FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant No. HR0011- 17-2-0049 awarded by the Department of Defense, and Grant Nos. HG009490, EB022376, GM118062, CA014051, and GM095450 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases, for example, those caused by point mutations. Point mutations represent the majority of known human genetic variants associated with disease (1). Developing robust methods to introduce and correct point mutations is therefore an important challenge to understand and treat diseases with a genetic component.

[0003] Engineered base editors have been recently developed (2, 3). Base editors are fusions of catalytically disabled Cas moiety and a nucleobase modification enzyme (e.g., natural or evolved nucleobase deaminases). In some cases, base editors may also include proteins that alter cellular DNA repair processes to increase the efficiency and stability of the resulting single-nucleotide change, e.g., a UGI domain (2, 3).

[0004] Two classes of base editors have been generally described to date: cytidine base editors convert target C G base pairs to T A base pairs, and adenine base editors convert A T base pairs to G C base pairs. Collectively, these two classes of base editors enable the targeted installation of all four transition mutations (C-to-T, G-to-A, A-to-G, and T-to-C), which collectively account for about 61% of known human pathogenic small nucleotide polymorphisms (SNPs) in the ClinVar database. In addition, base editors have been used widely in organisms ranging from prokaryotes to plants to amphibians to mammals, and have even been used to correct pathogenic mutations in human embryos (4- 18).

[0005] However, the utility of base editing is limited by several constraints, including the PAM requirement imposed by the particular Cas moiety used (e.g., naturally occurring Cas9 from S. pyogenes, or a modified version thereof, or a homolog thereof), off-target base editing of non-target nucleotides nearby the desired editing site, the production of undesired edited genomic byproducts (e.g., indels), and overall low editing efficiencies.

[0006] The development of“next-generation” base editors has begun to address some of these limitations, including base editors with different or expanded PAM compatibilities (19-21), highfidelity base editors with reduced off-target activity (20, 22-25), base editors with narrower editing windows (normally ~5 nucleotides wide) (19), and a cytidine base editor (BE4) with reduced by-products (6).

[0007] Nevertheless, despite these recent advances, the efficiency of base editing by base editors varies widely by among other factors, cell type and target locus. Thus, there continues to be a significant need in the art for the development of base editors with improved editing efficiencies, and in particular, wherein the improvements are aimed to address those fundamental underlying biological aspects which restrict the genome editing efficiencies of base editor systems. The present disclosure provides improved base editors which overcome the problems in the art.

SUMMARY OF THE INVENTION

[0008] The instant specification provides for improved base editors which overcome deficiencies of those in art. In particular, the specification provides base editors with improved editing efficiencies, for example, wherein the improvements address underlying biological aspects that limit the efficiency of genome editing achieved by existing base editor systems, including, for example, improved expression and/or nuclear localization. In addition, the instant specification provides for nucleic acid molecules encoding and/or expressing the improved base editors disclosed herein, as well as vectors for cloning and/or expressing the improved base editors described herein, host cells comprising said nucleic acid molecules and cloning and/or expression vectors, and compositions for delivering and/or administering nucleic acid-based embodiments described herein. In addition, the disclosure provides for improved base editors as described herein, as well as compositions comprising said improved base editors. Still further, the present disclosure provides for methods of making the base editors, as well as methods of using the improved base editors or nucleic acid molecules encoding the improved base editors in applications including editing a nucleic acid molecule, e.g., a genome, with improved efficiency as compared to base editor that forms the state of the art. The specification also provides methods for efficiently editing a target nucleic acid molecule, e.g., a single nucleobase of a genome, with a base editing system described herein (e.g., in the form of an improved base editor protein as described herein or a vector encoding same) and conducting based editing. Still further, the

specification provides therapeutic methods for treating a genetic disease and/or for altering or changing a genetic trait or condition by contacting a target nucleic acid molecule, e.g., a genome, with a base editing system (e.g., in the form of an isolated improved base editor protein or a vector encoding same) and conducting base editing to treat the genetic disease and/or change the genetic trait (e.g., eye color).

