guide RNAs Targeting to TCF4

[00109] In certain embodiments, the compositions comprise more than one gRNA. Each gRNA may contain a different guide sequence, such that the associated nuclease cleaves more than one target sequence. In some embodiments, the gRNAs may have the same or differing properties such as activity or stability within the RNP complex. In some embodiments involving vectors, where more than one gRNA is used, each gRNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one gRNA may be the same or different. In certain embodiments involving lipid nanoparticles, the two or more gRNAs may be formulated in the same lipid nanoparticle or in separate lipid nanoparticles.

[00110] In some embodiments, the guide sequence of each gRNA is complementary to a target sequence in the same strand of the TCF4 gene. In some embodiments, the guide sequence of each gRNA is complementary to a target sequence in the positive strand of the TCF4 gene. In some aspects, the guide sequences of each gRNA is complementary to a target sequence in the negative strand of the TCF4 gene. In some embodiments, the guide sequences of the gRNAs are

complementary to target sequences in opposite strands of the TCF4 gene.

[00111] In some aspects, the compositions comprise at least two gRNAs, wherein the at least two gRNAs comprise guide sequences that target nucleases to two different locations. In some embodiments, the two gRNAs may flank a TNR of the TCF4 gene (i.e., the two gRNAs are on either side of the TNR; said another way, one gRNA is 5' to the TNR and another gRNA is 3' to the TNR). In some embodiments, one gRNA is within a TNR of the TCF4 gene and the other gRNA is outside of the TNR (i.e., flanks the TNR) of the TCF4 gene.. In some embodiments, the two gRNAs target nucleases to target sequences that are about 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 150, 100, 50, or 30 nucleotides apart. In some embodiments, the nuclease cleaves each location and a DNA fragment comprising the TNR expansion region of intron 3 of TCF4 is excised. [00112] In some embodiments, only one gRNA is used. In some embodiments, a gRNA that targets to a sequence 5' of the TNRs of TCF4 is used. In some embodiments the guide sequence is complementary to the target sequence of SEQ ID NO: 1-93. In some embodiments, a gRNA that targets to a sequence 3' of the TNRs of TCF4 is used. In some embodiments, a guide complementary to the target sequence of SEQ ID NOs: 94-190 is used. In some embodiments, a gRNA that targets a sequence within the TNR repeat expansion in TCF4 is used. In some embodiments, use of a single guide leads to indel formation during NHEJ that reduces or eliminates the TNR sequence. In some embodiments, use of a single guide leads to indel formation during NHEJ that reduces or eliminates a part of the TNR sequence.

Guide RNAs for COL8A2

[00113] In some embodiments, the composition comprises at least one guide RNA (gRNA) comprising or consisting of a guide sequence complementary to any of the nucleic acids of SEQ ID NOs: 191-1084. In one aspect, the composition comprises at least one gRNA comprising or consisting of a guide sequence complementary to a target sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 191- 1084.

[00114] In other embodiments, the composition comprises at least two gRNA's comprising or consisting of at least two guide sequences complementary to any two or more of the nucleic acids of SEQ ID NOs: 191-1084. In some embodiments, the composition comprises at least two gRNA's comprising or consisting of at least two guide sequences complementary to any two or more of the nucleic acids that are at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a sequence of the nucleic acids of SEQ ID NOs: 191-1084.

[00115] In some embodiments, a gRNA that targets to a sequence in wild type COL8A2, without known mutations, is used. In some embodiments, a guide sequence complementary to a target sequence of SEQ ID NOs: 191-1063 is used.

[00116] In some embodiments, a gRNA that targets to a sequence corresponding to a mutation in COL8A2 known to produce a Gln455Lys mutation is used. In some embodiments, a guide sequence complementary to a target sequence of SEQ ID NOs: 1064-1069 is used, e.g., to selectively edit the Gln455Lys mutation, caused by the c. l364C>A nucleotide change.

[00117] In some embodiments, a gRNA that targets to a sequence corresponding to a mutation in COL8A2 known to produce a Gln455Val mutation is used. In some embodiments, a guide sequence complementary to a target sequence of SEQ ID NOs: 1070-1075 is used, e.g., to selectively edit the Gln455Val mutation caused by the c. l363-1364CA>GT nucleotide changes.

[00118] In some embodiments, a gRNA that targets to a sequence corresponding to a mutation in COL8A2 known to produce a Leu450Trp mutation is used. In some embodiments, a guide sequence complementary to a target sequence of SEQ ID NOs: 1076-1084 is used, e.g., to selectively edit the Leu450Trp mutation caused by the c. l 349T>G nucleotide change.

Target Sequences

[00119] In some embodiments, the guide RNA targets a nuclease to the COL8A2 gene. In some aspects, the crRNA comprises a guide sequence that is complementary to, and hybridizes with, a target sequence flanking the TNRs in the TCF4 gene. In some embodiments, two gRNAs are utilized. In such embodiments, the two gRNAs may flank a TNR of the TCF4 gene (i.e., the two gRNAs are on either side of the TNR). In some embodiments, one gRNA is within a TNR of the TCF4 gene and the other gRNA is outside of the TNR (i.e., flanks) the TNR of the TCF4 gene. In some embodiments the crRNA further comprises a flagpole region that is complementary to and hybridizes with a portion of a trRNA. In some embodiments, the crRNA may parallel the structure of a naturally occurring crRNA transcribed from a CRISPR locus of a bacteria, where the guide sequence acts as the "spacer" of the CRISPR/Cas9 system, and the flagpole corresponds to a portion of a repeat sequence flanking the spacers on the CRISPR locus.

Target Sequences for TCF4

[00120] The compositions of the present invention may be directed to and cleave a target sequence within or flanking TNRs in the TCF4 gene. For example, the TNR target sequence may be recognized and cleaved by the provided nuclease. In some embodiments, a Cas protein may be directed by a guide RNA to a target sequence flanking TNRs in the TCF4 gene, where the guide sequence of the guide RNA hybridizes with the target sequence or its reverse complement and directs a Cas protein to cleave the target sequence. In some embodiments, a Cas protein may be directed by a guide RNA to a target sequence within TNRs in the TCF4 gene. In some embodiments, a Cas protein may be directed by more than one guide RNA to two target sequences flanking TNRs in the TCF4 gene. In some embodiments, a Cas protein may be directed by more than one guide RNA to two target sequences, wherein one flanks TNRs in the TCF4 gene and another is within the TNRs in the TCF4 gene. [00121] In some embodiments, the selection of the one or more guide RNA is determined based on target sequences near TNRs in the TCF4 gene. For example, in some embodiments, the one or more guide RNA comprises a guide that is complementary to target sequences flanking TNRs in the TCF4 gene. In some embodiments, the crRNA sequence of the one or more guide RNA is complementary to and hybridizes to a target sequence chosen from SEQ ID NOs: 1-190.

[00122] In some embodiments, the target sequence may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100%

complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, where the total length of the guide sequence is about 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-6 mismatches where the guide sequence is about 20 nucleic acids. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1 or 2 mismatches where the guide sequence is about 20 nucleic acids.

[00123] The length of the target sequence may depend on the nuclease system used. For example, the target sequence for a CRISPR/Cas system may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides. In some embodiments, the target sequence may comprise 18-24 nucleotides. In some embodiments, the target sequence may comprise 19-21 nucleotides. In some embodiments, the target sequence may comprise 20 nucleotides. When nickases are used, the target sequence may comprise a pair of target sequences recognized by a pair of nickases on opposite strands of the DNA molecule.

Target Sequences for COL8A2

[00124] The compositions of the present invention may be directed to a target sequence in the

COL8A2 gene. For example, the COL8A2 target sequence may be recognized and cleaved by the provided nuclease. In some embodiments, a Cas protein may be directed by a guide RNA to a target sequence of COL8A2, where the guide sequence of the guide RNA hybridizes with and the Cas protein cleaves the target sequence. [00125] In some embodiments, the selection of the one or more guide RNA is determined based on target sequences in the COL8A2 gene. In some embodiments, the crRNA sequence of the one or more guide RNA is complementary to and hybridizes to a target sequence chosen from SEQ ID NOs: 191-1084.

[00126] In some embodiments, the selection of the one or more guide RNA is determined based on target sequences in the wild type COL8A2 gene, which does not have known mutations leading to abnormal function of the alpha subunit of collagen VIII (COL8A2). In some embodiments, the crRNA sequence of the one or more guide RNA is complementary to and hybridizes to a target sequence chosen from SEQ ID NOs: 191-1063.

[00127] In some embodiments, the selection of the one or more guide RNA is determined based on target sequences in the COL8A2 gene that correspond to Gln455Lys mutations in the COL8A2 protein, caused by the c. l364C>A nucleotide change. In some embodiments, the crRNA sequence of the one or more guide RNA is complementary to and hybridizes to a target sequence chosen from SEQ ID NOs: 1064-1069.

[00128] In some embodiments, the selection of the one or more guide RNA is determined based on target sequences in the COL8A2 gene that correspond to Gln455Val mutations in the COL8A2 protein, caused by the c. l 363-1364CA>GT nucleotide changes. In some embodiments, the crRNA sequence of the one or more guide RNA is complementary to and hybridizes to a target sequence chosen from SEQ ID NOs: 1070-1075.

[00129] In some embodiments, the selection of the one or more guide RNA is determined based on target sequences in the COL8A2 gene that correspond to Leu450Trp mutations in the COL8A2 protein, caused by the c. l349T>G nucleotide change. In some embodiments, the crRNA sequence of the one or more guide RNA is complementary to and hybridizes to a target sequence chosen from SEQ ID NOs: 1076-1084.

[00130] In some embodiments, the target sequence may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100%

complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches, where the total length of the guide sequence is about 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-6 mismatches where the guide sequence is about 20 nucleic acids. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1 or 2 mismatches where the guide sequence is about 20 nucleic acids.

[00131] The length of the target sequence may depend on the nuclease system used. For example, the target sequence for a CRISPR/Cas system may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides. In some embodiments, the target sequence may comprise 18-24 nucleotides. In some embodiments, the target sequence may comprise 19-21 nucleotides. In some embodiments, the target sequence may comprise 20 nucleotides. The target sequence may include a PAM. When nickases are used, the target sequence may comprise a pair of target sequences recognized by a pair of nickases on opposite strands of the DNA molecule.

Vectors

[00132] In certain embodiments of the invention, the compositions comprise DNA vectors encoding any of the guide RNAs described herein. In some embodiments, in addition to guide RNA sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding a nuclease such as Cas9. In some embodiments, the vector comprises a nucleotide sequence encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally- occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA.

[00133] In some embodiments, the crRNA and the trRNA are encoded by non-contiguous nucleic acids within one vector. In other embodiments, the crRNA and the trRNA may be encoded by a contiguous nucleic acid. In some embodiments, the crRNA and the trRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and the trRNA are encoded by the same strand of a single nucleic acid. In some embodiments, the vector encodes one or more sgRNAs. In other embodiments, the vector encodes two or more sgRNAs. Nuclease

[00134] In some embodiments, in addition to the at least one gRNA, the composition further comprises a nuclease. In some embodiments, the gRNA together with nuclease is called a ribonucleoprotein complex (RNP). In some embodiments, the nuclease is a Cas protein. In some embodiments, the gRNA together with a Cas protein is called a Cas RNP. In some embodiments, the Cas comprises Type-I, Type-II, or Type-Ill components. In some embodiments, the Cas protein is from the Type-I CRISPR/Cas system. In some embodiments, the Cas protein is from the Type-II CRISPR/Cas system. In some embodiments, the Cas protein is from the Type-Ill CRISPR/Cas system. In some embodiments, the Cas protein is Cas9. In some embodiments, the Cas protein is Cpf 1. In some embodiments, the Cas protein is the Cas9 protein from the Type-II CRISPR/Cas system. In some embodiment, the gRNA together with Cas9 is called a Cas9 RNP.

[00135] In embodiments encompassing a Cas nuclease, the Cas nuclease may be from a Type-

IIA, Type-IIB, or Type-IIC system. Non-limiting exemplary species that the Cas nuclease or other RNP components may be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Franci sella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene,

Rhodo spirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Strepto sporangium roseum,

Strepto sporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum,

Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus

chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp. , Lachnospiraceae bacterium ND2006, and Acaryochloris marina. In some embodiments, the Cas nuclease is the Cas9 protein from

Streptococcus pyogenes. In some embodiments, the Cas nuclease is the Cas9 protein from

Streptococcus thermophilus . In some embodiments, the Cas nuclease is the Cas9 protein from Neisseria meningitidis. In some embodiments, the Cas nuclease is the Cas9 protein is from

Staphylococcus aureus. In some embodiments, the Cas nuclease is the Cpfl protein from

Francisella novicida. In some embodiments, the Cas nuclease is the Cpfl protein from

Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpfl protein from

Lachnospiraceae bacterium ND2006.

[00136] Wild type Cas9 has two nuclease doacmains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9. In each of the composition and method embodiments, the Cas induces a double strand break in target DNA.

[00137] Modified versions of Cas9 having one catalytic domain, either RuvC or HNH, that is inactive are termed "nickases". Nickases cut only one strand on the target DNA, thus creating a single-strand break. A single-strand break may also be known as a "nick." In some embodiments, the compositions and methods comprise nickases. In some embodiments, the compositions and methods comprise a nickase Cas9 that induces a nick rather than a double strand break in the target DNA.

[00138] In some embodiments, the Cas protein may be modified to contain only one functional nuclease domain. For example, the Cas protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase Cas is used having a RuvC domain with reduced activity. In some embodiments, a nickase Cas is used having an inactive RuvC domain. In some embodiments, a nickase Cas is used having an HNH domain with reduced activity. In some embodiments, a nickase Cas is used having an inactive HNH domain.

[00139] In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas protein may comprise an amino acid substitution in the RuvC or RuvC -like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). In some embodiments, the Cas protein may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein).

[00140] In some embodiments, the composition comprises a nickase and a pair of guide

RNAs. In some embodiments, the pair of guide RNAs are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase Cas is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase Cas is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.

[00141] In some embodiments, chimeric Cas proteins are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fokl. In some

embodiments, a Cas protein may be a modified nuclease.

[00142] In some embodiments, a Cas9-deaminase fusion is used, wherein the Cas9 is not capable of cleaving double-stranded DNA (dCas9). The term "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is a cytidine deaminase that converts cytidine (C) to uracil (U), which then gets converted by the cell to thymidine (T). In some embodiments, the deaminase is a guanine deaminase that converts guanine (G) to xanthine, which then gets converted by the cell to adenine (A). In some embodiments, the deaminase is an APOBEC 1 family deaminase, an activation-induced cytidine deaminase (AID), and adenosine deaminase such as an AD AT family deaminase, or an adenosine deaminase acting on RNA (ADAR), that converts adenine (A) to hypoxanthine, which then gets converted by the cell to guanine (G).

[00143] In other embodiments, the Cas protein may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas protein may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas protein may be a Cas3 protein. In some embodiments, the Cas protein may be from a Type-Ill CRISPR/Cas system. In some embodiments, the Cas protein may have an RNA cleavage activity.

PAM

[00144] In some embodiments, the target sequence may be adjacent to a PAM. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4, nucleotides of the 3' end of the target sequence. The length and the sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or a particular PAM sequence for a specific Cas9 protein or Cas9 ortholog, including those disclosed in Figure 1 of Ran et al., Nature 520: 186- 191 (2015), which is incorporated herein by reference. In some embodiments, the PAM may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NAG, NGA, NGAG, NGCG, NNGRRT, TTN, NGGNG, NG, NAAAAN,

NNAAAAW, NNNNACA, GNNNCNNA, and NNNNGATT (wherein N is defined as any nucleotide, and W is defined as either A or T, and R is defined as either A or G). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be NNAAAAW.

Methods of excising TNRs

[00145] TNRs in TCF4 have been correlated with increased risk of FECD. Additionally, mutations in TCF4 have been associated with schizophrenia and PSC. Delivery of guide RNAs together with a Cas protein (or nucleic acid encoding a Cas protein) may be used as a treatment for these disorders, for example by excising TNRs (or a portion thereof) from the TCF4 gene.

Accordingly, certain embodiments provided herein involve methods of excising TNRs from TCF4. In some embodiments, the method of comprises delivering to a cell any one of the CRISPR/Cas compositions provided herein which comprise one or more gRNAs which direct a nuclease to a Target Sequence provided in Table 2 herein. In some embodiments, the method comprises delivering to a cell two gRNAs together with a Cas protein (or nucleic acid encoding a Cas protein), wherein a first gRNA comprises a guide sequence which targets a region 5' of the TNR and is selected from the group consisting of SEQ ID NOs: 1089-1181 and a second gRNA comprises a guide sequence which targets a region 3 ' of the TNR and is selected from the group consisting of SEQ ID NOs: 1182-1278. In some embodiments, the cell is a human cell, for example a human corneal endothelium cell. In some embodiments, the method results in a population of cells wherein some fraction of the population has the TNR excised from a TCF4 gene. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% or more of the cells within the population has the TNR excised from a TCF4 gene. Methods for measuring the percent of exision within a population of cells are known, and include those provided herein, e.g., next generation sequencing (NGS) methods, for example where the excision percentage is defined as the number of sequencing reads containing a deletion of the TNRs divided by the total number of reads overlapping the target region.

[00146] Use of the CRISPR/Cas system can lead to double-stranded breaks in the DNA, or single-stranded breaks in the DNA if a nickase enzyme is used.

[00147] NHEJ is a process whereby double-stranded breaks (DSBs) in the DNA are repaired via re-ligation of the break ends, which can produce errors in the form of insertion/deletion (indel) mutations. NHEJ can thus be a means to knockout or reduce levels of a specific gene product, as indels occurring within a coding exon can lead to frameshift mutations and premature stop codons.

[00148] HR and HDR are alternative major DNA repair pathways that can be leveraged to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. This can be used to correct single base changes, deletions, insertions, inversions, and other mutations. In some cases, a repair template is used that introduces silent (i.e.,

synonymous) nucleotide changes within the DNA that prevent recognition by the CRISPR nuclease used to initiate the repair process, thereby preventing indel formation within the corrected gene.

[00149] In some embodiments, the template may be used in HR, e.g., to modify a target gene such as TCF4 and/or COL8A2. In some embodiments, the HR may result in the integration of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided such that HR may occur at two or more target sites. For example, different templates may be provided to repair a single gene in a cell, or two different genes in a cell. In some embodiments, multiple copies of at least one template are provided to a cell. In some embodiments, the different templates may be provided in independent copy numbers or independent amounts.

[00150] In other embodiments, the template may be used in HDR, e.g., to modify a target gene such as TCF4 and/or COL8A2. HDR involves DNA strand invasion at the site of the cleavage in the nucleic acid. In some embodiments, the HDR may result in including the template sequence in the edited target nucleic acid molecule. In some embodiments, a single template may be provided. In other embodiments, two or more templates having different sequences may be used at two or more sites by HDR. For example, different templates may be provided to repair a single gene in a cell, or two different genes in a cell. In some embodiments, multiple copies of at least one template are provided to a cell. In some embodiments, the different templates may be provided in independent copy numbers or independent amounts. [00151] In yet other embodiments, the template may be used in gene editing mediated by

NHEJ, e.g., to modify a target gene such as TCF4 and/or COL8A2. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, a single template may be provided. In other embodiments, two or more templates having different sequences may be inserted at two or more sites by NHEJ. For example, different templates may be provided to insert a single template in a cell, or two different templates in a cell. In some embodiments, the different templates may be provided in independent copy numbers. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences.

[00152] The template may be of any suitable length. In some embodiments, the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template may be a single-stranded nucleic acid. The template can be double-stranded or partially double-stranded nucleic acid. In certain embodiments, the single stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template may comprise a nucleotide sequence that is complementary to a portion of the target nucleic acid molecule comprising the target sequence {i.e., a "homology arm"). In some embodiments, the template may comprise a homology arm that is complementary to the sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule. In some embodiments, the template may comprise a first homology arm and a second homology arm (also called a first and second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. Where a template contains two homology arms, each arm can be the same length or different lengths, and the sequence between the homology arms can be substantially similar or identical to the target sequence between the homology arms, or it can be entirely unrelated. In some embodiments, the degree of

complementarity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, may permit homologous recombination, such as, e.g. , high-fidelity homologous recombination, between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%.