[0009] The present inventors have surprisingly discovered various ways to improve the efficiency of base editing by recognizing that the fraction of cells expressing active base editors, and/or the amount of functional base editor protein produced by each cell, constitutes restrictions on the efficiency of base editing. In particular, the inventors have surprisingly discovered that by (a) improving nuclear localization of the expressed base editor or component thereof to the nucleus, (b) optimizing codon usage of the sequence encoding the base editor or component thereof, and (c) enhancing the expression of the sequence encoding the base editor or component thereof, or a combination thereof, e.g., by ancestral protein reconstruction (ASR), significantly improves the editing efficiencies of previously known base editors, e.g., cytidine base editors. Ancestral protein reconstruction uses an alignment of known protein sequences, an evolutionary model, and a resulting phylogenetic tree to infer ancestral protein sequences at the nodes of the phylogeny. See, Harms, M.J. et ah, “Evolutionary biochemistry: revealing the historical and physical causes of protein properties.” Nature reviews. Genetics 14, 559-571 (2013); the entire contents of which are incorporated herein by reference. Indeed, ASR has been shown to improve the expression of a variety of proteins while retaining wild-type levels of biochemical activity. See, Wheeler, L.C., et ah,“The thermostability and specificity of ancient proteins.” Curr Opin Struct Biol 38, 37-43 (2016); Nguyen, V. et ah,“Evolutionary drivers of thermoadaptation in enzyme catalysis.” Science 355, 289-294 (2017); Wilson, C. et al.“Kinase dynamics. Using ancient protein kinases to unravel a modern cancer drug's mechanism.” Science 347, 882-886 (2015); and Risso, V.A., et ah,“Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian beta-lactamases.” J Am Chem Soc 135, 2899-2902 (2013); the entire contents of each of which are incorporated herein by reference. [0010] These methods can be used to provide improved base editors that can be used to efficiently edit a nucleic acid molecule in a manner that is dramatically improved as compared to base editors known in the art. The improved base editors may be used to efficiently edit nucleic acid molecules, e.g., a genome, for example, by correcting a disease- causing point mutation.

[0011] Thus, in one aspect, the specification discloses a fusion protein comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp); (ii) a DNA effector domain; (iii) a first nuclear localization sequence; and (iv) a second nuclear localization sequence. In certain embodiments, the first nuclear localization sequence (NLS) and/or the second nuclear localization sequence is a bipartite nuclear localization sequence, for example a bipartite nuclear localization sequence that comprises the amino acid sequence of

KRT ADGS EFES PKKKRKV (SEQ ID NO: 1) or KRT ADGS EFEPKKKRKV (SEQ ID NO: 2). Nuclear localization sequences may be at the N-terminus, and/or the C-terminus of the fusion proteins (e.g., base editors) provided herein. For example, any of the fusion proteins provided herein may have an N-terminal and a C-terminal NLS.

[0012] It should be appreciated that any of the fusion proteins provided herein contain a nucleic acid programmable DNA binding protein, such as a Cas9 domain, in order to bring the fusion protein in proximity to a target nucleic acid sequence (e.g., for the purposes of base editing). The nucleic acid programmable DNA binding protein may be a Cas9 domain, such as a Cas9 nickase domain. For example, the Cas9 nickase domain may be a Cas9 nickase that cuts a nucleic acid target strand of a nucleotide duplex, where the nucleotide target strand is the strand that binds a gRNA. As one example, the Cas9 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of:

MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK VMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKY S LFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 3).

[0013] In some aspects, the fusion proteins provided herein include an effector domain that is capabable of making a modification to a nucleic acid (e.g., DNA). For example, the DNA effector domain may be a deaminase domain, such as a cytidine deaminase domain or an adenosine deaminase domain. In certain embodiments, the deaminase domain is a cytidine deaminase domain, such as an APOBEC or AID cytidine deaminase. For base editing proteins that are capable of deaminating a cytidine to a uridine, e.g., to induce a C to T mutation in a DNA molecule, the cytidine deaminase can be a deaminase from the apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. For example, the cytidine deaminase may comprise an APOBEC cytidine deaminase having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of:

S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKETCLLYEINW GGRHS IWRHT S QNTN KH VE VNFIEKFTTERYFCPNTRCS GG WFLS WS PC GEC S RAITEFLS R YPH VTLFIYIARL YHH ADPRNRQGLRDLIS S G VTIQIMTEQES G Y C WRNFVN Y S PS NE AHWPR YPHLW V RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 4). The cytidine deaminase may also be an ancestral cytidine deaminase, such as any of the Anc689, Anc687, Anc686, Anc655, or Anc733 ancestral cytidine deaminases provided herein (e.g., any one of SEQ ID NOs: 5-9)

[0014] The fusion proteins provided herein, e.g., those that comprise two or more NLSs, may further include one or more Uracil-DNA glycosylase inhibitor (UGI) domains, which are capable of inhibiting Uracil-DNA glycosylase, thereby improving base editing efficiency of C to T base editor proteins. As one example, any of the fusion proteins provided herein comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of

TNLS DIIEKET GKQLVIQES ILMLPEE VEE VIGNKPES DILVHT A YDES TDEN VMLLT S D APEYKPW ALVIQDSNGENKIKML (SEQ ID NO: 108).

[0015] It should be appreciated that the fusion proteins provided herein may be arranged in any conifguation, for example, the fusion protein may have the structure: NH ₂ - [first nuclear localization sequence] -[cytidine deaminase domain]-[Cas9 domain] -[first UGI domain]-[second UGI domain] -[second nuclear localization sequence] -COOH, and each instance of comprises an optional linker. Linker sequences that may be used to link certain domains of the fusion protein are provided herein and may be modified to enhance the properties of the fusion proteins herein, such as base editing efficiency or modulating a base editing window.

[0016] In some aspects, any of the fusion proteins provided herein have an effector domain that includes an adenosine deaminase. Such fusion proteins may be used as adenosine base editing proteins, e.g., for generating an A to G mutation in a DNA molecule. Accordingly, in certain embodiments, the effector domain comprises an adenosine deaminase, for example an adenosine deaminase that deaminates an adenine in DNA.

Adenosine deaminases that deaminate adenine in DNA have been described previously, for example in PCT/US2017/045381 (published as WO 2018/027078).

[0017] In certain embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15, and includes one or more substitutions that confers the ability of the adenosine deaminase to deaminate adenine in DNA. For example, In certain embodiments, said one or more substitutions comprise a group of substitutions selected from the groups of substitutions consisting of: (i) W23L, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, A142N, S 146C, D147Y, R152P, E155V, I156F, and K157N; (ii) W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N; (iii) H36L, P48S, R51L, L84F, A106V, D108N, H123Y, A142N, S146C, D147Y, E155V, I156F, and K157N; (iv) H36L, P48S, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N; (v) H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N; (vi) L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F; (vii) A106V, D108N, D147Y, and E155V; (viii) A106V and D108N; and (ix) D108N; of the amino acid sequence of SEQ ID NO: 15. It should be appreciated the the fusion proteins comprising an adenosine deaminase may further comprise a second adenosine deaminase, e.g., a TadA adenosine deaminase as set forth in SEQ ID NO: 15. Without wishing to be bound by any particular theory, dimerization of adenosine deaminase domains may improve base editing efficiency of any of the fusion proteins provided herein. As one example, the fusion protein may comprises the structure: NH ₂ -[first nuclear localization sequence] -[first adenosine deaminase] -[second adenosine deaminase]- [Cas9 domain]-[second nuclear localization sequence] -COOH, and each instance of comprises an optional linker.