[00153] In some embodiments, the template contains ssDNA or dsDNA containing flanking invert-terminal repeat (ITR) sequences. In some embodiments, the template is supplied as a plasmid, minicircle, nanocircle, or PCR product.

Excision fragments

[00154] Generation of excision fragments is a means to harness the power of CRISPR technology to precisely remove small regions of DNA between two target sequences through use of two guide RNAs complementary to these target sequences. In some embodiments, the two guide RNAs target nucleases to sequences that are about 3000, 2500, 2000, 1500, 1000, 500, 400, 300, 200, 150, 100, 50, or 30 nucleotides apart, leading to excision of a DNA fragment between the target sequences.

Treatment of FECD with CRISPR/Cas Compositions

[00155] Any of the compositions described herein may be administered to subjects to treat

FECD in individuals with genetic mutations leading to increased risk of FECD.

[00156] Any of the compositions described herein may be administered to subjects to treat

FECD in individuals with TNR expansion in intron 3 of TCF4. Methods of treating FECD comprising administering any of the compositions described herein are encompassed. In some aspects, the compositions are administered in therapeutically effective amounts. In some

embodiments, a method of excising, mutating, reducing copy number of, ameliorating, and/or eradicating TNRs of TCF4 is encompassed, comprising administering one or more of the

compositions described herein. In some embodiments, a method of excising, reducing copy number of, ameliorating, and/or eradicating the TNRs of one or both copies of TCF4 per cell in a subject is provided, comprising administering one or more of the compositions described herein. In some embodiments, the cell is a corneal endothelium cell.

[00157] In some aspects, a method of reducing, inhibiting, or ameliorating RNA toxicity of

TCF4 comprising administering one or more of the compositions described herein is encompassed. In some embodiments, a method of inhibiting RNA toxicity is encompassed comprising

administering one or more of the compositions described herein, wherein the level of toxic RNA products of TCF4 does not return to pre-administration levels after treatment, returning normal function to the corneal endothelial cells, and preventing cell death. [00158] In some embodiments, treatment may be with a vector and/or lipid nanoparticle comprising the appropriate guide or guides, delivered into the anterior chamber of the eye. In some embodiments, treatment may be with a vector and/or lipid nanoparticle comprising the appropriate guide or guides, delivered into the posterior chamber of the eye. In some embodiments, treatment may be with a vector and/or lipid nanoparticle comprising the appropriate guide or guides, delivered into the cornea itself. In some embodiments, treatment may be with a vector and/or lipid

nanoparticle comprising the appropriate guide or guides, delivered into the corneal stroma. In some embodiments, treatment may be with a vector and/or lipid nanoparticle comprising the appropriate guide or guides, delivered into the corneal limbus. In some embodiments, treatment may be with a vector and/or lipid nanoparticle comprising the appropriate guide or guides, delivered topically onto the epithelial surface of the cornea. In any of the preceding embodiments of this paragraph as well as other embodiments described herein, treatment further comprises delivery of a Cas protein (e.g., Cas9), for example using a lipid nanoparticle, or delivery of a nucleic acid encoding a Cas protein using a vector and/or lipid nanoparticle. In some embodiments, for example those using a lipid nanoparticle, the nucleic acid encoding the Cas protein is mRNA. In some embodiments, a Cas protein or a nucleic acid encoding a Cas protein is delivered via the same vector and/or lipid nanoparticle that is used to deliver the appropriate guide or guides. In some embodiments, a Cas protein or a nucleic acid encoding a Cas protein is delivered via a different vector and/or lipid nanoparticle that is used to deliver the appropriate guide or guides.

[00159] In some embodiments, a single administration of the CRISPR compositions of the invention may be sufficient to correct the underlying genetic defect or mutation associated with disease. In other embodiments, more than one administration of the CRISPR therapeutic may be beneficial, to maximize editing across all target cells and all alleles via cumulative effects.

[00160] Use of the compositions described herein for the preparation of a medicament for treating FECD are encompassed. In some embodiments, the patient with FECD, possible FECD, and/or a family history suggestive of FECD is screened for TNRs in TCF4 before initiation of treatment with the compositions of the invention. In some embodiments, treatment is initiated in a patient if 50 or more TNR are present in intron 3 of TCF4.

[00161] Mutations in COL8A2 have been correlated with an increased risk of FECD and

PPCD. Any of the compositions described herein may be administered to subjects to treat FECD in individuals with mutations in COL8A2 leading to gene products with amino acid mutations. In some embodiments, these amino acid mutations are Gln455Lys, Gln455Val, or Leu450Trp. [00162] Methods of treating FECD comprising administering any of the compositions described herein are encompassed. In some aspects, the compositions are administered in therapeutically effective amounts. In some embodiments, a method of cleaving, mutating, ameliorating, and/or eradicating mutations in COL8A2 is encompassed, comprising administering one or more of the compositions described herein. In some embodiments, use of CRISPR/Cas compositions is done together with a process of NHEJ, leading to generation of indels and loss of a COL8A2 allele. In some embodiments, use of CRISPR/Cas compositions is done together with either an exogenous template for HR/HDR, or using the endogenous normal allele as template for

HR/HDR, for the purpose of correcting a nucleic acid mutation that leads to an amino acid mutation in the alpha 2 subunit of collagen VIII. In some embodiments, the mutation in the COL8A2 gene that is corrected is the Gln455Lys mutation, caused by the c.1364C>A nucleotide change. In some embodiments, the mutation in the COL8A2 gene that is corrected is the Gln455Val mutation caused by the c. l 363-1364CA>GT nucleotide changes. In some embodiments, the mutation in the COL8A2 gene that is corrected is the Leu450Trp mutation caused by the c. l 349T>G nucleotide change. In some embodiments, use of a template together with a Cas RNP leads to correction of the nucleic acid sequence such that the mutation is no longer present. In some embodiments, the cell is a corneal endothelium cell.

[00163] In some aspects, a method of reducing, inhibiting, or ameliorating the abnormal collagen formed by mutant COL8A2, comprising administration of one or more of the compositions described herein is encompassed. In some embodiments, a method of inhibiting production of abnormal alpha subunit of collagen VIII (COL8A2) is encompassed comprising administration of one or more of the compositions described herein, wherein the level of abnormal COL8A2 does not return to pre-administration levels after treatment. In some embodiments, a method of correcting a genetic mutation with HR or HDR, such that only normal collagen is produced, is encompassed comprising administering one or more of the compositions described herein. Reduction or correction of the mutant form of collagen should prevent the abnormal collagen deposition seen in the cornea of FECD patients.

[00164] Use of the compositions described herein for the preparation of a medicament for treating FECD are encompassed. In some embodiments, the patient with FECD, possible FECD, and/or a family history suggestive of FECD is screened for mutation in COL8A2 before initiation of treatment with the compositions of the invention. In some embodiments, the patient with PPCD, possible PPCD, and/or a family history suggestive of PPCD is screened for mutation in COL8A2 before initiation of treatment with the compositions of the invention. In some embodiments, treatment is initiated in a patient if a mutation is present, such the Gln455Lys mutation caused by the c. l364C>A nucleotide change, the Gln455Val mutation caused by the c. l 363-1364CA>GT nucleotide changes, or the Leu450Trp mutation caused by the c. l 349T>G nucleotide change.

[00165] In some embodiments, a single administration of the CRISPR compositions of the invention may be sufficient to correct the underlying genetic defect or mutation associated with disease. In other embodiments, more than one administration of the CRISPR therapeutic may be beneficial, to maximize editing across all target cells and all alleles via cumulative effects.In some embodiments, the efficacy of treatment with the compositions of the invention is seen at 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years after delivery.

[00166] A number of different types of assessments may be used to determine efficacy of a treatment for FECD, see Eghrari and Gottsch, Expert Rev Ophthalmol. 5(2): 147-159 (2010). In some embodiments, efficacy of treatment with the compositions is based on assessment by slit-lamp microscopy over time. In some embodiments, efficacy of treatment with the compositions is based on quantitative measurement of disease progression by corneal pachymetry measurements of corneal thickness over time. In some embodiments, efficacy of treatment with the compositions is based on improvement, stabilization, or slowing of change in corneal pachymetry over time.

[00167] In some embodiments, efficacy of treatment with the compositions is based on assessment of visual acuity over time. In some embodiments, efficacy of treatment with the compositions is based on improvement, stabilization, or slowing of decline in visual acuity over time.

[00168] In some embodiments, efficacy of treatment with the compositions is based on specular microscopy. In some embodiments, this specular microscopy is used to document the presence of guttae. In some embodiments, efficacy of treatment with the compositions is based on a decrease in formation of new guttae. In some embodiments, efficacy of treatment with the compositions is based on a decrease in presence of existing guttae.

[00169] In some embodiments, efficacy of treatment with the compositions is based on the patient retaining acceptable visual acuity and avoiding need for a corneal transplant. In some embodiments, efficacy of treatment with the compositions is based on a delay in the time until a corneal transplant is needed. This corneal transplant may be a full corneal transplant or a transplant of the inner layer of the cornea.

[00170] In addition to being associated with FECD, genetic variants in the TCF4 gene have been associated with two other conditions, primary sclerosing cholangitis (PSC) and schizophrenia (see Forrest MP et al, Trends Mol Med. 2014 Jun;20(6):322-31). It remains unclear how noncoding variants in the TCF4 gene increase risk for PSC and schizophrenia. One possibility is that these variants serve as markers for a co-inherited expansion in the same TNR region within intron 3 that has been linked to RNA-mediated toxicity in FECD. While this hypothesis remains unproven, the variants associated with PSC and schizophrenia are located physically and haplotypically close to the TNR-containing region within intron 3, suggesting co-inheritance of variants in these neighboring regions. Moreover, the risk variants associated with PSC and schizophrenia have not been associated with changes in expression of the TCF4 gene, suggesting that another mechanism is involved, such as the RNA toxicity seen in patients with the TNR expansion in intron 3.

Combination Therapy

[00171] In some embodiments, the compositions of the invention are used as a single agent for the treatment of FECD, PPCD, PSC, and/or Schizophrenia.

[00172] In some embodiments, the compositions of the invention are used in combination with other therapies for FECD, PPCD, PSC, and/or Schizophrenia. In some embodiments, the combination therapy is soft contact lenses. In some embodiments, these soft contact lenses smooth out microscopic swelling on the surface of the eye. In some embodiments, the compositions of the invention are used in combination with eye drops or ointments that draw fluid out of the cornea. In some embodiments, these eye drops or ointments are Muro 128 ^® 5% (Sodium Chloride

Hypertonicity Ophthalmic Solution, 5%, Bausch and Lomb), Muro 128 5% Ointment (Sodium Chloride Hypertonicity Ophthalmic Ointment, 5%) (Bausch and Lomb), or other saline or tear replacements.

[00173] In some embodiments, glucocorticoids or corticosteroids are used together with the compositions of the invention to reduce the immune response to the therapeutic.

[00174] Combination treatments may be achieved by way of the simultaneous, sequential, or separate dosing of the individual components of the treatment. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination.

Delivery of CRISPR/Cas Compositions

[00175] In some embodiments, the CRISPR/Cas compositions described herein may be administered via a vector and/or lipid nanoparticle comprising the appropriate guide or guides. Viral Vectors

[00176] CRISPR/Cas composistions can be delivered by a vector system. In some

embodiments, the CRISPR/Cas composistions may be provided on one or more vectors. In some embodiments, the vector may be a DNA vector. In other embodiments, the vector may be an RNA vector. In some embodiments, the vector may be circular. In other embodiments, the vector may be linear. In some embodiments, the vector may be enclosed in a lipid nanoparticle, liposome, non-lipid nanoparticle, or viral capsid. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors, and expression vectors.

[00177] In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be genetically modified from its wild type counterpart. For example, the viral vector may comprise an insertion, deletion, or substitution of one or more nucleotides to facilitate cloning or such that one or more properties of the vector is changed. Such properties may include packaging capacity, transduction efficiency, immunogenicity, genome integration, replication, transcription, and translation. In some embodiments, a portion of the viral genome may be deleted such that the virus is capable of packaging exogenous sequences having a larger size. In some embodiments, the viral vector may have an enhanced transduction efficiency. In some embodiments, the immune response induced by the virus in a host may be reduced. In some embodiments, viral genes (such as, e.g., integrase) that promote integration of the viral sequence into a host genome may be mutated such that the virus becomes non-integrating. In some embodiments, the viral vector may be replication defective. In some embodiments, the viral vector may comprise exogenous transcriptional or translational control sequences to drive expression of coding sequences on the vector. In some embodiments, the virus may be helper-dependent. For example, the virus may need one or more helper virus to supply viral components (such as, e.g., viral proteins) required to amplify and package the vectors into viral particles. In such a case, one or more helper components, including one or more vectors encoding the viral components, may be introduced into a host cell along with the vector system described herein. In other embodiments, the virus may be helper-free. For example, the virus may be capable of amplifying and packaging the vectors without any helper virus. In some embodiments, the vector system described herein may also encode the viral components required for virus amplification and packaging.

[00178] Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vector, lentivirus vectors, adenovirus vectors, helper dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculovirus vectors, and retrovirus vectors. In some embodiments, the viral vector may be an AAV vector. In some embodiments, the AAV vector has a serotype of 2, 3, 5, 7, 8, 9, or rh.10. In other embodiments, the viral vector may a lentivirus vector. In some embodiments, the lentivirus may be non-integrating.

[00179] In some embodiments, the viral vector may be an adenovirus vector. In some embodiments, the adenovirus may be a high-cloning capacity or "gutless" adenovirus, where all coding viral regions apart from the 5' and 3' inverted terminal repeats (ITRs) and the packaging signal (T) are deleted from the virus to increase its packaging capacity. In yet other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, the HSV-1 -based vector is helper dependent, and in other embodiments it is helper independent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with structural components for packaging, while a 30kb-deleted HSV-1 vector that removes non-essential viral functions does not require helper virus. In additional embodiments, the viral vector may be bacteriophage T4. In some embodiments, the bacteriophage T4 may be able to package any linear or circular DNA or RNA molecules when the head of the virus is emptied. In further embodiments, the viral vector may be a baculovirus vector. In yet further embodiments, the viral vector may be a retrovirus vector. In embodiments using AAV or lentiviral vectors, which have smaller cloning capacity, it may be necessary to use more than one vector to deliver all the components of a vector system as disclosed herein. For example, one AAV vector may contain sequences encoding a Cas protein, while a second AAV vector may contain one or more guide sequences. However, in some embodiments, a single AAV vector may contain sequences encoding a Cas protein and one or more guide sequences. In some embodiments involving use of a single AAV to deliver CRISPR/Cas components described herein, a small Cas9 ortholog is used. In some embodiments, the small Cas9 ortholog is derived from Neisseria meningitidis, Campylobacter jejuni or Staphylococcus aureus.

[00180] In some embodiments, the vector may be capable of driving expression of one or more coding sequences in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus.

[00181] In some embodiments, the vector may comprise a nucleotide sequence encoding the nuclease described herein. In some embodiments, the nuclease encoded by the vector may be a Cas protein. In some embodiments, the vector system may comprise one copy of the nucleotide sequence encoding the nuclease. In other embodiments, the vector system may comprise more than one copy of the nucleotide sequence encoding the nuclease. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding the nuclease may be operably linked to at least one promoter.

[00182] In some embodiments, the promoter may be constitutive, inducible, or tissue- specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphogly cerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On ^® promoter (Clontech).

[00183] In some embodiments, the promoter may be a tissue-specific promoter, e.g., a promoter specific for expression in the corneal endothelium.

[00184] The vector may further comprise a nucleotide sequence encoding the guide RNA described herein. In some embodiments, the vector comprises one copy of the guide RNA. In other embodiments, the vector comprises more than one copy of the guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be non-identical such that they target different target sequences, or may be identical in that they target the same target sequence. In some embodiments where the vectors comprise more than one guide RNA, each guide RNA may have other different properties, such as activity or stability within the Cas RNP complex. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence, such as a promoter, a 3' UTR, or a 5' UTR. In one embodiment, the promoter may be a tRNA promoter, e.g., tRNA ^Lys3 , or a tRNA chimera. See Mefferd et al, RNA. 2015 21 : 1683-9; Scherer et al, Nucleic Acids Res. 2007 35: 2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and HI promoters. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human HI promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotide encoding the crRNA of the guide RNA and the nucleotide encoding the trRNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotide encoding the crRNA and the nucleotide encoding the trRNA may be driven by the same promoter. In some embodiments, the crRNA and trRNA may be transcribed into a single transcript. For example, the crRNA and trRNA may be processed from the single transcript to form a double-molecule guide RNA. Alternatively, the crRNA and trRNA may be transcribed into a single-molecule guide RNA. In other embodiments, the crRNA and the trRNA may be driven by their corresponding promoters on the same vector. In yet other embodiments, the crRNA and the trRNA may be encoded by different vectors.

[00185] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector comprising the nucleotide sequence encoding a Cas protein. In some embodiments, expression of the guide RNA and of the Cas protein may be driven by their own corresponding promoters. In some embodiments, expression of the guide RNA may be driven by the same promoter that drives expression of the Cas9 protein. In some embodiments, the guide RNA and the Cas protein transcript may be contained within a single transcript. For example, the guide RNA may be within an untranslated region (UTR) of the Cas protein transcript. In some embodiments, the guide RNA may be within the 5' UTR of the Cas protein transcript. In other embodiments, the guide RNA may be within the 3' UTR of the Cas protein transcript. In some embodiments, the intracellular half-life of the Cas protein transcript may be reduced by containing the guide RNA within its 3' UTR and thereby shortening the length of its 3' UTR. In additional embodiments, the guide RNA may be within an intron of the Cas protein transcript. In some embodiments, suitable splice sites may be added at the intron within which the guide RNA is located such that the guide RNA is properly spliced out of the transcript. In some embodiments, expression of the Cas protein and the guide RNA in close proximity on the same vector may facilitate more efficient formation of the CRISPR RNP complex.

[00186] In some embodiments, the compositions comprise a vector system, wherein the system comprises more than one vector. In some embodiments, the vector system may comprise one single vector. In other embodiments, the vector system may comprise two vectors. In additional embodiments, the vector system may comprise three vectors. When different guide RNAs are used for multiplexing, or when multiple copies of the guide RNA are used, the vector system may comprise more than three vectors.

[00187] In some embodiments, the vector system may comprise inducible promoters to start expression only after it is delivered to a target cell. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non- induced) expression level, such as, e.g., the Tet-On ^® promoter (Clontech).

[00188] In additional embodiments, the vector system may comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue.

[00189] The vector may be delivered by liposome, a nanoparticle, an exosome, or a microvesicle. The vector may also be delivered by a lipid nanoparticle; see e.g.,

PCT/US2017/024973, filed March 30, 2017, claiming priority to U.S.S.N. 62/315,602, filed March 30, 2016 and entitled "LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS

COMPONENTS," the contents of which are hereby incorporated by reference in their entirety.

[00190] In some embodiments, the vector may be delivered via a solution delivered directly to the cornea. Delivery may be accomplished via topical application, injection into the cornea itself, injection into the anterior chamber, injection into the posterior chamber, injection into the corneal limbus, or other means.

[00191] In some embodiments, the vector may be delivered systemically.

Lipid Nanoparticles (LNPs)

[00192] In some embodiments, the guide RNA compositions described herein, alone or encoded on one or more vectors, are administered via a lipid nanoparticle; see e.g.,

PCT/US2017/024973, filed March 30, 2017, claiming priority to U.S.S.N. 62/315,602, filed March 30, 2016 and entitled "LIPID NANOPARTICLE FORMULATIONS FOR CRISPR/CAS

COMPONENTS," the contents of which are hereby incorporated by reference in their entirety. Any lipid nanoparticle known to those of skill in the art to be capable of delivering nucleotides to subjects may be utilized to administer the guide RNAs described herein, as well as either mRNA encoding Cas or Cas-deaminase fusion protein or Cas9 or Cas9-deaminase fusion protein itself.