[0018] Some aspects of the disclosure provide nucleic acid sequences, e.g., DNA sequences encoding any of the fusion proteins, fusion protein domains (e.g., effector domains, napDNAbps, UGI domains) or linkers provided herein. In some embodiments, the DNA sequences are sequence optimized for expression in one or more cell types. For example, the DNA sequences may be optimized for expression in a mammalian cell (e.g., a HEK 293T cell). It should be appreciated that optimizing the codon usage of base editor constructs can greatly improve base editing efficiency. The DNA sequences may be codon optimized for expressin in a mammalian cell using Integrated DNA Technologies (IDT), GeneArt, Coller, and GenScript. Preferably, DNA sequences are codon optimized for expressin in a mammalian cell usingGenScript. As one example

[0019] In one aspect, the specification discloses a complex comprising any one of the presently disclosed fusion proteins and an RNA bound to the napDNAbp. In certain embodiments, the RNA is a guide RNA (gRNA). In certain embodiments, the RNA is a single guide RNA (sgRNA). In certain embodiments, the RNA comprises a nucleic acid sequence that targets SCN9a, MPDU1, or HBG

[0020] In one aspect, the specification discloses a method comprising contacting a nucleic acid (e.g., double stranded DNA) molecule with any of the presently disclosed complexes. The DNA may include a target sequence associated with a disease or disorder that may be corrected by contacting the complex with the DNA. In certain embodiments, the target sequence comprises a point mutation associated with a disease or disorder. For example, the target sequence may have a T to C point mutation associated with a disease or disorder, where the deamination of the mutant C base results in a sequence that is not associated with a disease or disorder. In certain embodiments, the target sequence comprises a G to A point mutation associated with a disease or disorder, where the deamination of the mutant A base results in a sequence that is not associated with a disease or disorder. The methods provided herein can be performed in vitro, such as in cell culture, or in vivo, such as in a subject.

[0021] In certain embodiments, the subject has been diagnosed with a disease or disorder. In certain embodiments, the disease or disorder is selected from the group consisting of congenital disorder of glycosylation type lf, familial erythromyalgia, paroxysomal extreme pain disorder, chronic insensitivity to pain, sickle cell anemia, and b- thalassemia. In certain embodiments, the disease or disorder is associated with a point mutation in a MDPU1 gene, a SCN9a gene or an HBG1 and/or an HBG2 gene.

[0022] In one aspect, the specification discloses pharmaceutical compositions comprising any of the presently disclosed fusion proteins, complexes, nucleic acids, and/or vectors. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, a lipid (e.g., a cationic lipid), and/or a polymer.

[0023] In one asepect, the disclosure provides ancestral cytidine deaminases, such as Anc689, Anc687 Anc686, Anc655, and Anc733. In another aspect, the specification provides ancestral cytidine deaminases that comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 5-9. In other embodiments, the ancestral cytidine deaminases comprise an amino acid sequence set forth in any one of SEQ ID NOs: 5-9. The application also provides for DNA sequences that encode such ancestral cytidine deaminases.