[00193] In some embodiments, the LNP comprises (i) a CCD lipid for encapsulation and for endosomal escape, (ii) a neutral lipid for stabilization, (iii) a helper lipid, also for stabilization, and (iv) a stealth lipid. The LNP carries cargo, which may include any or all of the following: an mRNA encoding a Cas nuclease or Cas-deaminase, such as Cas9 or Cas9-deaminase; one or more guide RNAs or a nucleic acids encoding one or more guide RNA; and one or more viral vectors encoding Cas9 or Cas9-deaminase, one or more guide RNAs, or both Cas9/Cas9-deaminase and guide RNAs. In one embodiment, the LNP comprises a CCD lipid, such as Lipid A, Lipid B, Lipid C, or Lipid D. In some aspects, the CCD lipid is Lipid A. In some aspects, the CCD lipid is Lipid B. In some embodiments, the LNP comprises a CCD lipid, a neutral lipid, a helper lipid, and a stealth lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In some embodiments, the stealth lipid is PEG2k-DMG. In additional embodiments, the LNP comprises a CCD lipid selected from Lipid A or Lipid B, cholesterol, DSPC, and PEG2k-DMG.

[00194] In some embodiments, suitable LNP formulations include a CCD lipid, along with a helper lipid, a neutral lipid, and a stealth lipid. By "lipid nanoparticle" is meant a particle that comprises a plurality of (i.e. more than one) lipid molecules physically associated with each other by intermolecular forces. The LNPs may be, e.g., microspheres (including unilamellar and

multilamellar vesicles, e.g., "liposomes"— lamellar phase lipid bilayers that, in some embodiments, are substantially spherical— and, in more particular embodiments, can comprise an aqueous core, e.g., comprising a substantial portion of RNA molecules), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Emulsions, micelles, and suspensions may be suitable compositions for local and/or topical delivery.

[00195] In some embodiments, the CCD lipid is Lipid A, which is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)car bonyl)oxy)methyl)propyl octadeca- 9,12-dienoate, also called 3-((4,44ois(octyloxy)butanoyl)oxy)-2-((((3-

(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Lipid A can be depicted as:

[00197] Lipid A may be synthesized according to WO2015/095340 (e.g. , pp. 84-86), incorporated by reference in its entirety.

[00198] In some embodiments, the CCD lipid is Lipid B, which is ((5-

((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane -8,l-diyl)bis(decanoate), also called ((5- ((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8, l-diyl) bis(decanoate). Lipid B can be depicted as:

[00200] Lipid B may be synthesized according to WO2014/136086 (e.g., pp. 107-09), incorporated by reference in its entirety.

[00201] In some embodiments, the CCD lipid is Lipid C, which is 2-((4-(((3-

(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)prop ane-l,3-diyl (9Z,9'Z,12Z,12'Z)- bis(octadeca-9,12-dienoate). Lipid C can be depicted as:

[00202] In some embodiments, the CCD lipid is Lipid D, which is 3-(((3-

(dimethylamino)propoxy)carbonyl)oxy)- 13 -(octanoyloxy)tridecyl 3 -octylundecanoate.

[00203] Lipid D can be depicted as:

[00205] Lipid C and Lipid D may be synthesized according to WO2015/095340, incorporated by reference in its entirety.

[00206] "Neutral lipids" suitable for use in a lipid composition include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-l,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), pohsphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), l-myristoyl-2- palmitoyl phosphatidylcholine (MPPC), 1 -palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), l-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine

distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC). Neutral lipids function to stabilize and improve processing of the LNPs.

[00207] "Helper lipids" are lipids that enhance transfection (e.g. transfection of the nanoparticle including the biologically active agent). The mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In certain embodiments, the helper lipid enhances membrane fusogenicity. Helper lipids include steroids, sterols, and alkyl resorcinols. Helper lipids suitable for use in the LNPs include, but are not limited to, cholesterol, 5- heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In some embodiments, the helper lipid may be cholesterol hemisuccinate.

[00208] "Stealth lipids" are lipids that alter the length of time the nanoparticles can exist in vivo {e.g., in the blood). Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition include, but are not limited to, stealth lipids having a hydrophilic head group linked to a lipid moiety. Stealth lipids suitable for use in a lipid composition of the present disclosure and information about the biochemistry of such lipids can be found in Romberg et al, Pharmaceutical Research, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al, Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed, e.g., in WO 2006/007712.

[00209] In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and poly [N-(2-hy droxypropyl)methacrylamide] .

[00210] Stealth lipids may comprise a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups.

[00211] Unless otherwise indicated, the term "PEG" as used herein means any polyethylene glycol or other polyalkylene ether polymer. In some embodiments, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In some embodiments, the term includes PEG copolymers such as PEG- polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000, in a sub-embodiment, about 150 to about 30,000, in a sub-embodiment, about 150 to about 20,000, in a sub-embodiment about 150 to about 15,000, in a sub-embodiment, about 150 to about 10,000, in a sub-embodiment, about 150 to about 6,000, in a sub-embodiment, about 150 to about 5,000, in a sub-embodiment, about 150 to about 4,000, in a sub-embodiment, about 150 to about 3,000, in a sub-embodiment, about 300 to about 3,000, in a sub-embodiment, about 1,000 to about 3,000, and in a sub-embodiment, about 1,500 to about 2,500.

[00212] In certain embodiments, the PEG (e.g. , conjugated to a lipid, such as a stealth lipid), is a "PEG-2K," also termed "PEG 2000," which has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I), wherei that the number averaged degree of polymerization comprises about 45 subunits

However, other PEG embodiments known in the art may be used, including, e.g. , those where the number-averaged degree of polymerization comprises about 23 subunits (n=23), and/or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

[00213] In any of the embodiments described herein, the stealth lipid may be selected from

PEG-dilauroylglycerol, PEG-dimynstoylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog # DSPE- 020CN, NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimynstylglycamide, PEG- dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (l-[8'-(Cholest-5-en-3 [beta]- oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl- poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (cat. #880150P from Avanti Polar Lipids, Alabaster, Alabama, USA), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #880120C from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypoly ethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), polyethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and l,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the stealth lipid may be PEG2k-DMG. In some embodiments, the stealth lipid may be PEG2k-DSG. In one embodiment, the stealth lipid may be PEG2k-DSPE. In one embodiment, the stealth lipid may be PEG2k-DMA. In one embodiment, the stealth lipid may be PEG2k-DSA. In one embodiment, the stealth lipid may be PEG2k-Cl 1. In some embodiments, the stealth lipid may be PEG2k-C14. In some embodiments, the stealth lipid may be PEG2k-C16. In some embodiments, the stealth lipid may be PEG2k-C18.

[00214] Embodiments of the present disclosure also provide lipid compositions described according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the mol-% of the CCD lipid may be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the CCD lipid may be from about 35 mol-% to about 55 mol-%. In one embodiment, the mol-% of the CCD lipid may be from about 40 mol-% to about 50 mol-%. In one embodiment, the mol-% of the CCD lipid may be from about 42 mol-% to about 47 mol-%. In one embodiment, the mol-% of the CCD lipid may be about 45%. In some embodiments, the CCD lipid mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[00215] In one embodiment, the mol-% of the helper lipid may be from about 30 mol-% to about 60 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 35 mol-% to about 55 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 40 mol-% to about 50 mol-%. In one embodiment, the mol-% of the helper lipid may be from about 41 mol-% to about 46 mol-%. In one embodiment, the mol-% of the helper lipid may be about 44 mol-%. In some embodiments, the helper mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[00216] In one embodiment, the mol-% of the neutral lipid may be from about 1 mol-% to about 20 mol-%. In one embodiment, the mol-% of the neutral lipid may be from about 5 mol-% to about 15 mol-%. In one embodiment, the mol-% of the neutral lipid may be from about 7 mol-% to about 12 mol-%. In one embodiment, the mol-% of the neutral lipid may be about 9 mol-%. In some embodiments, the neutral lipid mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

[00217] In one embodiment, the mol-% of the stealth lipid may be from about 1 mol-% to about 10 mol-%. In one embodiment, the mol-% of the stealth lipid may be from about 1 mol-% to about 5 mol-%. In one embodiment, the mol-% of the stealth lipid may be from about 1 mol-% to about 3 mol-%. In one embodiment, the mol-% of the stealth lipid may be about 2 mol-%. In one embodiment, the mol-% of the stealth lipid may be about 1 mol-%. In some embodiments, the stealth lipid mol-% of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol-%. In certain embodiments, LNP inter-lot variability will be less than 15%, less than 10% or less than 5%.

Location of Administration

[00218] In some embodiments, the compositions are delivered into the anterior chamber of the eye. In some embodiments, the compositions are delivered into the posterior chamber of the eye. In some embodiments, the compositions are delivered into the cornea itself. In some embodiments, the compositions are delivered into the corneal stroma. In some embodiments, the compositions are delivered into the corneal limbus. In some embodiments, the compositions are delivered onto the epithelial surface of the cornea. In any of the preceding embodiments of this paragraph as well as other embodiments described herein, treatment further comprises delivery of a Cas protein (e.g., Cas9), for example using a lipid nanoparticle, or delivery of a nucleic acid encoding a Cas protein using a vector and/or lipid nanoparticle. In some embodiments, for example those using a lipid nanoparticle, the nucleic acid encoding the Cas protein is mRNA. In some embodiments, a Cas protein or a nucleic acid encoding a Cas protein is delivered via the same vector and/or lipid nanoparticle that is used to deliver the appropriate guide or guides. In some embodiments, a Cas protein or a nucleic acid encoding a Cas protein is delivered via a different vector and/or lipid nanoparticle that is used to deliver the appropriate guide or guides.

[00219] Any of the compositions described herein may be administered to subjects to excise a portion or all of the TNR expansion in intron 3 of TCF4. Methods of treating FECD comprising administering any of the compositions described herein are encompassed. In some aspects, the compositions are administered in therapeutically effective amounts. In some embodiments, a method of excising, mutating, reducing copy number of, ameliorating, and/or eradicating TNRs of TCF4 is encompassed, comprising administering one or more of the compositions described herein. In some embodiments, a method of cleaving, mutating, reducing copy number of, ameliorating, and/or eradicating the TNRs of one or both copies of TCF4 per cell in a subject is provided, comprising administering one or more of the compositions described herein. In some embodiments, the cell is a corneal endothelium cell.

[00220] In some embodiments, two gRNAs are used to excise all of the TNRs in TCF4. In some embodiments, a first guide that is 5' to the TNR is provided with a second guide that is 3' to the TNR, or vice versa. Where two gRNAs are contemplated, a composition comprising any of the following combinations of guides is provided:

Combination 01 : In some embodiments, a composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1089, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 02: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1090, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 03: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1091, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 04: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1092, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 05: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1093, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 06: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1094, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 07: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1095, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 08: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1096, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 09: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1097, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 10: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1098, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 11 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1099, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 12: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1100, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 13: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1101, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 14: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1102, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 15: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1103, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 16: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1104, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 17: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1105, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 18: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1106, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 19: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1107, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 20: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1108, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 21 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1109, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 22: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1110, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 23: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1111, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 24: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1112, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 25: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1113, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 26: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1114, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 27: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1115, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 28: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1116, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 29: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1117, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 30: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1118, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 31 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1119, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 32: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1120, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 33: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1121, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 34: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1122, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 35: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1123, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 36: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1124, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 37: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1125, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 38: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1126, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 39: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1127, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 40: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1128, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 41 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1129, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 42: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1130, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 43: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1131, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 44: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1132, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 45: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1133, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 46: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1134, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 47: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1135, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 48: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1136, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 49: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1137, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 50: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1138, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 51 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1139, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 52: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1140, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 53: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1141, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 54: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1142, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 55: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1143, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 56: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1144, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 57: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1145, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 58: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1146, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 59: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1147, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 60: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1148, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 61 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1149, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 62: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1150, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 63: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1151, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 64: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1152, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 65: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1153, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 66: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1154, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 67: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1155, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 68: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1156, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 69: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1157, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 70: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1158, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 71 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1159, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 72: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1160, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 73: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1161, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 74: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1162, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 75: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1163, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 76: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1164, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 77: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1165, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 78: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1166, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 79: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1167, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 80: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1168, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 81 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1169, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 82: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1170, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 83: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1171, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 84: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1172, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 85: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1173, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 86: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1174, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 87: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1175, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 88: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1176, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 89: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1177, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 90: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1178, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278. Combination 91 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1179, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 92: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1180, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 93: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1181, and a second gRNA comprising a sequence selected from SEQ ID NOs: 1182-1278.

Combination 94: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1182 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 95: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1183 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 96: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1184 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 97: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1185 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 98: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1186 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 99: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1187 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 100: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1188 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181. Combination 101 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1189 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 102: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1190 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 103: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1191 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 104: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1192 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 105: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1193 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 106: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1194 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 107: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1195 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 108: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1196 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 109: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1197 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 110: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1198 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181. Combination 111 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1199 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 112: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1200 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 113: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1201 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 114: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1202 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 115: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1203 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 116: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1204 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 117: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1205 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 118: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1206 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 119: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1207 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 120: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1208 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181. Combination 121 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1209 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 122: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1210 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 123: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1211 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 124: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1212 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 125: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1213 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 126: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1214 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 127: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1215 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 128: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1216 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 129: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1217 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 130: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1218 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181. Combination 131 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1219 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 132: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1220 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 133: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1221 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 134: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1222 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 135: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1223 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 136: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1224 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 137: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1225 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 138: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1226 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 139: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1227 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 140: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1228 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181. Combination 141 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1229 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 142: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1230 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 143: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1231 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 144: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1232 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 145: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1233 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 146: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1234 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 147: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1235 and a second gRNA comprising a sequence selected from SEQ ID NOs: 1089-1181.

Combination 148: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1236 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 149: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1237 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 150: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1238 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181. Combination 151 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1239 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 152: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1240 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 153: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1241 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 154: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1242 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 155: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1243 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 156: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1244 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 157: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1245 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 158: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1246 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 159: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1247 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 160: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1248 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181. Combination 161 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1249 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 162: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1250 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 163: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1251 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 164: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1252 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 165: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1253 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 166: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1254 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 167: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1255 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 168: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1256 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 169: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1257 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 170: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1258 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181. Combination 171 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1259 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 172: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1260 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 173: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1261 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 174: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1262 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 175: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1263 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 176: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1264 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 177: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1265 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 178: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1266 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 179: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1267 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 180: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1268 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181. Combination 181 : In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1269 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 182: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1270 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 183: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1271 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 184: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1272 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 185: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1273 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 186: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1274 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 187: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1275 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 188: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1276 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 189: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1277 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181.

Combination 190: In some embodiments, the composition comprises two gRNAs comprising a first gRNA comprising SEQ ID NO: 1278 and a second gRNA comprising a sequence selected from SEQ ID Nos: 1089-1181. EXAMPLES

Example 1. Use of pairs of gRNAs to excise TNR expansions from TCF4

[00221] To remove the TNRs from TCF4 and limit the production of toxic RNAs, CRISPR guides have been designed to simultaneously cut on either side of the expansion using specific target sequences. These gRNAs have been designed to work with wild type S. pyogenes Cas9 ("Spy

Cas9"). Other gRNAs, suitable for use with other CRISPR nucleases, could be designed in a similar manner.

[00222] Target sequences were selected using the sequence of the TCF4 intron 3 sequence with flanking exons (SEQ ID NO: 1085). This sequence is based on UCSC Genome browser, Human, February 2009 (GRCh37/hgl9) assembly. This sequence contains a set of 24 CTG repeats (TNRs) at range 53253387-53253458 within the intron position chrl8:53252584-53254275. The exact range of CTG repeats in this intron will vary based on the number of repeats, where a number of repeats > 40 is associated with increased risk for developing disease. In the hg38 build, the repeats are located at chrl8:55, 586,156-55, 586,228, within the intron spanning chrl8:55, 585,280- 55,587,136. Target sequences and corresponding guide sequences are listed in Table 2 (SEQ ID NOs: 1-190 (target sequences) and SEQ ID NOs: 1089-1278 (guide sequences)). The particular forms of the crRNAs and trRNAs used in this Example 1 are provided in Table 1 as SEQ ID NO: 1087 and SEQ ID NO: 1088, respectively. The target sequence for the 5' guide sequences (SEQ ID NOs:

1089-1181) is located between Chrl8:55, 585,285-55, 586,153 and is upstream of the location of the TNRs. The target sequence for the 3' guide sequences (SEQ ID NOs: 94-190) is located between Chrl8:55586225-55587203 and is downstream of the location of the TNRs. Table 2 lists SEQ ID NOs: 1-190 (target sequences) and SEQ ID NOs: 1089-1278 (guide sequences that direct a nuclease to a corresponding target sequence and bind to the reverse compliment of the target sequences). Cutting Frequency Determination (CFD) scores were generated for each guide sequence in silico, according to the methodology reported by Doench et al., Nat Biotechnol. 2016 Feb; 34(2): 184-191. These scores (which have been multiplied by a factor of 100 to convert to decimals as compared to how Doench et al report scores) provide a measure of the off-target potential for a given gRNA.

[00223] gRNAs having guide sequences provided in Table 2 were screened in a 96- well format to determine their editing (e.g., indel forming) efficiency. To this end, a HEK293 cell line constitutively expressing Spy Cas9 ("HEK293_Cas9") was cultured in DMEM media supplemented with 10% fetal bovine serum and 500 μg/ml G418. Cells were plated at a density of 10,000 cells/well in a 96-well plate 20 hours prior to transfection. Cells were transfected with Lipofectamine RNAiMAX (ThermoFisher, Cat. 13778150) according to the manufacturer's protocol. Cells were transfected with a lipoplex containing individual crRNA (25 nM), trRNA (25 nM), Lipofectamine RNAiMAX (0.3 μΙ,ΛνβΙΙ) and OptiMem. Genomic DNA was extracted from each well using 50 Buccal Amp DNA Extraction solution (Epicentre, Cat. QE09050) according to manufacturer's protocol.

[00224] To quantitatively determine the efficiency of editing at the target location in the genome, deep sequencing was utilized to identify the presence of insertions and deletions ("indels") introduced by gene editing. PCR primers were designed around the target sites and the genomic area of interest was amplified. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to the human reference genome after eliminating those having low quality scores. The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion, substitution, or deletion was calculated. The editing percentage (e.g., the "editing efficiency" or "percent editing") is defined as the total number of sequence reads with insertions or deletions over the total number of sequence reads, including wild type. The editing efficiency numbers for each gRNA used are reported in Table 2.

[00225] After completing the initial evaluation above to identify those with optimal editing efficiency, pairs of gRNAs were screened to determine pairs capable of removing the intervening section of DNA containing the TNR, as shown in Figure 1. Following excision of the intervening section, the break will then be repaired by the cell through the nonhomologous end joining (NHEJ) DNA repair pathway, which is highly efficient even in non- dividing cells such as those in the corneal endothelium. This process follows excision of the DNA fragment between the two guide sequences, which can occur at high frequency even when the guide sequences are >3000 nucleotides apart. No additional homologous template DNA is required for this editing approach, greatly simplifying the process. As the deleted range is contained within an intron, no effect on the gene product of TCF4 would be expected as the intron does not affect the final mRNA product or the protein product.

[00226] After removal of the TNR repeat, the TCF4 RNA transcript should no longer aggregate within the cell, nor sequester the splicing factors that are required for normal cellular function. Removal of the relevant region within intron 3 is unlikely to have any detrimental effects on RNA stability or the expression of the TCF4 gene itself, because this intron would normally be removed by RNA splicing during maturation of the final RNA product. Thus, the region of DNA within intron 3 is not be contained within the final RNA product used for translation of the TCF4 protein. Without the TNR, the mRNA and gene product of TCF4 should function normally, much the same as a normal allele with minimal TNR expansion. Moreover, as corneal endothelial cells are essentially non-dividing, correction of the cells once should result in a permanent amelioration of the disease.

Treatment should halt the abnormal deposition of collagen (i.e., guttae) characteristic of the disease, and may over time lead to resorption of existing guttae. It is also proposed that treatment of individuals with a known predisposition to FECD, such as those with family histories of the disease and who are confirmed to have TNR expansion of intron 3 of TCF4, using this technology may prevent development of disease.

[00227] To demonstrate excision of the TNR, pairs of RNPs were formed, each having a gRNA targeting one side of the TNR. Brifely, a 50 uM solution of pre-annealed gRNA (e.g., a dgRNA having a crRNA and trRNA) was prepared by heating crRNA and trRNA at eqimolar amounts in water at 95°C for 2 minutes, and allowing them to cool at room temperature. The pre-annleaed gRNA was added to Spy Cas9 protein (at 50 uM concentration) and was incubated at room temperature for 10 minutes, giving a final RNP solution having gRNA at 3.33 uM and Cas9 protein at 1.66 μΜ. HEK293 cells which do not constitutively express Cas9 were plated in SF electroporation buffer (Lonza) in 96-well format at -50,000 cells/well in a volume of 20 uL. 5 μΐ of each RNP solution (e.g., for each pair being tested) was added to the wells and the cells were electroporated using a Lonza Amaxa instrument. After electroporation, 80 y^L of cell culture media was added to the wells and the cells were transferred to a 96-well flat bottom tissue culture plate and incubated at 37°C for 24 hours. The cells were then lysed and genomic DNA was extracted as described above. [00228] To determine efficiciences of TNR excision, a similar NGS analysis was performed as described above for editing efficiency. Brifely, deep sequencing was performed to identify deletions caused by gene editing of two locations flanking the TNRs. PCR primers were designed around the target site (the TNR in intron 3 of TCF4), and the genomic area of interest was amplified. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing. The resulting amplicons were sequenced on an Illumina MiSeq instrument. Reads were filtered to eliminate those with low quality scores, and the resulting reads were mapped to the reference genome. Reads overlapping the target region were further filtered and locally realigned to identify large deletions. The number of reads containing deletions spanning the two targeted regions was calculated. The excision percentage is defined as the number of sequencing reads containing a deletion of the TNRs divided by the total number of reads overlapping the target region. The excision percentages for each pair tested are reported in Table 7.

[00229] As shown in Table 7 and Figure 2, 93 pairs of gRNAs were tested, with some pairs achieving greater than 80% excision, with one pair in particular achieving over 88% excision (e.g., using gRNAs having guide sequences directing a nuclease to a target sequence comprising SEQ ID NO: 83 and SEQ ID NO: 109; corresponding to guide RNAs comprising SEQ ID NO: 1177 and SEQ ID NO: 1197, respectively).

Table 7

SEQ ID NOs (5' Target SEQ ID NOs (3' Target Excision Percent Sequence) Sequence)

64 106 66.1

85 114 65.86

86 114 61.58

83 114 59.88

53 114 43.8

83 112 27.6

74 114 20.7

85 108 7.35

83 107 6.69

85 115 6.44

58 109 5.69

86 108 5.57

83 96 5.17

74 109 4.46

77 115 4.45

53 96 4.44

83 108 4.4

74 125 4.3

85 94 4.17

86 96 3.53

53 107 3.42

83 94 3.21

71 115 3.21

77 96 3.12

58 112 3.11

77 109 3.08

85 95 3

53 94 2.9

77 95 2.82 SEQ ID NOs (5' Target SEQ ID NOs (3' Target Excision Percent Sequence) Sequence)

86 115 2.75

85 96 2.65

58 94 2.61

58 115 2.61

71 96 2.56

58 107 2.53

83 95 2.43

58 96 2.36

77 94 2.24

56 94 2.21

77 108 2.17

77 112 2.16

86 94 2.08

77 107 1.9

86 95 1.87

56 96 1.87

54 94 1.72

71 94 1.69

77 114 1.65

71 114 1.64

56 95 1.63

58 95 1.5

53 112 1.32

71 109 1.3

74 112 1.28

54 96 1.17

58 114 1.15

74 108 1.09

53 108 0.79 SEQ ID NOs (5' Target SEQ ID NOs (3' Target Excision Percent Sequence) Sequence)

74 107 0.62

74 94 0.61

71 107 0.56

71 95 0.55

71 112 0.55

74 96 0.47

74 95 0.46

74 115 0.41

54 95 0.37

53 95 0.35

77 125 0.33

54 112 0.09

56 114 0.01

73 101 0.01

54 109 0

54 114 0

54 107 0

54 108 0

54 115 0

56 109 0

56 107 0

56 108 0

56 112 0

56 115 0

56 125 0

53 125 0 Example 2. Use of gRNAs to treat mutations in COL8A2

[00230] Three mutations in COL8A2, Gln455Lys, Gln455Val, and Leu450Trp, have been associated with early-onset FECD and posterior polymorphous corneal dystrophy (PPDC), and knock-in animal studies have shown a pathology consistent with human early- onset FECD. These models are associated with abnormal intracellular accumulation of mutant collagen VIII peptides with altered stability of the triple helical structure. Therefore, decreasing mutant collagen VIII in patients with diagnosis or family history of mutations in COL8A2 may improve disease course. Alternatively, selectively reducing levels of COL8A2 with mutation at either Gln455Lys, Gln455Val, or Leu450Trp may reduce levels of mutant collagen VIII peptides and improve disease course. Another approach would be to correct mutations in the DNA leading to amino acid mutations in the alpha subunit 2 of collagen VIII (COL8A2) and thereby remove the abnormal gene product.

[00231] Target sequences were selected for developing Cas RNP therapies using

NCBI Reference Sequence NM_005202.3 of transcript variant 1 of the COL8A2 gene. This sequence does not contain mutations known to occur at positions 455 and 450 in the amino acid sequence of the collagen VIII gene product and may be termed the "wild type COL8A2 sequence." Target sequences were selected between Chrl :36097532-36100270 (hg38 version), as listed in Table 3 (SEQ ID NOs: 191-1063). Guide sequences complementary to the target sequences can be used to generate gRNAs for use with RNPs to target COL8A2.