[0024] It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non limiting embodiments when considered in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIGs. 1A-1E. Probing the factors that limit base editing efficiency in human cells. (FIG. 1A), BE4 (left) and ABE (right) induce the deamination of target C or target A nucleotides. They also nick the non-edited strand to direct DNA repair processes to replace that strand using the deaminated C (uracil, U) or the deaminated A (inosine, I) as a template. The result is BE4-mediated conversion of a target C.G base pair to a T·A base pair, and ABE-mediated conversion of a target A·T base pair to a G.C base pair. (FIG. 1B) Base pair changes required to correct pathogenic SNPs in the ClinVar database. The“1” wedge (47%) require conversion of the type mediated by ABE, while the“2” wedge (14%) require conversion of the type mediated by BE4. (FIG. 1C) Three base editor and fluorescent protein construct pairs used to elucidate the relationship between base editor expression and editing efficiency in human cells. All samples were transfected with an mCherry expression plasmid as a transfection control. In addition, cells were transfected with a BE4 expression plasmid (“in trans”), with a plasmid co-expressing both BE4 and GFP on separate promoters (“in cis”), or with a plasmid expressing BE4-P2A-GFP, where P2A is a self-cleaving peptide that liberates free BE4 and free GFP during translation of a single mRNA (“P2A”). (FIG. 1D) Percent mCherry-positive or GFP-positive HEK293T cells 3 days after transfection of the construct pairs in (FIG. 1C). (FIG. 1E) Target OG-to-T*A editing efficiency for unsorted HEK293T cells and sorted populations of HEK293T cells. The sorted in trans cells were mCherry-positive, while the sorted in cis and P2A cells were dual mCherry-positive and GFP-positive. Values and error bars in (FIG. 1D) and (FIG. 1E) represent the mean and standard deviation of three biological replicates 3 days after transfection.

[0026] FIGs. 2A-2E. Optimization of the BE4 OG-to-T.A base editor by improving nuclear localization, improving codon usage, and performing ancestral protein reconstruction of cytidine deaminases. (FIG. 2A) BE4 architecture and effects of six NLS configurations on BE4 base editing efficiency at five endogenous genomic loci in HEK293T cells. (FIG. 2B) Effects of five codon usage methods on base editing efficiency of bis-bpNLS-BE4 at five endogenous genomic loci in HEK293T cells. Codon optimizations are as follows: IDT, Integrated DNA Technologies; JC, Jeff Coller; GA, GeneArt; GS, GenScript; IDT-GS, IDT APOBEC + GenScript Cas9 nickase. (FIG. 2C) Phylogenetic tree for ancestral APOBEC reconstruction. Numbered dots denote ancestral APOBEC sequences assayed for base editing activity in (FIG. 2D). (FIG. 2D) Base editing activity of bis-bpNLS-BE4 constructs with GenScript codon optimization using the ancestral APOBEC domains in (FIG. 2C) at five endogenous genomic loci in HEK293T cells. (FIG. 2E) Comparison of BE4, bis-bpNLS-BE4 with GenScript codons (BE4max), and bis-bpNLS-BE4 with ancestral Anc689 APOBEC and GenScript codons (AncBE4max) at three endogenous genomic loci in HEK293T cells across eight different plasmid doses. Values and error bars in (FIGs. 2A-2B) and (FIGs. 2D-2E) represent the mean and standard deviation of three biological replicates 3 days after transfection, except two replicates were used for wild-type rat APOBEC 1 in (FIG. 2D).

[0027] FIGs. 3A-3B. Optimization of the ABE 7.10 A»T-to-G*C base editor by improving nuclear localization and improving codon usage. (FIG. 3A) ABE architecture, effects of NLS configuration (SV40 versus bis-bpNLS), and effects of codon usage (IDT versus GenScript) on ABE base editing efficiency at five endogenous genomic loci in HEK293T cells. BP: bis-bpNLS; GS: GenScript codon usage. (FIG. 3B) Comparison of previously reported ABE 7.10, bis-bpNLS-ABE with IDT codons, and bis-bpNLS-ABE with GenScript codons (ABEmax) at three endogenous genomic loci in HEK293T cells across eight different plasmid doses. Values and error bars represent the mean and standard deviation of three biological replicates 3 days after transfection, except two biological replicates were obtained for the 750 ng dose of bis-bpNLS ABE 7.10 at Site 5 and Site 13 in FIG. 3B.

[0028] FIGs. 4A-4D. Comparison of optimized AncBE4max, BE4max, and ABEmax base editors with previously reported BE4 and ABE 7.10 for the correction of pathogenic SNPs. (FIG. 4A) OG to T·A editing outcomes for the correction of the Leul l9Pro T>C mutation in MDPU1 driving congenital disorder of glycosylation (CDG) type lf by BE4, BE4max, or AncBE4max, in unsorted or sorted patient-derived fibroblasts. Among sorted cells, BE4 samples were sorted for mCherry-positive cells, while BE4max-P2A-GFP and AncBE4max-P2A-GFP samples were sorted for GFP-positive cells. All other OG to T·A edits in the editing window are silent and are not shown. (FIG. 4B) G to T·A editing outcomes for editing the 3’ splice acceptor in intron 6 of SCN9a in mouse N2a cells, unsorted or sorted as described in (FIG. 4A). (FIG. 4C) A·T to G*C editing outcomes for the installation of activating mutations at protospacer positions A5 and A8 (-116 A to G and -113 A to G) in the Bell la binding sites of both HBG1 and HBG2 fetal hemoglobin promoters by ABE 7.10 or ABEmax in unsorted or sorted HEK293T cells. Among sorted cells, ABE samples were sorted for mCherry-positive cells, and ABEmax-P2A-GFP samples were sorted for GFP-positive cells. (FIG. 4D) A·T to G*C editing outcomes for the installation of a mutation at protospacer position A3 (HBG -175 T to C) thought to be the strongest SNP known to mediate the activation of fetal hemoglobin expression ⁴⁸ by ABE 7.10 or ABEmax in HEK293T cells, unsorted or sorted as described in (FIG. 4C). Values and error bars represent the mean and standard deviation of three biological replicates 3 days after transfection, except two biological replicates were obtained for AncBE4max for the SCN9a target.

[0029] FIGs. 5A-5B. Unpaired two-sided t-test p-values for NFS and codon optimizations. (FIG. 5A) Unpaired two-sided t-test p-values for previously reported BE4 (C- terminal SV40 NFS, IDT codons) compared to bis-bpNFS BE4 (IDT codons) at all Cs within the activity window across five endogenous genomic loci in HEK293T cells transfected with 750 ng of base editor plasmid and 250 ng of sgRNA plasmid. (FIG. 5B) Unpaired two-sided t-test p-values for BE4 bis-bpNFS using IDE codons compared to GenScript codons at all Cs within the activity window across five endogenous genomic loci in HEK293T cells transfected with 750 ng of base editor plasmid and 250 ng of sgRNA plasmid. *p<0.05; **p<0.0l.

[0030] FIGs. 6A-6B. C*G-to-T»A base editing outcomes for BE4 variants with chimeric codon usages. (FIG. 6A) C*G-to-T»A base editing outcomes of bis-bpNFS BE4 variants using full-length GenScript codon optimization (BE4max) compared to chimeric constructs in which the APOBEC1 and Cas9 nickase components are constructed with different codon usages in HEK293T cells. Chimeras include (APOBECl-Cas9 nickase): GenScript-IDT, GenScript-Jin Soo Kim, IDT-Jin Soo Kim. (FIG. 6B) Comparison of BE4max and bis-bpNFS BE4 with chimeric IDT-GenScript codon usage at three endogenous genomic loci in HEK293T cells across eight different plasmid doses. Values and error bars represent the mean and standard deviation of three biological replicates 3 days after transfection.

[0031] FIG. 7. Multiple sequence alignment of rat APOBEC1 and reconstructed ancestral cytidine deaminases. Residues are shaded based on the degree of conservation. The sequences from top to bottom correspond to SEQ ID NOs: 4-9.