Table 3: Target sequences for wild type COL8A2

SEQ ID Chromosomal Strand Target sequence

No location

191 Chrl : 36097532- + GGGGAGGAGGCCAGGGCAGCAGG

36097554

192 Chrl : 36097545- + GGGCAGCAGGACCCCCCCCGCGG

36097567

193 Chrl : 36097546- + GGCAGCAGGACCCCCCCCGCGGG

36097568

194 Chrl : 36097554- + GACCCCCCCCGCGGGTTATGTGG

36097576

195 Chrl : 36097555- + ACCCCCCCCGCGGGTTATGTGGG

36097577

196 Chrl : 36097556- + CCCCCCCCGCGGGTTATGTGGGG

36097578

197 Chrl : 36097556- ^— CCCCACATAACCCGCGGGGGGGG

36097578

198 Chrl : 36097557- ^— GCCCCACATAACCCGCGGGGGGG

36097579

199 Chrl : 36097558- ^— TGCCCCACATAACCCGCGGGGGG

36097580

200 Chrl : 36097559- ^— CTGCCCCACATAACCCGCGGGGG

36097581

201 Chrl : 36097560- ^— TCTGCCCCACATAACCCGCGGGG

36097582

202 Chrl : 36097561- ^— CTCTGCCCCACATAACCCGCGGG

36097583

203 Chrl : 36097562- — GCTCTGCCCCACATAACCCGCGG

36097584

204 Chrl : 36097578- + GCAGAGCAAGAATCCTGAAAAGG

36097600

205 Chrl : 36097581- + GAGCAAGAATCCTGAAAAGGAGG

36097603

206 Chrl : 36097586- + AGAATCCTGAAAAGGAGGAGTGG

36097608

207 Chrl : 36097591- ^— TACATCCACTCCTCCTTTTCAGG

36097613

208 Chrl : 36097599- + GGAGGAGTGGATGTACTCCGTGG

36097621

209 Chrl : 36097607- + GGATGTACTCCGTGGAGTAGAGG

36097629

210 Chrl : 36097614- + CTCCGTGGAGTAGAGGCCGTTGG

36097636

211 Chrl : 36097616- ^— GGCCAACGGCCTCTACTCCACGG

36097638

212 Chrl : 36097619- + TGGAGTAGAGGCCGTTGGCCTGG

36097641 213 Chrl : 36097627- + AGGCCGTTGGCCTGGTCCGACGG 36097649

214 Chrl : 36097630- ^— ATGCCGTCGGACCAGGCCAACGG

36097652

215 Chrl : 36097637- ^— GGTGCAGATGCCGTCGGACCAGG

36097659

216 Chrl : 36097643- ^— GGTCTGGGTGCAGATGCCGTCGG

36097665

217 Chrl : 36097646- + ACGGCATCTGCACCCAGACCTGG

36097668

218 Chrl : 36097653- + CTGCACCCAGACCTGGTCGTTGG

36097675

219 Chrl : 36097654- + TGCACCCAGACCTGGTCGTTGGG

36097676

220 Chrl : 36097658- ^— GCGGCCCAACGACCAGGTCTGGG

36097680

221 Chrl : 36097659- ^— TGCGGCCCAACGACCAGGTCTGG

36097681

222 Chrl : 36097664- + CCTGGTCGTTGGGCCGCAGCTGG

36097686

223 Chrl : 36097664- ^— CCAGCTGCGGCCCAACGACCAGG

36097686

224 Chrl : 36097671- + GTTGGGCCGCAGCTGGAGCACGG

36097693

225 Chrl : 36097677- ^— GTGGGGCCGTGCTCCAGCTGCGG

36097699

226 Chrl : 36097688- + GCACGGCCCCACCAGATGCCTGG

36097710

227 Chrl : 36097694- + CCCCACCAGATGCCTGGTCCAGG

36097716

228 Chrl : 36097694- ^— CCTGGACCAGGCATCTGGTGGGG

36097716

229 Chrl : 36097695- ^— ACCTGGACCAGGCATCTGGTGGG

36097717

230 Chrl : 36097696- ^— TACCTGGACCAGGCATCTGGTGG

36097718

231 Chrl : 36097699- ^— GGCTACCTGGACCAGGCATCTGG

36097721

232 Chrl : 36097706- ^— CAAGAAGGGCTACCTGGACCAGG

36097728

233 Chrl : 36097712- ^— TGAGTACAAGAAGGGCTACCTGG

36097734

234 Chrl : 36097719- + GCCCTTCTTGTACTCATCGTAGG

36097741

235 Chrl : 36097720- — ACCTACGATGAGTACAAGAAGGG

36097742 236 Chrl : 36097721- ^— TACCTACGATGAGTACAAGAAGG 36097743

237 Chrl : 36097725- + CTTGTACTCATCGTAGGTATAGG

36097747

238 Chrl : 36097728- + GTACTCATCGTAGGTATAGGTGG

36097750

239 Chrl : 36097732- + TCATCGTAGGTATAGGTGGCCGG

36097754

240 Chrl : 36097751- + CCGGCACGTTGTTCTTGTACAGG

36097773

241 Chrl : 36097751- ^— CCTGTACAAGAACAACGTGCCGG

36097773

242 Chrl : 36097752- + CGGCACGTTGTTCTTGTACAGGG

36097774

243 Chrl : 36097767- + GTACAGGGCCACCCACACGTTGG

36097789

244 Chrl : 36097775- ^— CAAGGGCACCAACGTGTGGGTGG

36097797

245 Chrl : 36097778- ^— CGTCAAGGGCACCAACGTGTGGG

36097800

246 Chrl : 36097779- ^— ACGTCAAGGGCACCAACGTGTGG

36097801

247 Chrl : 36097787- + TGGTGCCCTTGACGTGCACATGG

36097809

248 Chrl : 36097792- ^— GCTTACCATGTGCACGTCAAGGG

36097814

249 Chrl : 36097793- ^— TGCTTACCATGTGCACGTCAAGG

36097815

250 Chrl : 36097816- + AAGTAGTAGACGCCGCCCACAGG

36097838

251 Chrl : 36097817- + AGTAGTAGACGCCGCCCACAGGG

36097839

252 Chrl : 36097821- + GTAGACGCCGCCCACAGGGCAGG

36097843

253 Chrl : 36097828- ^— ATCTTCACCTGCCCTGTGGGCGG

36097850

254 Chrl : 36097831- ^— GGCATCTTCACCTGCCCTGTGGG

36097853

255 Chrl : 36097832- ^— TGGCATCTTCACCTGCCCTGTGG

36097854

256 Chrl : 36097836- + AGGGCAGGTGAAGATGCCAGTGG

36097858

257 Chrl : 36097840- + CAGGTGAAGATGCCAGTGGCTGG

36097862

258 Chrl : 36097841- + AGGTGAAGATGCCAGTGGCTGGG

36097863 259 Chrl : 36097852- ^— AGCGGCTACAACCCAGCCACTGG 36097874

260 Chrl : 36097856- + TGGCTGGGTTGTAGCCGCTGTGG

36097878

261 Chrl : 36097870- ^— ACTCTCTACAATGGCCACAGCGG

36097892

262 Chrl : 36097874- + TGTGGCCATTGTAGAGAGTCCGG

36097896

263 Chrl : 36097879- ^— TTTGACCGGACTCTCTACAATGG

36097901

264 Chrl : 36097887- + GAGAGTCCGGTCAAATTTCACGG

36097909

265 Chrl : 36097888- + AGAGTCCGGTCAAATTTCACGGG

36097910

266 Chrl : 36097893- ^— GCATGCCCGTGAAATTTGACCGG

36097915

267 Chrl : 36097899- + AAATTTCACGGGCATGCCCGAGG

36097921

268 Chrl : 36097902- + TTTCACGGGCATGCCCGAGGCGG

36097924

269 Chrl : 36097903- + TTCACGGGCATGCCCGAGGCGGG

36097925

270 Chrl : 36097904- + TCACGGGCATGCCCGAGGCGGGG

36097926

271 Chrl : 36097908- + GGGCATGCCCGAGGCGGGGAAGG

36097930

272 Chrl : 36097909- + GGCATGCCCGAGGCGGGGAAGGG

36097931

273 Chrl : 36097914- + GCCCGAGGCGGGGAAGGGCGAGG

36097936

274 Chrl : 36097915- ^— ACCTCGCCCTTCCCCGCCTCGGG

36097937

275 Chrl : 36097916- ^— CACCTCGCCCTTCCCCGCCTCGG

36097938

276 Chrl : 36097932- + CGAGGTGAGCACCGCAGTGAAGG

36097954

277 Chrl : 36097936- + GTGAGCACCGCAGTGAAGGCCGG

36097958

278 Chrl : 36097941- + CACCGCAGTGAAGGCCGGTGTGG

36097963

279 Chrl : 36097943- ^— TGCCACACCGGCCTTCACTGCGG

36097965

280 Chrl : 36097946- + CAGTGAAGGCCGGTGTGGCATGG

36097968

281 Chrl : 36097947- + AGTGAAGGCCGGTGTGGCATGGG

36097969 282 Chrl : 36097955- ^— GCTGTCTGCCCATGCCACACCGG 36097977

283 Chrl : 36097975- + AGCTCGCCCAGCCCAAACTGTGG

36097997

284 Chrl : 36097981- ^— GGCAAGCCACAGTTTGGGCTGGG

36098003

285 Chrl : 36097982- ^— GGGCAAGCCACAGTTTGGGCTGG

36098004

286 Chrl : 36097986- ^— AGGGGGGCAAGCCACAGTTTGGG

36098008

287 Chrl : 36097987- ^— AAGGGGGGCAAGCCACAGTTTGG

36098009

288 Chrl : 36097998- + CTTGCCCCCCTTGCCCAGCACGG

36098020

289 Chrl : 36098002- ^— GGTGCCGTGCTGGGCAAGGGGGG

36098024

290 Chrl : 36098003- ^— GGGTGCCGTGCTGGGCAAGGGGG

36098025

291 Chrl : 36098004- ^— AGGGTGCCGTGCTGGGCAAGGGG

36098026

292 Chrl : 36098005- ^— GAGGGTGCCGTGCTGGGCAAGGG

36098027

293 Chrl : 36098006- ^— GGAGGGTGCCGTGCTGGGCAAGG

36098028

294 Chrl : 36098011- ^— GGTGTGGAGGGTGCCGTGCTGGG

36098033

295 Chrl : 36098012- ^— CGGTGTGGAGGGTGCCGTGCTGG

36098034

296 Chrl : 36098019- + GGCACCCTCCACACCGCCGTTGG

36098041

297 Chrl : 36098020- + GCACCCTCCACACCGCCGTTGGG

36098042

298 Chrl : 36098023- ^— CTGCCCAACGGCGGTGTGGAGGG

36098045

299 Chrl : 36098024- + CCTCCACACCGCCGTTGGGCAGG

36098046

300 Chrl : 36098024- ^— CCTGCCCAACGGCGGTGTGGAGG

36098046

301 Chrl : 36098027- ^— GCACCTGCCCAACGGCGGTGTGG

36098049

302 Chrl : 36098032- ^— GGCTTGCACCTGCCCAACGGCGG

36098054

303 Chrl : 36098035- ^— GCAGGCTTGCACCTGCCCAACGG

36098057

304 Chrl : 36098053- — TTCGATGAGACTGGCATCGCAGG

36098075 305 Chrl : 36098055- + TGCGATGCCAGTCTCATCGAAGG 36098077

306 Chrl : 36098062- + CCAGTCTCATCGAAGGCCCCAGG

36098084

307 Chrl : 36098062- ^— CCTGGGGCCTTCGATGAGACTGG

36098084

308 Chrl : 36098063- + CAGTCTCATCGAAGGCCCCAGGG

36098085

309 Chrl : 36098064- + AGTCTCATCGAAGGCCCCAGGGG

36098086

310 Chrl : 36098071- + TCGAAGGCCCCAGGGGCACCAGG

36098093

311 Chrl : 36098072- + CGAAGGCCCCAGGGGCACCAGGG

36098094

312 Chrl : 36098073- + GAAGGCCCCAGGGGCACCAGGGG

36098095

313 Chrl : 36098074- + AAGGCCCCAGGGGCACCAGGGGG

36098096

314 Chrl : 36098078- ^— GGGACCCCCTGGTGCCCCTGGGG

36098100

315 Chrl : 36098079- ^— CGGGACCCCCTGGTGCCCCTGGG

36098101

316 Chrl : 36098080- + CCAGGGGCACCAGGGGGTCCCGG

36098102

317 Chrl : 36098080- ^— CCGGGACCCCCTGGTGCCCCTGG

36098102

318 Chrl : 36098081- + CAGGGGCACCAGGGGGTCCCGGG

36098103

319 Chrl : 36098082- + AGGGGCACCAGGGGGTCCCGGGG

36098104

320 Chrl : 36098083- + GGGGCACCAGGGGGTCCCGGGGG

36098105

321 Chrl : 36098088- + ACCAGGGGGTCCCGGGGGCCCGG

36098110

322 Chrl : 36098089- + CCAGGGGGTCCCGGGGGCCCGGG

36098111

323 Chrl : 36098089- ^— CCCGGGCCCCCGGGACCCCCTGG

36098111

324 Chrl : 36098092- + GGGGGTCCCGGGGGCCCGGGAGG

36098114

325 Chrl : 36098098- + CCCGGGGGCCCGGGAGGCCCCGG

36098120

326 Chrl : 36098098- ^— CCGGGGCCTCCCGGGCCCCCGGG

36098120

327 Chrl : 36098099- — TCCGGGGCCTCCCGGGCCCCCGG

36098121 328 Chrl : 36098101- + GGGGGCCCGGGAGGCCCCGGAGG 36098123

329 Chrl : 36098102- + GGGGCCCGGGAGGCCCCGGAGGG

36098124

330 Chrl : 36098106- ^— CGGGCCCTCCGGGGCCTCCCGGG

36098128

331 Chrl : 36098107- ^— ACGGGCCCTCCGGGGCCTCCCGG

36098129

332 Chrl : 36098115- ^— CTGGAATCACGGGCCCTCCGGGG

36098137

333 Chrl : 36098116- + CCCGGAGGGCCCGTGATTCCAGG

36098138

334 Chrl : 36098116- ^— CCTGGAATCACGGGCCCTCCGGG

36098138

335 Chrl : 36098117- + CCGGAGGGCCCGTGATTCCAGGG

36098139

336 Chrl : 36098117- ^— CCCTGGAATCACGGGCCCTCCGG

36098139

337 Chrl : 36098118- + CGGAGGGCCCGTGATTCCAGGGG

36098140

338 Chrl : 36098125- + CCCGTGATTCCAGGGGAGCCAGG

36098147

339 Chrl : 36098125- ^— CCTGGCTCCCCTGGAATCACGGG

36098147

340 Chrl : 36098126- + CCGTGATTCCAGGGGAGCCAGGG

36098148

341 Chrl : 36098126- ^— CCCTGGCTCCCCTGGAATCACGG

36098148

342 Chrl : 36098134- + CCAGGGGAGCCAGGGACCCCTGG

36098156

343 Chrl : 36098134- ^— CCAGGGGTCCCTGGCTCCCCTGG

36098156

344 Chrl : 36098135- + CAGGGGAGCCAGGGACCCCTGGG

36098157

345 Chrl : 36098136- + AGGGGAGCCAGGGACCCCTGGGG

36098158

346 Chrl : 36098137- + GGGGAGCCAGGGACCCCTGGGGG

36098159

347 Chrl : 36098143- ^— ACGGGGCCCCCAGGGGTCCCTGG

36098165

348 Chrl : 36098145- + AGGGACCCCTGGGGGCCCCGTGG

36098167

349 Chrl : 36098146- + GGGACCCCTGGGGGCCCCGTGGG

36098168

350 Chrl : 36098150- — TGGGCCCACGGGGCCCCCAGGGG

36098172 351 Chrl : 36098151- ^— CTGGGCCCACGGGGCCCCCAGGG 36098173

352 Chrl : 36098152- ^— GCTGGGCCCACGGGGCCCCCAGG

36098174

353 Chrl : 36098160- ^— CTGGCACGGCTGGGCCCACGGGG

36098182

354 Chrl : 36098161- + CCCGTGGGCCCAGCCGTGCCAGG

36098183

355 Chrl : 36098161- ^— CCTGGCACGGCTGGGCCCACGGG

36098183

356 Chrl : 36098162- ^— ACCTGGCACGGCTGGGCCCACGG

36098184

357 Chrl : 36098169- ^— CAGGGGAACCTGGCACGGCTGGG

36098191

358 Chrl : 36098170- ^— GCAGGGGAACCTGGCACGGCTGG

36098192

359 Chrl : 36098174- ^— GAGAGCAGGGGAACCTGGCACGG

36098196

360 Chrl : 36098179- ^— GAGGGGAGAGCAGGGGAACCTGG

36098201

361 Chrl : 36098185- + TCCCCTGCTCTCCCCTCTCCAGG

36098207

362 Chrl : 36098186- + CCCCTGCTCTCCCCTCTCCAGGG

36098208

363 Chrl : 36098186- ^— CCCTGGAGAGGGGAGAGCAGGGG

36098208

364 Chrl : 36098187- + CCCTGCTCTCCCCTCTCCAGGGG

36098209

365 Chrl : 36098187- ^— CCCCTGGAGAGGGGAGAGCAGGG

36098209

366 Chrl : 36098188- + CCTGCTCTCCCCTCTCCAGGGGG

36098210

367 Chrl : 36098188- ^— CCCCCTGGAGAGGGGAGAGCAGG

36098210

368 Chrl : 36098194- + CTCCCCTCTCCAGGGGGCCCTGG

36098216

369 Chrl : 36098196- ^— TGCCAGGGCCCCCTGGAGAGGGG

36098218

370 Chrl : 36098197- ^— CTGCCAGGGCCCCCTGGAGAGGG

36098219

371 Chrl : 36098198- + CCTCTCCAGGGGGCCCTGGCAGG

36098220

372 Chrl : 36098198- ^— CCTGCCAGGGCCCCCTGGAGAGG

36098220

373 Chrl : 36098203- + CCAGGGGGCCCTGGCAGGCCTGG

36098225 374 Chrl : 36098203- ^— CCAGGCCTGCCAGGGCCCCCTGG 36098225

375 Chrl : 36098211- ^— AGGGGGAACCAGGCCTGCCAGGG

36098233

376 Chrl : 36098212- ^— AAGGGGGAACCAGGCCTGCCAGG

36098234

377 Chrl : 36098216- + GCAGGCCTGGTTCCCCCTTCAGG

36098238

378 Chrl : 36098221- + CCTGGTTCCCCCTTCAGGCCCGG

36098243

379 Chrl : 36098221- ^— CCGGGCCTGAAGGGGGAACCAGG

36098243

380 Chrl : 36098225- + GTTCCCCCTTCAGGCCCGGCAGG

36098247

381 Chrl : 36098228- ^— AGGCCTGCCGGGCCTGAAGGGGG

36098250

382 Chrl : 36098229- ^— AAGGCCTGCCGGGCCTGAAGGGG

36098251

383 Chrl : 36098230- ^— CAAGGCCTGCCGGGCCTGAAGGG

36098252

384 Chrl : 36098231- + CCTTCAGGCCCGGCAGGCCTTGG

36098253

385 Chrl : 36098231- ^— CCAAGGCCTGCCGGGCCTGAAGG

36098253

386 Chrl : 36098232- + CTTCAGGCCCGGCAGGCCTTGGG

36098254

387 Chrl : 36098233- + TTCAGGCCCGGCAGGCCTTGGGG

36098255

388 Chrl : 36098239- ^— ATTGGGCCCCAAGGCCTGCCGGG

36098261

389 Chrl : 36098240- ^— TATTGGGCCCCAAGGCCTGCCGG

36098262

390 Chrl : 36098242- + GGCAGGCCTTGGGGCCCAATAGG

36098264

391 Chrl : 36098243- + GCAGGCCTTGGGGCCCAATAGGG

36098265

392 Chrl : 36098248- ^— GCTGGCCCTATTGGGCCCCAAGG

36098270

393 Chrl : 36098251- + TGGGGCCCAATAGGGCCAGCTGG

36098273

394 Chrl : 36098256- ^— AGGGTCCAGCTGGCCCTATTGGG

36098278

395 Chrl : 36098257- ^— CAGGGTCCAGCTGGCCCTATTGG

36098279

396 Chrl : 36098258- + CAATAGGGCCAGCTGGACCCTGG

36098280 397 Chrl : 36098266- + CCAGCTGGACCCTGGAGTCCTGG 36098288

398 Chrl : 36098266- ^— CCAGGACTCCAGGGTCCAGCTGG

36098288

399 Chrl : 36098267- + CAGCTGGACCCTGGAGTCCTGGG

36098289

400 Chrl : 36098275- ^— TCAGGAATCCCAGGACTCCAGGG

36098297

401 Chrl : 36098276- ^— CTCAGGAATCCCAGGACTCCAGG

36098298

402 Chrl : 36098277- + CTGGAGTCCTGGGATTCCTGAGG

36098299

403 Chrl : 36098278- + TGGAGTCCTGGGATTCCTGAGGG

36098300

404 Chrl : 36098284- ^— AGGGGTCCCTCAGGAATCCCAGG

36098306

405 Chrl : 36098288- + GGATTCCTGAGGGACCCCTCAGG

36098310

406 Chrl : 36098293- + CCTGAGGGACCCCTCAGGCCAGG

36098315

407 Chrl : 36098293- ^— CCTGGCCTGAGGGGTCCCTCAGG

36098315

408 Chrl : 36098302- + CCCCTCAGGCCAGGCTGCCCAGG

36098324

409 Chrl : 36098302- ^— CCTGGGCAGCCTGGCCTGAGGGG

36098324

410 Chrl : 36098303- + CCCTCAGGCCAGGCTGCCCAGGG

36098325

411 Chrl : 36098303- ^— CCCTGGGCAGCCTGGCCTGAGGG

36098325

412 Chrl : 36098304- ^— TCCCTGGGCAGCCTGGCCTGAGG

36098326

413 Chrl : 36098311- ^— TTGGGGCTCCCTGGGCAGCCTGG

36098333

414 Chrl : 36098319- ^— AAGGTGACTTGGGGCTCCCTGGG

36098341

415 Chrl : 36098320- ^— AAAGGTGACTTGGGGCTCCCTGG

36098342

416 Chrl : 36098328- ^— TGGGGCAGAAAGGTGACTTGGGG

36098350

417 Chrl : 36098329- ^— CTGGGGCAGAAAGGTGACTTGGG

36098351

418 Chrl : 36098330- + CCAAGTCACCTTTCTGCCCCAGG

36098352

419 Chrl : 36098330- — CCTGGGGCAGAAAGGTGACTTGG

36098352 420 Chrl : 36098331- + CAAGTCACCTTTCTGCCCCAGGG 36098353

421 Chrl : 36098338- ^— GCAGGAGCCCTGGGGCAGAAAGG

36098360

422 Chrl : 36098346- ^— CAGGGGTGGCAGGAGCCCTGGGG

36098368

423 Chrl : 36098347- + CCCAGGGCTCCTGCCACCCCTGG

36098369

424 Chrl : 36098347- ^— CCAGGGGTGGCAGGAGCCCTGGG

36098369

425 Chrl : 36098348- ^— ACCAGGGGTGGCAGGAGCCCTGG

36098370

426 Chrl : 36098356- + CCTGCCACCCCTGGTCCTCCAGG

36098378

427 Chrl : 36098356- ^— CCTGGAGGACCAGGGGTGGCAGG

36098378

428 Chrl : 36098357- + CTGCCACCCCTGGTCCTCCAGGG

36098379

429 Chrl : 36098360- ^— TCGCCCTGGAGGACCAGGGGTGG

36098382

430 Chrl : 36098363- ^— GGGTCGCCCTGGAGGACCAGGGG

36098385

431 Chrl : 36098364- ^— CGGGTCGCCCTGGAGGACCAGGG

36098386

432 Chrl : 36098365- ^— ACGGGTCGCCCTGGAGGACCAGG

36098387

433 Chrl : 36098371- ^— GGTTTCACGGGTCGCCCTGGAGG

36098393

434 Chrl : 36098374- + CCAGGGCGACCCGTGAAACCCGG

36098396

435 Chrl : 36098374- ^— CCGGGTTTCACGGGTCGCCCTGG

36098396

436 Chrl : 36098383- ^— AAGGGTGAGCCGGGTTTCACGGG

36098405

437 Chrl : 36098384- ^— CAAGGGTGAGCCGGGTTTCACGG

36098406

438 Chrl : 36098385- + CGTGAAACCCGGCTCACCCTTGG

36098407

439 Chrl : 36098386- + GTGAAACCCGGCTCACCCTTGGG

36098408

440 Chrl : 36098392- ^— ACTGGGCCCAAGGGTGAGCCGGG

36098414

441 Chrl : 36098393- ^— AACTGGGCCCAAGGGTGAGCCGG

36098415

442 Chrl : 36098395- + GGCTCACCCTTGGGCCCAGTTGG

36098417 443 Chrl : 36098401- + CCCTTGGGCCCAGTTGGTCCAGG 36098423

444 Chrl : 36098401- ^— CCTGGACCAACTGGGCCCAAGGG

36098423

445 Chrl : 36098402- + CCTTGGGCCCAGTTGGTCCAGGG

36098424

446 Chrl : 36098402- ^— CCCTGGACCAACTGGGCCCAAGG

36098424

447 Chrl : 36098403- + CTTGGGCCCAGTTGGTCCAGGGG

36098425

448 Chrl : 36098404- + TTGGGCCCAGTTGGTCCAGGGGG

36098426

449 Chrl : 36098409- ^— ATGGACCCCCTGGACCAACTGGG

36098431

450 Chrl : 36098410- ^— CATGGACCCCCTGGACCAACTGG

36098432

451 Chrl : 36098411- + CAGTTGGTCCAGGGGGTCCATGG

36098433

452 Chrl : 36098412- + AGTTGGTCCAGGGGGTCCATGGG

36098434

453 Chrl : 36098419- + CCAGGGGGTCCATGGGCCCCAGG

36098441

454 Chrl : 36098419- ^— CCTGGGGCCCATGGACCCCCTGG

36098441

455 Chrl : 36098428- ^— AGGGGACTTCCTGGGGCCCATGG

36098450

456 Chrl : 36098435- ^— AGGTGAGAGGGGACTTCCTGGGG

36098457

457 Chrl : 36098436- ^— CAGGTGAGAGGGGACTTCCTGGG

36098458

458 Chrl : 36098437- + CCAGGAAGTCCCCTCTCACCTGG

36098459

459 Chrl : 36098437- ^— CCAGGTGAGAGGGGACTTCCTGG

36098459

460 Chrl : 36098438- + CAGGAAGTCCCCTCTCACCTGGG

36098460

461 Chrl : 36098446- + CCCCTCTCACCTGGGACCCCTGG

36098468

462 Chrl : 36098446- ^— CCAGGGGTCCCAGGTGAGAGGGG

36098468

463 Chrl : 36098447- ^— ACCAGGGGTCCCAGGTGAGAGGG

36098469

464 Chrl : 36098448- ^— AACCAGGGGTCCCAGGTGAGAGG

36098470

465 Chrl : 36098455- — GCTGGGAAACCAGGGGTCCCAGG

36098477 466 Chrl : 36098459- + GGACCCCTGGTTTCCCAGCCAGG 36098481

467 Chrl : 36098462- ^— TGGCCTGGCTGGGAAACCAGGGG

36098484

468 Chrl : 36098463- ^— GTGGCCTGGCTGGGAAACCAGGG

36098485

469 Chrl : 36098464- ^— AGTGGCCTGGCTGGGAAACCAGG

36098486

470 Chrl : 36098467- + GGTTTCCCAGCCAGGCCACTAGG

36098489

471 Chrl : 36098472- ^— AGGGGCCTAGTGGCCTGGCTGGG

36098494

472 Chrl : 36098473- ^— CAGGGGCCTAGTGGCCTGGCTGG

36098495

473 Chrl : 36098474- + CAGCCAGGCCACTAGGCCCCTGG

36098496

474 Chrl : 36098477- ^— TGACCAGGGGCCTAGTGGCCTGG

36098499

475 Chrl : 36098482- ^— CGAGGTGACCAGGGGCCTAGTGG

36098504

476 Chrl : 36098490- ^— CTGGCATTCGAGGTGACCAGGGG

36098512

477 Chrl : 36098491- + CCCTGGTCACCTCGAATGCCAGG

36098513

478 Chrl : 36098491- ^— CCTGGCATTCGAGGTGACCAGGG

36098513

479 Chrl : 36098492- ^— GCCTGGCATTCGAGGTGACCAGG

36098514

480 Chrl : 36098500- + CCTCGAATGCCAGGCACTCCTGG

36098522

481 Chrl : 36098500- ^— CCAGGAGTGCCTGGCATTCGAGG

36098522

482 Chrl : 36098501- + CTCGAATGCCAGGCACTCCTGGG

36098523

483 Chrl : 36098502- + TCGAATGCCAGGCACTCCTGGGG

36098524

484 Chrl : 36098503- + CGAATGCCAGGCACTCCTGGGGG

36098525

485 Chrl : 36098509- ^— GGAGGACCCCCAGGAGTGCCTGG

36098531

486 Chrl : 36098512- + GGCACTCCTGGGGGTCCTCCAGG

36098534

487 Chrl : 36098518- ^— GCAGGGCCTGGAGGACCCCCAGG

36098540

488 Chrl : 36098527- — AAGGGTGAGGCAGGGCCTGGAGG

36098549 489 Chrl : 36098530- + CCAGGCCCTGCCTCACCCTTAGG 36098552

490 Chrl : 36098530- ^— CCTAAGGGTGAGGCAGGGCCTGG

36098552

491 Chrl : 36098535- ^— CTGGGCCTAAGGGTGAGGCAGGG

36098557

492 Chrl : 36098536- + CCTGCCTCACCCTTAGGCCCAGG

36098558

493 Chrl : 36098536- ^— CCTGGGCCTAAGGGTGAGGCAGG

36098558

494 Chrl : 36098537- + CTGCCTCACCCTTAGGCCCAGGG

36098559

495 Chrl : 36098538- + TGCCTCACCCTTAGGCCCAGGGG

36098560

496 Chrl : 36098539- + GCCTCACCCTTAGGCCCAGGGGG

36098561

497 Chrl : 36098540- ^— GCCCCCTGGGCCTAAGGGTGAGG

36098562

498 Chrl : 36098545- ^— CGTGGGCCCCCTGGGCCTAAGGG

36098567

499 Chrl : 36098546- ^— ACGTGGGCCCCCTGGGCCTAAGG

36098568

500 Chrl : 36098553- ^— CTGGCAGACGTGGGCCCCCTGGG

36098575

501 Chrl : 36098554- + CCAGGGGGCCCACGTCTGCCAGG

36098576

502 Chrl : 36098554- ^— CCTGGCAGACGTGGGCCCCCTGG

36098576

503 Chrl : 36098562- ^— CAGGGCTTCCTGGCAGACGTGGG

36098584

504 Chrl : 36098563- ^— GCAGGGCTTCCTGGCAGACGTGG

36098585

505 Chrl : 36098572- + CCAGGAAGCCCTGCAGACCCAGG

36098594

506 Chrl : 36098572- ^— CCTGGGTCTGCAGGGCTTCCTGG

36098594

507 Chrl : 36098580- ^— CTGGACTTCCTGGGTCTGCAGGG

36098602

508 Chrl : 36098581- + CCTGCAGACCCAGGAAGTCCAGG

36098603

509 Chrl : 36098581- ^— CCTGGACTTCCTGGGTCTGCAGG

36098603

510 Chrl : 36098582- + CTGCAGACCCAGGAAGTCCAGGG

36098604

511 Chrl : 36098583- + TGCAGACCCAGGAAGTCCAGGGG

36098605 512 Chrl : 36098584- + GCAGACCCAGGAAGTCCAGGGGG 36098606

513 Chrl : 36098589- ^— GGGGTCCCCCTGGACTTCCTGGG

36098611

514 Chrl : 36098590- ^— GGGGGTCCCCCTGGACTTCCTGG

36098612

515 Chrl : 36098599- ^— CAGGGTCTTGGGGGTCCCCCTGG

36098621

516 Chrl : 36098602- + GGGGGACCCCCAAGACCCTGTGG

36098624

517 Chrl : 36098603- + GGGGACCCCCAAGACCCTGTGGG

36098625

518 Chrl : 36098608- ^— CAGGGCCCACAGGGTCTTGGGGG

36098630

519 Chrl : 36098609- ^— GCAGGGCCCACAGGGTCTTGGGG

36098631

520 Chrl : 36098610- ^— AGCAGGGCCCACAGGGTCTTGGG

36098632

521 Chrl : 36098611- ^— GAGCAGGGCCCACAGGGTCTTGG

36098633

522 Chrl : 36098617- + CCCTGTGGGCCCTGCTCCCCTGG

36098639

523 Chrl : 36098617- ^— CCAGGGGAGCAGGGCCCACAGGG

36098639

524 Chrl : 36098618- ^— GCCAGGGGAGCAGGGCCCACAGG

36098640

525 Chrl : 36098626- ^— GATGGGGAGCCAGGGGAGCAGGG

36098648

526 Chrl : 36098627- ^— GGATGGGGAGCCAGGGGAGCAGG

36098649

527 Chrl : 36098633- ^— AGGGGAGGATGGGGAGCCAGGGG

36098655

528 Chrl : 36098634- ^— CAGGGGAGGATGGGGAGCCAGGG

36098656

529 Chrl : 36098635- + CCTGGCTCCCCATCCTCCCCTGG

36098657

530 Chrl : 36098635- ^— CCAGGGGAGGATGGGGAGCCAGG

36098657

531 Chrl : 36098642- ^— GGGTGAGCCAGGGGAGGATGGGG

36098664

532 Chrl : 36098643- ^— GGGGTGAGCCAGGGGAGGATGGG

36098665

533 Chrl : 36098644- ^— AGGGGTGAGCCAGGGGAGGATGG

36098666

534 Chrl : 36098648- — GGACAGGGGTGAGCCAGGGGAGG

36098670 535 Chrl : 36098651- ^— GGGGGACAGGGGTGAGCCAGGGG 36098673

536 Chrl : 36098652- ^— TGGGGGACAGGGGTGAGCCAGGG

36098674

537 Chrl : 36098653- ^— TTGGGGGACAGGGGTGAGCCAGG

36098675

538 Chrl : 36098662- + CCCCTGTCCCCCAAGAGTCCTGG

36098684

539 Chrl : 36098662- ^— CCAGGACTCTTGGGGGACAGGGG

36098684

540 Chrl : 36098663- + CCCTGTCCCCCAAGAGTCCTGGG

36098685

541 Chrl : 36098663- ^— CCCAGGACTCTTGGGGGACAGGG

36098685

542 Chrl : 36098664- ^— TCCCAGGACTCTTGGGGGACAGG

36098686

543 Chrl : 36098669- ^— TGGGGTCCCAGGACTCTTGGGGG

36098691

544 Chrl : 36098670- ^— CTGGGGTCCCAGGACTCTTGGGG

36098692

545 Chrl : 36098671- ^— GCTGGGGTCCCAGGACTCTTGGG

36098693

546 Chrl : 36098672- ^— AGCTGGGGTCCCAGGACTCTTGG

36098694

547 Chrl : 36098674- + AAGAGTCCTGGGACCCCAGCTGG

36098696

548 Chrl : 36098675- + AGAGTCCTGGGACCCCAGCTGGG

36098697

549 Chrl : 36098680- ^— AGGGGCCCAGCTGGGGTCCCAGG

36098702

550 Chrl : 36098687- ^— GGGGGACAGGGGCCCAGCTGGGG

36098709

551 Chrl : 36098688- ^— AGGGGGACAGGGGCCCAGCTGGG

36098710

552 Chrl : 36098689- ^— AAGGGGGACAGGGGCCCAGCTGG

36098711

553 Chrl : 36098691- + AGCTGGGCCCCTGTCCCCCTTGG

36098713

554 Chrl : 36098692- + GCTGGGCCCCTGTCCCCCTTGGG

36098714

555 Chrl : 36098693- + CTGGGCCCCTGTCCCCCTTGGGG

36098715

556 Chrl : 36098698- + CCCCTGTCCCCCTTGGGGCCTGG

36098720

557 Chrl : 36098698- — CCAGGCCCCAAGGGGGACAGGGG

36098720 558 Chrl : 36098699- ^— GCCAGGCCCCAAGGGGGACAGGG 36098721

559 Chrl : 36098700- ^— TGCCAGGCCCCAAGGGGGACAGG

36098722

560 Chrl : 36098705- ^— AGGACTGCCAGGCCCCAAGGGGG

36098727

561 Chrl : 36098706- ^— CAGGACTGCCAGGCCCCAAGGGG

36098728

562 Chrl : 36098707- + CCCTTGGGGCCTGGCAGTCCTGG

36098729

563 Chrl : 36098707- ^— CCAGGACTGCCAGGCCCCAAGGG

36098729

564 Chrl : 36098708- ^— GCCAGGACTGCCAGGCCCCAAGG

36098730

565 Chrl : 36098716- ^— TATGGGATGCCAGGACTGCCAGG

36098738

566 Chrl : 36098724- + TCCTGGCATCCCATAGCCAGTGG

36098746

567 Chrl : 36098725- + CCTGGCATCCCATAGCCAGTGGG

36098747

568 Chrl : 36098725- ^— CCCACTGGCTATGGGATGCCAGG

36098747

569 Chrl : 36098726- + CTGGCATCCCATAGCCAGTGGGG

36098748

570 Chrl : 36098733- ^— TGATAGGCCCCACTGGCTATGGG

36098755

571 Chrl : 36098734- ^— CTGATAGGCCCCACTGGCTATGG

36098756

572 Chrl : 36098740- + CCAGTGGGGCCTATCAGCCCAGG

36098762

573 Chrl : 36098740- ^— CCTGGGCTGATAGGCCCCACTGG

36098762

574 Chrl : 36098741- + CAGTGGGGCCTATCAGCCCAGGG

36098763

575 Chrl : 36098742- + AGTGGGGCCTATCAGCCCAGGGG

36098764

576 Chrl : 36098743- + GTGGGGCCTATCAGCCCAGGGGG

36098765

577 Chrl : 36098744- + TGGGGCCTATCAGCCCAGGGGGG

36098766

578 Chrl : 36098749- ^— CGGGGCCCCCCTGGGCTGATAGG

36098771

579 Chrl : 36098750- + CTATCAGCCCAGGGGGGCCCCGG

36098772

580 Chrl : 36098751- + TATCAGCCCAGGGGGGCCCCGGG

36098773 581 Chrl : 36098757- ^— CAGGGACCCGGGGCCCCCCTGGG 36098779

582 Chrl : 36098758- + CCAGGGGGGCCCCGGGTCCCTGG

36098780

583 Chrl : 36098758- ^— CCAGGGACCCGGGGCCCCCCTGG

36098780

584 Chrl : 36098767- ^— AAAGGGGAGCCAGGGACCCGGGG

36098789

585 Chrl : 36098768- ^— CAAAGGGGAGCCAGGGACCCGGG

36098790

586 Chrl : 36098769- + CCGGGTCCCTGGCTCCCCTTTGG

36098791

587 Chrl : 36098769- ^— CCAAAGGGGAGCCAGGGACCCGG

36098791

588 Chrl : 36098775- ^— CAGGGGCCAAAGGGGAGCCAGGG

36098797

589 Chrl : 36098776- ^— TCAGGGGCCAAAGGGGAGCCAGG

36098798

590 Chrl : 36098779- + GGCTCCCCTTTGGCCCCTGATGG

36098801

591 Chrl : 36098780- + GCTCCCCTTTGGCCCCTGATGGG

36098802

592 Chrl : 36098783- ^— GGGCCCATCAGGGGCCAAAGGGG

36098805

593 Chrl : 36098784- ^— AGGGCCCATCAGGGGCCAAAGGG

36098806

594 Chrl : 36098785- ^— CAGGGCCCATCAGGGGCCAAAGG

36098807

595 Chrl : 36098788- + TTGGCCCCTGATGGGCCCTGTGG

36098810

596 Chrl : 36098792- ^— AGGACCACAGGGCCCATCAGGGG

36098814

597 Chrl : 36098793- ^— CAGGACCACAGGGCCCATCAGGG

36098815

598 Chrl : 36098794- + CCTGATGGGCCCTGTGGTCCTGG

36098816

599 Chrl : 36098794- ^— CCAGGACCACAGGGCCCATCAGG

36098816

600 Chrl : 36098803- ^— GCAGGGTTGCCAGGACCACAGGG

36098825

601 Chrl : 36098804- ^— AGCAGGGTTGCCAGGACCACAGG

36098826

602 Chrl : 36098812- + CCTGGCAACCCTGCTGCCCCTGG

36098834

603 Chrl : 36098812- — CCAGGGGCAGCAGGGTTGCCAGG

36098834 604 Chrl : 36098813- + CTGGCAACCCTGCTGCCCCTGGG 36098835

605 Chrl : 36098820- ^— TGGGAGTCCCAGGGGCAGCAGGG

36098842

606 Chrl : 36098821- ^— GTGGGAGTCCCAGGGGCAGCAGG

36098843

607 Chrl : 36098828- ^— AGACGGTGTGGGAGTCCCAGGGG

36098850

608 Chrl : 36098829- ^— TAGACGGTGTGGGAGTCCCAGGG

36098851

609 Chrl : 36098830- ^— GTAGACGGTGTGGGAGTCCCAGG

36098852

610 Chrl : 36098836- + ACTCCCACACCGTCTACTCCAGG

36098858

611 Chrl : 36098839- + CCCACACCGTCTACTCCAGGAGG

36098861

612 Chrl : 36098839- ^— CCTCCTGGAGTAGACGGTGTGGG

36098861

613 Chrl : 36098840- ^— ACCTCCTGGAGTAGACGGTGTGG

36098862

614 Chrl : 36098845- ^— AAAGGACCTCCTGGAGTAGACGG

36098867

615 Chrl : 36098848- + TCTACTCCAGGAGGTCCTTTTGG

36098870

616 Chrl : 36098849- + CTACTCCAGGAGGTCCTTTTGGG

36098871

617 Chrl : 36098854- ^— GTGGGCCCAAAAGGACCTCCTGG

36098876

618 Chrl : 36098863- + CCTTTTGGGCCCACAGCTCCTGG

36098885

619 Chrl : 36098863- ^— CCAGGAGCTGTGGGCCCAAAAGG

36098885

620 Chrl : 36098872- ^— AGGGGGGAGCCAGGAGCTGTGGG

36098894

621 Chrl : 36098873- ^— CAGGGGGGAGCCAGGAGCTGTGG

36098895

622 Chrl : 36098874- + CACAGCTCCTGGCTCCCCCCTGG

36098896

623 Chrl : 36098875- + ACAGCTCCTGGCTCCCCCCTGGG

36098897

624 Chrl : 36098876- + CAGCTCCTGGCTCCCCCCTGGGG

36098898

625 Chrl : 36098881- + CCTGGCTCCCCCCTGGGGCCTGG

36098903

626 Chrl : 36098881- — CCAGGCCCCAGGGGGGAGCCAGG

36098903 627 Chrl : 36098888- ^— TGGAGTTCCAGGCCCCAGGGGGG 36098910

628 Chrl : 36098889- ^— CTGGAGTTCCAGGCCCCAGGGGG

36098911

629 Chrl : 36098890- + CCCCTGGGGCCTGGAACTCCAGG

36098912

630 Chrl : 36098890- ^— CCTGGAGTTCCAGGCCCCAGGGG

36098912

631 Chrl : 36098891- ^— TCCTGGAGTTCCAGGCCCCAGGG

36098913

632 Chrl : 36098892- ^— CTCCTGGAGTTCCAGGCCCCAGG

36098914

633 Chrl : 36098893- + CTGGGGCCTGGAACTCCAGGAGG

36098915

634 Chrl : 36098899- ^— TCTGGGCCTCCTGGAGTTCCAGG

36098921

635 Chrl : 36098908- ^— AAGGGTGAGTCTGGGCCTCCTGG

36098930

636 Chrl : 36098916- ^— CAGGAGACAAGGGTGAGTCTGGG

36098938

637 Chrl : 36098917- + CCAGACTCACCCTTGTCTCCTGG

36098939

638 Chrl : 36098917- ^— CCAGGAGACAAGGGTGAGTCTGG

36098939

639 Chrl : 36098918- + CAGACTCACCCTTGTCTCCTGGG

36098940

640 Chrl : 36098919- + AGACTCACCCTTGTCTCCTGGGG

36098941

641 Chrl : 36098926- + CCCTTGTCTCCTGGGGCCCCAGG

36098948

642 Chrl : 36098926- ^— CCTGGGGCCCCAGGAGACAAGGG

36098948

643 Chrl : 36098927- ^— TCCTGGGGCCCCAGGAGACAAGG

36098949

644 Chrl : 36098935- ^— GATGGGCTTCCTGGGGCCCCAGG

36098957

645 Chrl : 36098942- ^— TGGTTTGGATGGGCTTCCTGGGG

36098964

646 Chrl : 36098943- ^— CTGGTTTGGATGGGCTTCCTGGG

36098965

647 Chrl : 36098944- + CCAGGAAGCCCATCCAAACCAGG

36098966

648 Chrl : 36098944- ^— CCTGGTTTGGATGGGCTTCCTGG

36098966

649 Chrl : 36098952- — TAGGCAAACCTGGTTTGGATGGG

36098974 650 Chrl : 36098953- ^— TTAGGCAAACCTGGTTTGGATGG 36098975

651 Chrl : 36098957- ^— TGGCTTAGGCAAACCTGGTTTGG

36098979

652 Chrl : 36098962- + CCAGGTTTGCCTAAGCCAGCTGG

36098984

653 Chrl : 36098962- ^— CCAGCTGGCTTAGGCAAACCTGG

36098984

654 Chrl : 36098968- + TTGCCTAAGCCAGCTGGACCAGG

36098990

655 Chrl : 36098969- + TGCCTAAGCCAGCTGGACCAGGG

36098991

656 Chrl : 36098971- ^— CTCCCTGGTCCAGCTGGCTTAGG

36098993

657 Chrl : 36098972- + CTAAGCCAGCTGGACCAGGGAGG

36098994

658 Chrl : 36098976- + GCCAGCTGGACCAGGGAGGCCGG

36098998

659 Chrl : 36098977- + CCAGCTGGACCAGGGAGGCCGGG

36098999

660 Chrl : 36098977- ^— CCCGGCCTCCCTGGTCCAGCTGG

36098999

661 Chrl : 36098978- + CAGCTGGACCAGGGAGGCCGGGG

36099000

662 Chrl : 36098979- + AGCTGGACCAGGGAGGCCGGGGG

36099001

663 Chrl : 36098980- + GCTGGACCAGGGAGGCCGGGGGG

36099002

664 Chrl : 36098981- + CTGGACCAGGGAGGCCGGGGGGG

36099003

665 Chrl : 36098985- + ACCAGGGAGGCCGGGGGGGCCGG

36099007

666 Chrl : 36098986- + CCAGGGAGGCCGGGGGGGCCGGG

36099008

667 Chrl : 36098986- ^— CCCGGCCCCCCCGGCCTCCCTGG

36099008

668 Chrl : 36098987- + CAGGGAGGCCGGGGGGGCCGGGG

36099009

669 Chrl : 36098988- + AGGGAGGCCGGGGGGGCCGGGGG

36099010

670 Chrl : 36098995- ^— GGGGGTGCCCCCGGCCCCCCCGG

36099017

671 Chrl : 36099004- + CCGGGGGCACCCCCCTGCCCTGG

36099026

672 Chrl : 36099004- — CCAGGGCAGGGGGGTGCCCCCGG

36099026 673 Chrl : 36099005- + CGGGGGCACCCCCCTGCCCTGGG 36099027

674 Chrl : 36099006- + GGGGGCACCCCCCTGCCCTGGGG

36099028

675 Chrl : 36099013- + CCCCCCTGCCCTGGGGCCCCAGG

36099035

676 Chrl : 36099013- ^— CCTGGGGCCCCAGGGCAGGGGGG

36099035

677 Chrl : 36099014- ^— GCCTGGGGCCCCAGGGCAGGGGG

36099036

678 Chrl : 36099015- ^— TGCCTGGGGCCCCAGGGCAGGGG

36099037

679 Chrl : 36099016- ^— CTGCCTGGGGCCCCAGGGCAGGG

36099038

680 Chrl : 36099017- ^— GCTGCCTGGGGCCCCAGGGCAGG

36099039

681 Chrl : 36099021- + CCCTGGGGCCCCAGGCAGCCCGG

36099043

682 Chrl : 36099021- ^— CCGGGCTGCCTGGGGCCCCAGGG

36099043

683 Chrl : 36099022- + CCTGGGGCCCCAGGCAGCCCGGG

36099044

684 Chrl : 36099022- ^— CCCGGGCTGCCTGGGGCCCCAGG

36099044

685 Chrl : 36099026- + GGGCCCCAGGCAGCCCGGGCTGG

36099048

686 Chrl : 36099029- ^— GGGCCAGCCCGGGCTGCCTGGGG

36099051

687 Chrl : 36099030- ^— TGGGCCAGCCCGGGCTGCCTGGG

36099052

688 Chrl : 36099031- ^— GTGGGCCAGCCCGGGCTGCCTGG

36099053

689 Chrl : 36099039- ^— ATAATGGAGTGGGCCAGCCCGGG

36099061

690 Chrl : 36099040- ^— GATAATGGAGTGGGCCAGCCCGG

36099062

691 Chrl : 36099049- ^— CTCAAGGGGGATAATGGAGTGGG

36099071

692 Chrl : 36099050- + CCACTCCATTATCCCCCTTGAGG