[0032] FIGs. 8A-8C. Unpaired two-sided t-test p-values for BE4, BE4max, and AncBE4max editing at three genomic loci. (FIG. 8A) Unpaired two-sided t-test p-values for BE4 compared to BE4max at HEK3 (C5), RNF2 (C6), and EMX1 (C4) across eight different base editor plasmid doses in HEK293T cells. (FIG. 8B) Unpaired two-sided t-test p-values for BE4 compared to AncBE4max at HEK3 (C5), RNF2 (C6), and EMX1 (C4) across eight different base editor plasmid doses in HEK293T cells. (FIG. 8C) Unpaired two-sided t-test p- values for BE4max compared to AncBE4max at HEK3 (C5), RNF2 (C6), and EMX1 (C4) across eight different base editor plasmid doses in HEK293T cells. *p<0.05; **p<0.0l;

***p<0.00l; ****p<0.000l.

[0033] FIG. 9. Indel frequencies for BE4, BE4max, and AncBE4 at three genomic loci. Indel frequencies are shown for BE4, BE4max, and AncBE4max at HEK3, RNF2, and EMX1 sites across eight different plasmid doses in HEK293T cells. % Indel and Stdev values represent the mean and standard deviation of three biological replicates 3 days after transfection.

[0034] FIGs. 10A-10D. Improved mRNA levels, protein levels, and base editing from BE4max and AncBE4max compared with BE4. (FIG. 10A) BE4max and AncBE4max result in 3.7- and 5.2-fold higher mRNA levels, respectively, than BE4 in HEK293T cells 3 days after base editor and guide RNA plasmid transfection as determined by qRT-PCR. Base editor mRNA levels were normalized to b-actin levels by AACt. Normalized values were adjusted for transfection efficiency as determined by qPCR amplification of the bGH terminator sequence present on BE4 plasmids. (FIG. 10B) Western blot of C-terminal HA- tagged BE4, BE4max, and AncBE4max in HEK293T cells 3 days after plasmid transfection, visualizing with anti-HA (top) or anti-actin (bottom) antibodies. The mock sample is cells transfected with guide RNA plasmid alone. (FIG. 10C) BE4max-P2A-GFP and

AncBE4max-P2A-GFP show higher GFP and mCherry double-positive cell populations compared to BE4-P2A-GFP for three genomic loci 3 days after transfection. (FIG. 10D) OG-to-T*A base editing outcomes for GFP and mCherry dual-positive cells expressing BE4- P2A-GFP, BE4max-P2A-GFP, and AncBE4max-P2A-GFP at three genomic loci tested. Values and error bars in (FIG. 10A), (FIG. 10C), and (FIG. 10D) represent the mean and standard deviation of three biological replicates 3 days after transfection.

[0035] FIGs. 11A-11B. Unpaired two-sided t-test p-values editing by ABE and ABEmax. (FIG. 11 A) Unpaired two-sided t-test p-values comparing ABE and ABEmax at five genomic loci tested with 750ng of ABE editor and 250ng gRNA in HEK293T cells.

(FIG. 11B) Unpaired two-sided t-test p-values for ABE and ABEmax at Site 5 (A7), Site 13 (A5), and Site 16 (A5) across eight different plasmid doses in HEK293T cells. *p<0.05; **p<0.0l; ***p<0.00l; ****p<0.000l.

[0036] FIG. 12. Indel frequencies for ABE versus ABEmax at three genomic loci. Indel frequencies are shown for ABE and ABEmax at Site 5, Site 13, and Site 16 across eight different plasmid doses in HEK293T cells. % Indel and Stdev values represent the mean and standard deviation of three biological replicates 3 days after transfection.

[0037] FIG. 13. C*G-to-T»A base editing of the 3’ splice acceptor of SCN9a intron 6 in sorted N2a cells. N2a cells were nucleofected with plasmids encoding AncBE4max-P2A- GFP and the targeting sgRNA. Following a 3 day incubation, GFP-positive cells were isolated by FACS and analyzed by HTS. The protospacer of the non-transcribed strand is shown here; C7 corresponds to +1 G and C8 corresponds to -1 G of the 3’ splice acceptor. The PAM is shown as the last GGG in the sequence. The sequence corresponds to SEQ ID NO: 71.