36099072

693 Chrl : 36099050- ^— CCTCAAGGGGGATAATGGAGTGG

36099072

694 Chrl : 36099055- ^— CGAGGCCTCAAGGGGGATAATGG

36099077

695 Chrl : 36099062- — AGGTGATCGAGGCCTCAAGGGGG

36099084 696 Chrl : 36099063- ^— CAGGTGATCGAGGCCTCAAGGGG 36099085

697 Chrl : 36099064- + CCCTTGAGGCCTCGATCACCTGG

36099086

698 Chrl : 36099064- ^— CCAGGTGATCGAGGCCTCAAGGG

36099086

699 Chrl : 36099065- + CCTTGAGGCCTCGATCACCTGGG

36099087

700 Chrl : 36099065- ^— CCCAGGTGATCGAGGCCTCAAGG

36099087

701 Chrl : 36099066- + CTTGAGGCCTCGATCACCTGGGG

36099088

702 Chrl : 36099067- + TTGAGGCCTCGATCACCTGGGGG

36099089

703 Chrl : 36099073- + CCTCGATCACCTGGGGGCCCAGG

36099095

704 Chrl : 36099073- ^— CCTGGGCCCCCAGGTGATCGAGG

36099095

705 Chrl : 36099082- ^— CAGGGGGAGCCTGGGCCCCCAGG

36099104

706 Chrl : 36099083- + CTGGGGGCCCAGGCTCCCCCTGG

36099105

707 Chrl : 36099084- + TGGGGGCCCAGGCTCCCCCTGGG

36099106

708 Chrl : 36099085- + GGGGGCCCAGGCTCCCCCTGGGG

36099107

709 Chrl : 36099090- ^— CAGGGCCCCAGGGGGAGCCTGGG

36099112

710 Chrl : 36099091- + CCAGGCTCCCCCTGGGGCCCTGG

36099113

711 Chrl : 36099091- ^— CCAGGGCCCCAGGGGGAGCCTGG

36099113

712 Chrl : 36099098- ^— GGGGGAACCAGGGCCCCAGGGGG

36099120

713 Chrl : 36099099- ^— AGGGGGAACCAGGGCCCCAGGGG

36099121

714 Chrl : 36099100- ^— CAGGGGGAACCAGGGCCCCAGGG

36099122

715 Chrl : 36099101- + CCTGGGGCCCTGGTTCCCCCTGG

36099123

716 Chrl : 36099101- ^— CCAGGGGGAACCAGGGCCCCAGG

36099123

717 Chrl : 36099108- ^— CAGGATTCCAGGGGGAACCAGGG

36099130

718 Chrl : 36099109- + CCTGGTTCCCCCTGGAATCCTGG

36099131 719 Chrl : 36099109- ^— CCAGGATTCCAGGGGGAACCAGG 36099131

720 Chrl : 36099110- + CTGGTTCCCCCTGGAATCCTGGG

36099132

721 Chrl : 36099111- + TGGTTCCCCCTGGAATCCTGGGG

36099133

722 Chrl : 36099112- + GGTTCCCCCTGGAATCCTGGGGG

36099134

723 Chrl : 36099116- ^— AGGGCCCCCAGGATTCCAGGGGG

36099138

724 Chrl : 36099117- ^— CAGGGCCCCCAGGATTCCAGGGG

36099139

725 Chrl : 36099118- + CCCTGGAATCCTGGGGGCCCTGG

36099140

726 Chrl : 36099118- ^— CCAGGGCCCCCAGGATTCCAGGG

36099140

727 Chrl : 36099119- ^— GCCAGGGCCCCCAGGATTCCAGG

36099141

728 Chrl : 36099127- ^— CAAGGGGTGCCAGGGCCCCCAGG

36099149

729 Chrl : 36099128- + CTGGGGGCCCTGGCACCCCTTGG

36099150

730 Chrl : 36099129- + TGGGGGCCCTGGCACCCCTTGGG

36099151

731 Chrl : 36099135- ^— CAGGTGCCCAAGGGGTGCCAGGG

36099157

732 Chrl : 36099136- + CCTGGCACCCCTTGGGCACCTGG

36099158

733 Chrl : 36099136- ^— CCAGGTGCCCAAGGGGTGCCAGG

36099158

734 Chrl : 36099143- ^— TGGAAAACCAGGTGCCCAAGGGG

36099165

735 Chrl : 36099144- ^— CTGGAAAACCAGGTGCCCAAGGG

36099166

736 Chrl : 36099145- + CCTTGGGCACCTGGTTTTCCAGG

36099167

737 Chrl : 36099145- ^— CCTGGAAAACCAGGTGCCCAAGG

36099167

738 Chrl : 36099146- + CTTGGGCACCTGGTTTTCCAGGG

36099168

739 Chrl : 36099154- ^— ATTACTATCCCTGGAAAACCAGG

36099176

740 Chrl : 36099162- + TCCAGGGATAGTAATGCCTGAGG

36099184

741 Chrl : 36099163- + CCAGGGATAGTAATGCCTGAGGG

36099185 742 Chrl : 36099163- ^— CCCTCAGGCATTACTATCCCTGG 36099185

743 Chrl : 36099164- + CAGGGATAGTAATGCCTGAGGGG

36099186

744 Chrl : 36099169- + ATAGTAATGCCTGAGGGGCCCGG

36099191

745 Chrl : 36099170- + TAGTAATGCCTGAGGGGCCCGGG

36099192

746 Chrl : 36099173- + TAATGCCTGAGGGGCCCGGGAGG

36099195

747 Chrl : 36099178- + CCTGAGGGGCCCGGGAGGCCAGG

36099200

748 Chrl : 36099178- ^— CCTGGCCTCCCGGGCCCCTCAGG

36099200

749 Chrl : 36099179- + CTGAGGGGCCCGGGAGGCCAGGG

36099201

750 Chrl : 36099180- + TGAGGGGCCCGGGAGGCCAGGGG

36099202

751 Chrl : 36099181- + GAGGGGCCCGGGAGGCCAGGGGG

36099203

752 Chrl : 36099187- + CCCGGGAGGCCAGGGGGTCCTGG

36099209

753 Chrl : 36099187- ^— CCAGGACCCCCTGGCCTCCCGGG

36099209

754 Chrl : 36099188- + CCGGGAGGCCAGGGGGTCCTGGG

36099210

755 Chrl : 36099188- ^— CCCAGGACCCCCTGGCCTCCCGG

36099210

756 Chrl : 36099189- + CGGGAGGCCAGGGGGTCCTGGGG

36099211

757 Chrl : 36099190- + GGGAGGCCAGGGGGTCCTGGGGG

36099212

758 Chrl : 36099196- ^— CGGGGACCCCCAGGACCCCCTGG

36099218

759 Chrl : 36099197- + CAGGGGGTCCTGGGGGTCCCCGG

36099219

760 Chrl : 36099200- + GGGGTCCTGGGGGTCCCCGGAGG

36099222

761 Chrl : 36099205- ^— CAGGGCCTCCGGGGACCCCCAGG

36099227

762 Chrl : 36099206- + CTGGGGGTCCCCGGAGGCCCTGG

36099228

763 Chrl : 36099214- ^— CGAGGGGACCAGGGCCTCCGGGG

36099236

764 Chrl : 36099215- — ACGAGGGGACCAGGGCCTCCGGG

36099237 765 Chrl : 36099216- ^— TACGAGGGGACCAGGGCCTCCGG 36099238

766 Chrl : 36099223- + CCCTGGTCCCCTCGTATTCCTGG

36099245

767 Chrl : 36099223- ^— CCAGGAATACGAGGGGACCAGGG

36099245

768 Chrl : 36099224- ^— GCCAGGAATACGAGGGGACCAGG

36099246

769 Chrl : 36099230- ^— GGGGGAGCCAGGAATACGAGGGG

36099252

770 Chrl : 36099231- ^— GGGGGGAGCCAGGAATACGAGGG

36099253

771 Chrl : 36099232- ^— CGGGGGGAGCCAGGAATACGAGG

36099254

772 Chrl : 36099241- + CCTGGCTCCCCCCGAAGCCCCGG

36099263

773 Chrl : 36099241- ^— CCGGGGCTTCGGGGGGAGCCAGG

36099263

774 Chrl : 36099248- ^— AGGGCAGCCGGGGCTTCGGGGGG

36099270

775 Chrl : 36099249- ^— CAGGGCAGCCGGGGCTTCGGGGG

36099271

776 Chrl : 36099250- + CCCCGAAGCCCCGGCTGCCCTGG

36099272

777 Chrl : 36099250- ^— CCAGGGCAGCCGGGGCTTCGGGG

36099272

778 Chrl : 36099251- ^— ACCAGGGCAGCCGGGGCTTCGGG

36099273

779 Chrl : 36099252- ^— CACCAGGGCAGCCGGGGCTTCGG

36099274

780 Chrl : 36099253- + CGAAGCCCCGGCTGCCCTGGTGG

36099275

781 Chrl : 36099258- ^— TCGGGCCACCAGGGCAGCCGGGG

36099280

782 Chrl : 36099259- ^— GTCGGGCCACCAGGGCAGCCGGG

36099281

783 Chrl : 36099260- ^— GGTCGGGCCACCAGGGCAGCCGG

36099282

784 Chrl : 36099267- ^— CTGGCAAGGTCGGGCCACCAGGG

36099289

785 Chrl : 36099268- + CCTGGTGGCCCGACCTTGCCAGG

36099290

786 Chrl : 36099268- ^— CCTGGCAAGGTCGGGCCACCAGG

36099290

787 Chrl : 36099269- + CTGGTGGCCCGACCTTGCCAGGG

36099291 788 Chrl : 36099276- ^— CAGGGCTCCCTGGCAAGGTCGGG 36099298

789 Chrl : 36099277- + CCGACCTTGCCAGGGAGCCCTGG

36099299

790 Chrl : 36099277- ^— CCAGGGCTCCCTGGCAAGGTCGG

36099299

791 Chrl : 36099278- + CGACCTTGCCAGGGAGCCCTGGG

36099300

792 Chrl : 36099279- + GACCTTGCCAGGGAGCCCTGGGG

36099301

793 Chrl : 36099280- + ACCTTGCCAGGGAGCCCTGGGGG

36099302

794 Chrl : 36099281- ^— TCCCCCAGGGCTCCCTGGCAAGG

36099303

795 Chrl : 36099286- ^— GCTGGTCCCCCAGGGCTCCCTGG

36099308

796 Chrl : 36099294- ^— TGGGCAAGGCTGGTCCCCCAGGG

36099316

797 Chrl : 36099295- ^— ATGGGCAAGGCTGGTCCCCCAGG

36099317

798 Chrl : 36099299- + GGGGACCAGCCTTGCCCATCCGG

36099321

799 Chrl : 36099300- + GGGACCAGCCTTGCCCATCCGGG

36099322

800 Chrl : 36099304- ^— TTCTCCCGGATGGGCAAGGCTGG

36099326

801 Chrl : 36099308- ^— TGGCTTCTCCCGGATGGGCAAGG

36099330

802 Chrl : 36099310- + TTGCCCATCCGGGAGAAGCCAGG

36099332

803 Chrl : 36099311- + TGCCCATCCGGGAGAAGCCAGGG

36099333

804 Chrl : 36099312- + GCCCATCCGGGAGAAGCCAGGGG

36099334

805 Chrl : 36099313- + CCCATCCGGGAGAAGCCAGGGGG

36099335

806 Chrl : 36099313- ^— CCCCCTGGCTTCTCCCGGATGGG

36099335

807 Chrl : 36099314- ^— GCCCCCTGGCTTCTCCCGGATGG

36099336

808 Chrl : 36099318- ^— CTGGGCCCCCTGGCTTCTCCCGG

36099340

809 Chrl : 36099322- + GAGAAGCCAGGGGGCCCAGCAGG

36099344

810 Chrl : 36099323- + AGAAGCCAGGGGGCCCAGCAGGG

36099345 811 Chrl : 36099328- + CCAGGGGGCCCAGCAGGGCCAGG 36099350

812 Chrl : 36099328- ^— CCTGGCCCTGCTGGGCCCCCTGG

36099350

813 Chrl : 36099336- ^— ATGGGCAGCCTGGCCCTGCTGGG

36099358

814 Chrl : 36099337- ^— CATGGGCAGCCTGGCCCTGCTGG

36099359

815 Chrl : 36099338- + CAGCAGGGCCAGGCTGCCCATGG

36099360

816 Chrl : 36099346- + CCAGGCTGCCCATGGAGTCCTGG

36099368

817 Chrl : 36099346- ^— CCAGGACTCCATGGGCAGCCTGG

36099368

818 Chrl : 36099354- ^— TGGGAAAGCCAGGACTCCATGGG

36099376

819 Chrl : 36099355- ^— ATGGGAAAGCCAGGACTCCATGG

36099377

820 Chrl : 36099361- + AGTCCTGGCTTTCCCATGCCTGG

36099383

821 Chrl : 36099364- ^— AAACCAGGCATGGGAAAGCCAGG

36099386

822 Chrl : 36099370- + TTTCCCATGCCTGGTTTTCCTGG

36099392

823 Chrl : 36099371- + TTCCCATGCCTGGTTTTCCTGGG

36099393

824 Chrl : 36099373- ^— TTCCCAGGAAAACCAGGCATGGG

36099395

825 Chrl : 36099374- ^— CTTCCCAGGAAAACCAGGCATGG

36099396

826 Chrl : 36099379- + CCTGGTTTTCCTGGGAAGCCAGG

36099401

827 Chrl : 36099379- ^— CCTGGCTTCCCAGGAAAACCAGG

36099401

828 Chrl : 36099380- + CTGGTTTTCCTGGGAAGCCAGGG

36099402

829 Chrl : 36099381- + TGGTTTTCCTGGGAAGCCAGGGG

36099403

830 Chrl : 36099382- + GGTTTTCCTGGGAAGCCAGGGGG

36099404

831 Chrl : 36099383- + GTTTTCCTGGGAAGCCAGGGGGG

36099405

832 Chrl : 36099388- + CCTGGGAAGCCAGGGGGGCCAGG

36099410

833 Chrl : 36099388- — CCTGGCCCCCCTGGCTTCCCAGG

36099410 834 Chrl : 36099389- + CTGGGAAGCCAGGGGGGCCAGGG 36099411

835 Chrl : 36099390- + TGGGAAGCCAGGGGGGCCAGGGG

36099412

836 Chrl : 36099391- + GGGAAGCCAGGGGGGCCAGGGGG

36099413

837 Chrl : 36099397- ^— CGGGGTCCCCCTGGCCCCCCTGG

36099419

838 Chrl : 36099400- + GGGGGGCCAGGGGGACCCCGAGG

36099422

839 Chrl : 36099405- + GCCAGGGGGACCCCGAGGCCCGG

36099427

840 Chrl : 36099406- + CCAGGGGGACCCCGAGGCCCGGG

36099428

841 Chrl : 36099406- ^— CCCGGGCCTCGGGGTCCCCCTGG

36099428

842 Chrl : 36099415- + CCCCGAGGCCCGGGCTTCCCAGG

36099437

843 Chrl : 36099415- ^— CCTGGGAAGCCCGGGCCTCGGGG

36099437

844 Chrl : 36099416- + CCCGAGGCCCGGGCTTCCCAGGG

36099438

845 Chrl : 36099416- ^— CCCTGGGAAGCCCGGGCCTCGGG

36099438

846 Chrl : 36099417- + CCGAGGCCCGGGCTTCCCAGGGG

36099439

847 Chrl : 36099417- ^— CCCCTGGGAAGCCCGGGCCTCGG

36099439

848 Chrl : 36099418- + CGAGGCCCGGGCTTCCCAGGGGG

36099440

849 Chrl : 36099419- + GAGGCCCGGGCTTCCCAGGGGGG

36099441

850 Chrl : 36099423- + CCCGGGCTTCCCAGGGGGGCCGG

36099445

851 Chrl : 36099423- ^— CCGGCCCCCCTGGGAAGCCCGGG

36099445

852 Chrl : 36099424- + CCGGGCTTCCCAGGGGGGCCGGG

36099446

853 Chrl : 36099424- ^— CCCGGCCCCCCTGGGAAGCCCGG

36099446

854 Chrl : 36099432- ^— AGGGAGAGCCCGGCCCCCCTGGG

36099454

855 Chrl : 36099433- ^— AAGGGAGAGCCCGGCCCCCCTGG

36099455

856 Chrl : 36099437- + GGGGGCCGGGCTCTCCCTTCAGG

36099459 857 Chrl : 36099442- ^— ATGGACCTGAAGGGAGAGCCCGG 36099464

858 Chrl : 36099445- + GGCTCTCCCTTCAGGTCCATCGG

36099467

859 Chrl : 36099451- ^— CTGCTGCCGATGGACCTGAAGGG

36099473

860 Chrl : 36099452- ^— GCTGCTGCCGATGGACCTGAAGG

36099474

861 Chrl : 36099454- + TTCAGGTCCATCGGCAGCAGCGG

36099476

862 Chrl : 36099460- + TCCATCGGCAGCAGCGGTAGAGG

36099482

863 Chrl : 36099461- ^— GCCTCTACCGCTGCTGCCGATGG

36099483

864 Chrl : 36099485- + TTTCTGAGAAAGAAAGAGAAAGG

36099507

865 Chrl : 36099486- + TTCTGAGAAAGAAAGAGAAAGGG

36099508

866 Chrl : 36099487- + TCTGAGAAAGAAAGAGAAAGGGG

36099509

867 Chrl : 36099495- + AGAAAGAGAAAGGGGCAGTCAGG

36099517

868 Chrl : 36099496- + GAAAGAGAAAGGGGCAGTCAGGG

36099518

869 Chrl : 36099497- + AAAGAGAAAGGGGCAGTCAGGGG

36099519

870 Chrl : 36099509- + GCAGTCAGGGGCCTGAACTGTGG

36099531

871 Chrl : 36099510- + CAGTCAGGGGCCTGAACTGTGGG

36099532

872 Chrl : 36099511- + AGTCAGGGGCCTGAACTGTGGGG

36099533

873 Chrl : 36099516- + GGGGCCTGAACTGTGGGGACAGG

36099538

874 Chrl : 36099517- + GGGCCTGAACTGTGGGGACAGGG

36099539

875 Chrl : 36099518- + GGCCTGAACTGTGGGGACAGGGG

36099540

876 Chrl : 36099520- ^— GTCCCCTGTCCCCACAGTTCAGG

36099542

877 Chrl : 36099542- ^— AATGGGGGAATGGGTAGATGGGG

36099564

878 Chrl : 36099543- ^— GAATGGGGGAATGGGTAGATGGG

36099565

879 Chrl : 36099544- — GGAATGGGGGAATGGGTAGATGG

36099566 880 Chrl : 36099551- ^— TCATACTGGAATGGGGGAATGGG 36099573

881 Chrl : 36099552- ^— CTCATACTGGAATGGGGGAATGG

36099574

882 Chrl : 36099553- + CATTCCCCCATTCCAGTATGAGG

36099575

883 Chrl : 36099557- ^— TGTACCTCATACTGGAATGGGGG

36099579

884 Chrl : 36099558- ^— GTGTACCTCATACTGGAATGGGG

36099580

885 Chrl : 36099559- ^— CGTGTACCTCATACTGGAATGGG

36099581

886 Chrl : 36099560- + CCATTCCAGTATGAGGTACACGG

36099582

887 Chrl : 36099560- ^— CCGTGTACCTCATACTGGAATGG

36099582

888 Chrl : 36099561- + CATTCCAGTATGAGGTACACGGG

36099583

889 Chrl : 36099565- ^— CTCTCCCGTGTACCTCATACTGG

36099587

890 Chrl : 36099566- + CAGTATGAGGTACACGGGAGAGG

36099588

891 Chrl : 36099574- + GGTACACGGGAGAGGAAGAATGG

36099596

892 Chrl : 36099575- + GTACACGGGAGAGGAAGAATGGG

36099597

893 Chrl : 36099576- + TACACGGGAGAGGAAGAATGGGG

36099598

894 Chrl : 36099598- + GCTGCCCCTTCCTGCTCTCATGG

36099620

895 Chrl : 36099602- ^— TCTTCCATGAGAGCAGGAAGGGG

36099624

896 Chrl : 36099603- ^— ATCTTCCATGAGAGCAGGAAGGG

36099625

897 Chrl : 36099604- ^— CATCTTCCATGAGAGCAGGAAGG

36099626

898 Chrl : 36099605- + CTTCCTGCTCTCATGGAAGATGG

36099627

899 Chrl : 36099606- + TTCCTGCTCTCATGGAAGATGGG

36099628

900 Chrl : 36099607- + TCCTGCTCTCATGGAAGATGGGG

36099629

901 Chrl : 36099608- ^— ACCCCATCTTCCATGAGAGCAGG

36099630

902 Chrl : 36099612- + CTCTCATGGAAGATGGGGTTTGG

36099634 903 Chrl : 36099613- + TCTCATGGAAGATGGGGTTTGGG 36099635

904 Chrl : 36099614- + CTCATGGAAGATGGGGTTTGGGG

36099636

905 Chrl : 36099615- + TCATGGAAGATGGGGTTTGGGGG

36099637

906 Chrl : 36099618- + TGGAAGATGGGGTTTGGGGGTGG

36099640

907 Chrl : 36099624- + ATGGGGTTTGGGGGTGGCCCAGG

36099646

908 Chrl : 36099625- + TGGGGTTTGGGGGTGGCCCAGGG

36099647

909 Chrl : 36099626- + GGGGTTTGGGGGTGGCCCAGGGG

36099648

910 Chrl : 36099635- + GGGTGGCCCAGGGGACATCTTGG

36099657

911 Chrl : 36099636- + GGTGGCCCAGGGGACATCTTGGG

36099658

912 Chrl : 36099637- + GTGGCCCAGGGGACATCTTGGGG

36099659

913 Chrl : 36099638- + TGGCCCAGGGGACATCTTGGGGG

36099660

914 Chrl : 36099641- ^— TTGCCCCCAAGATGTCCCCTGGG

36099663

915 Chrl : 36099642- ^— GTTGCCCCCAAGATGTCCCCTGG

36099664

916 Chrl : 36099645- + GGGGACATCTTGGGGGCAACAGG

36099667

917 Chrl : 36099646- + GGGACATCTTGGGGGCAACAGGG

36099668

918 Chrl : 36099660- + GCAACAGGGTGTCCTCCTTAAGG

36099682

919 Chrl : 36099661- + CAACAGGGTGTCCTCCTTAAGGG

36099683

920 Chrl : 36099672- ^— GGTGTTAGGAGCCCTTAAGGAGG

36099694

921 Chrl : 36099675- ^— TTGGGTGTTAGGAGCCCTTAAGG

36099697

922 Chrl : 36099685- + TCCTAACACCCAACCTACCTAGG

36099707

923 Chrl : 36099686- ^— GCCTAGGTAGGTTGGGTGTTAGG

36099708

924 Chrl : 36099689- + AACACCCAACCTACCTAGGCTGG

36099711

925 Chrl : 36099690- + ACACCCAACCTACCTAGGCTGGG

36099712 926 Chrl : 36099693- ^— AGGCCCAGCCTAGGTAGGTTGGG 36099715

927 Chrl : 36099694- ^— GAGGCCCAGCCTAGGTAGGTTGG

36099716

928 Chrl : 36099698- ^— GGAGGAGGCCCAGCCTAGGTAGG

36099720

929 Chrl : 36099702- ^— TCATGGAGGAGGCCCAGCCTAGG

36099724

930 Chrl : 36099708- + CTGGGCCTCCTCCATGAGCCTGG

36099730

931 Chrl : 36099713- ^— ATCAGCCAGGCTCATGGAGGAGG

36099735

932 Chrl : 36099716- ^— AGAATCAGCCAGGCTCATGGAGG

36099738

933 Chrl : 36099719- ^— GTGAGAATCAGCCAGGCTCATGG

36099741

934 Chrl : 36099726- ^— ATGAGAGGTGAGAATCAGCCAGG

36099748

935 Chrl : 36099741- ^— TCAGGTCATGCAGGGATGAGAGG

36099763

936 Chrl : 36099744- + CTCATCCCTGCATGACCTGAAGG

36099766

937 Chrl : 36099747- + ATCCCTGCATGACCTGAAGGTGG

36099769

938 Chrl : 36099749- ^— CTCCACCTTCAGGTCATGCAGGG

36099771

939 Chrl : 36099750- ^— ACTCCACCTTCAGGTCATGCAGG

36099772

940 Chrl : 36099752- + TGCATGACCTGAAGGTGGAGTGG

36099774

941 Chrl : 36099759- ^— CTGGTGGCCACTCCACCTTCAGG

36099781

942 Chrl : 36099760- + CTGAAGGTGGAGTGGCCACCAGG

36099782

943 Chrl : 36099763- + AAGGTGGAGTGGCCACCAGGTGG

36099785

944 Chrl : 36099775- ^— GGGCTGCTGGTGCCACCTGGTGG

36099797

945 Chrl : 36099778- ^— GGTGGGCTGCTGGTGCCACCTGG

36099800

946 Chrl : 36099788- ^— CGGGCTCTAAGGTGGGCTGCTGG

36099810

947 Chrl : 36099791- + GCAGCCCACCTTAGAGCCCGTGG

36099813

948 Chrl : 36099792- + CAGCCCACCTTAGAGCCCGTGGG

36099814 949 Chrl : 36099795- ^— GCTCCCACGGGCTCTAAGGTGGG 36099817

950 Chrl : 36099796- ^— TGCTCCCACGGGCTCTAAGGTGG

36099818

951 Chrl : 36099799- ^— CTCTGCTCCCACGGGCTCTAAGG

36099821

952 Chrl : 36099807- ^— AGGTGGGGCTCTGCTCCCACGGG

36099829

953 Chrl : 36099808- ^— GAGGTGGGGCTCTGCTCCCACGG

36099830

954 Chrl : 36099822- ^— AACTGGGAAGTTGGGAGGTGGGG

36099844

955 Chrl : 36099823- ^— GAACTGGGAAGTTGGGAGGTGGG

36099845

956 Chrl : 36099824- ^— TGAACTGGGAAGTTGGGAGGTGG

36099846

957 Chrl : 36099827- ^— AGATGAACTGGGAAGTTGGGAGG

36099849

958 Chrl : 36099830- ^— GGGAGATGAACTGGGAAGTTGGG

36099852

959 Chrl : 36099831- ^— GGGGAGATGAACTGGGAAGTTGG

36099853

960 Chrl : 36099836- + TTCCCAGTTCATCTCCCCCTTGG

36099858

961 Chrl : 36099838- ^— TTCCAAGGGGGAGATGAACTGGG

36099860

962 Chrl : 36099839- ^— CTTCCAAGGGGGAGATGAACTGG

36099861

963 Chrl : 36099850- ^— GCACAGGTGGTCTTCCAAGGGGG

36099872

964 Chrl : 36099851- ^— GGCACAGGTGGTCTTCCAAGGGG

36099873

965 Chrl : 36099852- ^— TGGCACAGGTGGTCTTCCAAGGG

36099874

966 Chrl : 36099853- ^— CTGGCACAGGTGGTCTTCCAAGG

36099875

967 Chrl : 36099863- ^— GTGCAGTTAGCTGGCACAGGTGG

36099885

968 Chrl : 36099866- ^— ACGGTGCAGTTAGCTGGCACAGG

36099888

969 Chrl : 36099872- ^— CTGGAAACGGTGCAGTTAGCTGG

36099894

970 Chrl : 36099873- + CAGCTAACTGCACCGTTTCCAGG

36099895

971 Chrl : 36099881- + TGCACCGTTTCCAGGCCCTCTGG

36099903 972 Chrl : 36099882- + GCACCGTTTCCAGGCCCTCTGGG 36099904

973 Chrl : 36099883- + CACCGTTTCCAGGCCCTCTGGGG

36099905

974 Chrl : 36099885- ^— TACCCCAGAGGGCCTGGAAACGG

36099907

975 Chrl : 36099890- + TCCAGGCCCTCTGGGGTATTAGG

36099912

976 Chrl : 36099891- ^— TCCTAATACCCCAGAGGGCCTGG

36099913

977 Chrl : 36099896- ^— GTTTTTCCTAATACCCCAGAGGG

36099918

978 Chrl : 36099897- ^— TGTTTTTCCTAATACCCCAGAGG

36099919

979 Chrl : 36099904- + GGTATTAGGAAAAACACTGAAGG

36099926

980 Chrl : 36099908- + TTAGGAAAAACACTGAAGGTAGG

36099930

981 Chrl : 36099916- + AACACTGAAGGTAGGAAAATTGG

36099938

982 Chrl : 36099919- + ACTGAAGGTAGGAAAATTGGTGG

36099941

983 Chrl : 36099920- + CTGAAGGTAGGAAAATTGGTGGG

36099942

984 Chrl : 36099921- + TGAAGGTAGGAAAATTGGTGGGG

36099943

985 Chrl : 36099928- + AGGAAAATTGGTGGGGAATGAGG

36099950

986 Chrl : 36099936- + TGGTGGGGAATGAGGAGCTGTGG

36099958

987 Chrl : 36099939- + TGGGGAATGAGGAGCTGTGGAGG

36099961

988 Chrl : 36099940- + GGGGAATGAGGAGCTGTGGAGGG

36099962

989 Chrl : 36099949- + GGAGCTGTGGAGGGCGCCTGAGG

36099971

990 Chrl : 36099958- + GAGGGCGCCTGAGGATCTGATGG

36099980

991 Chrl : 36099965- ^— CTGAGAGCCATCAGATCCTCAGG

36099987

992 Chrl : 36099966- + CTGAGGATCTGATGGCTCTCAGG

36099988

993 Chrl : 36099967- + TGAGGATCTGATGGCTCTCAGGG

36099989

994 Chrl : 36099970- + GGATCTGATGGCTCTCAGGGAGG

36099992 995 Chrl : 36099974- + CTGATGGCTCTCAGGGAGGCAGG 36099996

996 Chrl : 36099975- + TGATGGCTCTCAGGGAGGCAGGG

36099997

997 Chrl : 36099976- + GATGGCTCTCAGGGAGGCAGGGG

36099998

998 Chrl : 36099982- + TCTCAGGGAGGCAGGGGATTTGG

36100004

999 Chrl : 36099983- + CTCAGGGAGGCAGGGGATTTGGG

36100005

1000 Chrl : 36099984- + TCAGGGAGGCAGGGGATTTGGGG

36100006

1001 Chrl : 36099985- + CAGGGAGGCAGGGGATTTGGGGG

36100007

1002 Chrl : 36099989- + GAGGCAGGGGATTTGGGGGCTGG

36100011

1003 Chrl : 36099990- + AGGCAGGGGATTTGGGGGCTGGG

36100012

1004 Chrl : 36100002- + TGGGGGCTGGGAGCGATTTGAGG

36100024

1005 Chrl : 36100010- + GGGAGCGATTTGAGGCACTGTGG

36100032

1006 Chrl : 36100011- + GGAGCGATTTGAGGCACTGTGGG

36100033

1007 Chrl : 36100012- + GAGCGATTTGAGGCACTGTGGGG

36100034

1008 Chrl : 36100017- + ATTTGAGGCACTGTGGGGTGAGG

36100039

1009 Chrl : 36100020- + TGAGGCACTGTGGGGTGAGGAGG

36100042

1010 Chrl : 36100032- + GGGTGAGGAGGCTCTCACCCAGG

36100054

1011 Chrl : 36100038- + GGAGGCTCTCACCCAGGTACTGG

36100060

1012 Chrl : 36100049- ^— GAGGGCAAAGGCCAGTACCTGGG

36100071

1013 Chrl : 36100050- ^— TGAGGGCAAAGGCCAGTACCTGG

36100072

1014 Chrl : 36100053- + GGTACTGGCCTTTGCCCTCACGG

36100075

1015 Chrl : 36100057- + CTGGCCTTTGCCCTCACGGAAGG

36100079

1016 Chrl : 36100058- + TGGCCTTTGCCCTCACGGAAGGG

36100080

1017 Chrl : 36100061- + CCTTTGCCCTCACGGAAGGGCGG

36100083 1018 Chrl : 36100061- ^— CCGCCCTTCCGTGAGGGCAAAGG 36100083

1019 Chrl : 36100067- ^— GTGGGACCGCCCTTCCGTGAGGG

36100089

1020 Chrl : 36100068- ^— TGTGGGACCGCCCTTCCGTGAGG

36100090

1021 Chrl : 36100070- + TCACGGAAGGGCGGTCCCACAGG

36100092

1022 Chrl : 36100084- + TCCCACAGGTCCTTTCTGCATGG

36100106

1023 Chrl : 36100085- + CCCACAGGTCCTTTCTGCATGGG

36100107

1024 Chrl : 36100085- ^— CCCATGCAGAAAGGACCTGTGGG

36100107

1025 Chrl : 36100086- ^— GCCCATGCAGAAAGGACCTGTGG

36100108

1026 Chrl : 36100089- + CAGGTCCTTTCTGCATGGGCTGG

36100111

1027 Chrl : 36100094- ^— TACATCCAGCCCATGCAGAAAGG

36100116

1028 Chrl : 36100103- + ATGGGCTGGATGTACTTCACTGG

36100125

1029 Chrl : 36100104- + TGGGCTGGATGTACTTCACTGGG

36100126

1030 Chrl : 36100105- + GGGCTGGATGTACTTCACTGGGG

36100127

1031 Chrl : 36100126- + GGCATAGCCCGCCGCCCCACCGG

36100148

1032 Chrl : 36100133- ^— GGCGGGGCCGGTGGGGCGGCGGG

36100155

1033 Chrl : 36100134- ^— TGGCGGGGCCGGTGGGGCGGCGG

36100156

1034 Chrl : 36100137- ^— TGGTGGCGGGGCCGGTGGGGCGG

36100159

1035 Chrl : 36100140- ^— CTCTGGTGGCGGGGCCGGTGGGG

36100162

1036 Chrl : 36100141- + CCCACCGGCCCCGCCACCAGAGG

36100163

1037 Chrl : 36100141- ^— CCTCTGGTGGCGGGGCCGGTGGG

36100163

1038 Chrl : 36100142- ^— TCCTCTGGTGGCGGGGCCGGTGG

36100164

1039 Chrl : 36100145- ^— GCGTCCTCTGGTGGCGGGGCCGG

36100167

1040 Chrl : 36100149- — GCGGGCGTCCTCTGGTGGCGGGG

36100171 1041 Chrl : 36100150- ^— CGCGGGCGTCCTCTGGTGGCGGG 36100172

1042 Chrl : 36100151- + CCGCCACCAGAGGACGCCCGCGG

36100173

1043 Chrl : 36100151- ^— CCGCGGGCGTCCTCTGGTGGCGG

36100173

1044 Chrl : 36100154- ^— GGGCCGCGGGCGTCCTCTGGTGG

36100176

1045 Chrl : 36100157- ^— TGTGGGCCGCGGGCGTCCTCTGG

36100179

1046 Chrl : 36100167- ^— GGTGCTGGGGTGTGGGCCGCGGG

36100189

1047 Chrl : 36100168- ^— TGGTGCTGGGGTGTGGGCCGCGG

36100190

1048 Chrl : 36100174- ^— TGGTGCTGGTGCTGGGGTGTGGG

36100196

1049 Chrl : 36100175- ^— CTGGTGCTGGTGCTGGGGTGTGG

36100197

1050 Chrl : 36100180- ^— TGCTACTGGTGCTGGTGCTGGGG

36100202

1051 Chrl : 36100181- ^— CTGCTACTGGTGCTGGTGCTGGG

36100203

1052 Chrl : 36100182- ^— GCTGCTACTGGTGCTGGTGCTGG

36100204

1053 Chrl : 36100188- ^— GCTGCTGCTGCTACTGGTGCTGG

36100210

1054 Chrl : 36100194- ^— TTCGCTGCTGCTGCTGCTACTGG

36100216

1055 Chrl : 36100200- + GCAGCAGCAGCAGCGAAGACAGG

36100222

1056 Chrl : 36100201- + CAGCAGCAGCAGCGAAGACAGGG

36100223

1057 Chrl : 36100202- + AGCAGCAGCAGCGAAGACAGGGG

36100224

1058 Chrl : 36100222- + GGGTGTCAGAGTCCCCAGCATGG

36100244

1059 Chrl : 36100231- + AGTCCCCAGCATGGCGTCCGTGG

36100253

1060 Chrl : 36100234- ^— CGTCCACGGACGCCATGCTGGGG

36100256

1061 Chrl : 36100235- ^— ACGTCCACGGACGCCATGCTGGG

36100257

1062 Chrl : 36100236- ^— CACGTCCACGGACGCCATGCTGG

36100258

1063 Chrl : 36100248- — TCTTCTTTGCAGCACGTCCACGG

36100270 [00232] Use of gRNAs comprising guide sequences complementary to SEQ ID NOs: 191-1063, or that bind the reverse compliment of SEQ ID NOs: 191-1063 would be expected to target an nuclease (e.g., Cas9 or Cas9 RNP)to sequences of COL8A2. As heterozygous mutants of COL8A2 have been characterized in early -onset FECD, targeting a Cas RNP with a gRNA comprising a guide sequence complementary to a target sequence of SEQ ID NOs: 191-1063 could lead to the creation of indels via NHEJ. The generation of indels could decrease the expression of COL8A2, thereby decreasing the resulting toxic alpha-2 subunit of the collagen-8 protein. A decrease in the toxic COL8A2 product may improve the disease course of early-onset FECD, as other forms of collagen may take the place of the alpha-2 subunit. Certain guides may also be useful for excising the region of the COL8A2 gene that contains known disease-associated mutations, or changing the splicing pattern to favor isoforms that do not contain such mutations. Knockout of the COL8A2 gene using certain guides could also be used in conjunction with a wild type COL8A2 replacement strategy. For example the wild type COL8A2 coding sequence could be expressed via transgenic means, after removing expression of the endogenous, dominant-negative mutant form.

[00233] Based on the differences in nucleotide sequences for the mutant alleles, target sequences specific to the mutant alleles were also identified.

[00234] Table 4 lists target sequences specific for mutations leading to Gln455Lys, caused by the c. l364C>A nucleotide change. Use of gRNA comprising guide sequences complementary to SEQ ID NOs: 1064-1069 would target to the mutant allele, while not targeting or targeting less efficiently to the wild type allele. As individuals with the Gln455Lys mutation usually have only one affected allele, selective generation of indels due to NHEJ mediated by a Cas RNP targeted to the mutant allele of COL8A2 would be expected to only cause loss of this allele while preserving the other wild type COL8A2 allele. Alternatively, a gRNA comprising guide sequences complementary to SEQ ID NOs: 1064-1069, or guide sequences that bind to the reverse compliment of SEQ ID NOs: 1064-1069 also could be used together with a template to mediate correction of the mutation.

Table 4: Target sequences for COL8A2with Gln455Lys Mutation

SEQ ID No Target Target

Location Strand Target Sequence

1064 Chrl : 36098302

-36098324 + CCCCTCAGGCCAGGCTTCCCAGG

1065 Chrl : 36098302

-36098324 CCTGGGAAGCCTGGCCTGAGGGG

1066 Chrl : 36098303

-36098325 + CCCTCAGGCCAGGTTGCCCAGGG

1067 Chrl : 36098303

-36098325 CCCTGGGAAGCCTGGCCTGAGGG

1068 Chrl : 36098304

-36098326 TCCCTGGGAAGCCTGGCCTGAGG

1069 Chrl : 36098311

-36098333 TTGGGGCTCCCTGGGAAGCCTGG

[00235] Table 5 lists target sequences specific for a point mutation leading to Gln455Val, caused by the c. l 363-1364CA>GT nucleotide changes. Use of gRNA comprising guide sequences that directs a nuclease to SEQ ID NOs: 1070-1075 would target to the mutant allele, while not targeting or targeting less efficiently to the wild type allele. As individuals with the Gln455Val mutation usually have only one affected allele, selective generation of indels due to NHEJ mediated by a nuclease (e.g., Cas RNP) targeted to the mutant allele of COL8A2 would be expected to only cause loss of this allele while preserving the other wild type COL8A2 allele. Alternatively, a gRNA comprising guide sequences complementary to SEQ ID NOs: 1070-1075 also could be used together with a template to mediate correction of the mutation.

Table 5: Target sequences for COL8A2with Gln455Val Mutation

SEQ ID Target Target

No Location Strand Target Sequence

1070 Chrl : 36098302- 36098324 + CCCCTCAGGCCAGGCACCCCAGG

1071 Chrl : 36098302- 36098324 CCTGGGGTGCCTGGCCTGAGGGG

1072 Chrl : 36098303- 36098325 + CCCTCAGGCCAGGCACCCCAGGG

1073 Chrl : 36098303- 36098325 CCCTGGGGTGCCTGGCCTGAGGG

1074 Chrl : 36098304- 36098326 TCCCTGGGGTGCCTGGCCTGAGG

1075 Chrl : 36098311- 36098333 TTGGGGCTCCCTGGGGTGCCTGG

[00236] Table 6 lists target sequences specific for a point mutation leading to Leu450Trp, caused by the c. l 349T>G nucleotide change. Use of gRNA comprising guide sequences complementary to SEQ ID NOs: 1076-1084 would target to the mutant allele, while not targeting or targeting less efficiently to the wild type allele. As individuals with the Leu450Trp mutation usually have only one affected allele, selective generation of indels due to NHEJ mediated by a Cas RNP targeted to the mutant allele of COL8A2 would be expected to only cause loss of this allele while preserving the other wild type COL8A2 allele. Alternatively, a gRNA comprising guide sequences complementary to SEQ ID NOs: 1076-1084 also could be used together with a template to mediate correction of the mutation.

Table 6: Target sequences for COL8A2with Leu450Trp Mutation

SEQ Target Location Target

ID Strand

No Target Sequence

1076 Chrl : 36098311-36098333 _ TGGGGGCTCCCTGGGCAGCCTGG

1077 Chrl : 36098319-36098341 _ AAGGTGACTGGGGGCTCCCTGGG

1078 Chrl : 36098320-36098342 _ AAAGGTGACTGGGGGCTCCCTGG

1079 Chrl : 36098328-36098350 TGGGGCAGAAAGGTGACTGGGGG

1080 Chrl : 36098329-36098351 CTGGGGCAGAAAGGTGACTGGGG

1081 Chrl : 36098330-36098352 CCCAGTCACCTTTCTGCCCCAGG

1082 Chrl : 36098330-36098352 CCTGGGGCAGAAAGGTGACTGGG

1083 Chrl : 36098331-36098353 CCAGTCACCTTTCTGCCCCAGGG

1084 Chrl : 36098331-36098353 - CCCTGGGGCAGAAAGGTGACTGG

[00237] A template could be used together with a Cas RNP to correct a nucleotide mutation that leads to generation of collagen VIII with either a Gln455Lys, Gln455Val, or Leu450Trp mutation. In this way, the Cas RNP could target to the mutation, initiate NHEJ, and then mediate correction of the mutation based on an exogenous template. Targeting of a Cas RNP to correct mutations leading to expression of a Gln455Lys product could be done using a gRNA comprising a guide sequence complementary to a target sequence of SEQ ID NOs: 1064-1069 together with a template. Targeting of a Cas RNP to correct mutations leading to expression of a Gln455Val product could be done using a gRNA comprising a guide sequence complementary to a target sequence of SEQ ID NOs: 1070-1075 together with a template. Targeting of a Cas RNP to correct mutations leading to expression of a Leu450Trp gene product could be done using a gRNA comprising a guide sequence complementary to a target sequence of SEQ ID NOs: 1076-1084 together with a template. In this manner, selective editing of the mutant allele could be performed to correct defective collagen VIII caused by either Gln455Lys, Gln455Val, or Leu450Trp.

[00238] Thus, use of Cas RNP comprising gRNAs comprising guide sequences complementary to target sequences of COL8A2 may be novel means to treat FECD or PPCD. Target sequences include those to wild type COL8A2 as well as target sequences specific to mutations that can cause a mutant allele of COL8A2 and lead to gene products with Gln455Lys, Gln455Val, or Leu450Trp mutations. Mutation-specific target sequences listed in Tables 4, 5, and 6 can be used to develop guide RNAs for use with Cas (e.g., in Cas RNPs)with specificity for introducing further mutations in the mutant allele to eliminate its function or, alternatively, to use together with a template to correct the causative nucleotide mutation in COL8A2.

EQUIVALENTS

[00239] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and Examples detail certain embodiments and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the embodiment may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[00240] As used herein, the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.