[0038] FIGs. 14A-14C. Nucleofection and transfection efficiencies in type lf fibroblasts, N2a cells, and HEK293T cells for disease-associated targets. (FIG. 14A)

Congenital disorder of glycosylation type lf fibroblasts were nucleofected with a plasmid encoding mCherry, a plasmid encoding the targeting sgRNA, and a plasmid encoding BE4, BE4max-P2A-GFP, or AncBE4max-P2A-GFP, then sorted after 3 days. (FIG. 14B) N2a cells were transfected with a plasmid encoding mCherry, a plasmid encoding the targeting sgRNA, and a plasmid encoding BE4, BE4max-P2A-GFP, or AncBE4max-P2A-GFP, then sorted after 3 days. (FIG. 14C) HEK293T cells were transfected with a plasmid encoding mCherry, a plasmid encoding the targeting sgRNA, and a plasmid encoding ABE or

ABEmax-P2A-GFP, then sorted after 3 days. Values and error bars represent the mean and standard deviation of three biological replicates 3 days after transfection, except two biological replicates were used for AncBE4max-P2A-GFP in N2a cells.

[0039] FIG. 15. 468-taxa unrooted phylogeny of APOBEC homologs. The tree is shaded according to the UNIPROT annotation of sequences within each clade. Dotted lines indicate sequences used as an outgroup to root the tree in FIG. 2C.

[0040] FIG. 16. Flow Sorting HEK293T negative control from FIGs. 1A-1E.

[0041] FIG. 17. Flow Sorting HEK293T GFP+ control from FIGs. 1A-1E.

[0042] FIG. 18. Flow Sorting HEK293T mCherry-i- control from FIGs. 1A-1E.

[0043] FIG. 19. Flow Sorting Example HEK293T In trans sort from FIGs. 1A-1E. [0044] FIG. 20 Flow Sorting Example HEK293T In cis sort from FIGs. 1A-1E.

[0045] FIG. 21. Flow Sorting Example HEK293T P2A sort (B E4-P2 A-GFP) from FIGs. 1A-1E.

[0046] FIG. 22. Flow Sorting Example HEK293T P2A sort (BE4max-P2 A-GFP) from FIGs. 1A-1E.

[0047] FIG. 23. Flow Sorting Example HEK293T P2A sort (AncBE4max-P2 A-GFP) from FIGs. 1A-1E.

[0048] FIG. 24. Flow Sorting N2A negative control, SCN9a sites 1 and 2, from FIGs.

4A-4D.

[0049] FIG. 25. Flow Sorting N2A GFP+ control, SCN9a sites 1 and 2, from FIGs.

4A-4D.

[0050] FIG. 26. Flow Sorting N2A mCherry+ control, SCN9a sites 1 and 2, from FIGs. 4A-4D.

[0051] FIG. 27. Flow Sorting Example N2A BE4 mCherry In trans sort, SCN9a sites 1 and 2, from FIGs. 4A-4D.

[0052] FIG. 28. Flow Sorting Example N2A P2A sort (BE4max-P2 A-GFP) , SCN9a sites 1 and 2, from FIGs. 4A-4D.

[0053] FIG. 29. Flow Sorting Example N2A P2A sort (AncBE4max-P2 A-GFP) , SCN9a sites 1 and 2, from FIGs. 4A-4D.

[0054] FIG. 30. Flow Sorting CGD Type 1F Fibroblast negative control from FIGs.

4A-4D.

[0055] FIG. 31. Flow Sorting CGD Type 1F Fibroblast GFP+ control from FIGs. 4A- 4D.

[0056] FIG. 32. Flow Sorting CGD Type 1F Fibroblast mCherry+ control from FIGs.

4A-4D.

[0057] FIG. 33. Flow Sorting CGD Type 1F Example Fibroblast In trans sort (BE4) from FIGs. 4A-4D.

[0058] FIG. 34. Flow Sorting CGD Type 1F Example Fibroblast P2A sort (BE4max- P2A-GFP) from FIG. 4.

[0059] FIG. 35. Flow Sorting CGD Type 1F Example Fibroblast P2A sort

(AncBE4max-P2A-GFP) from FIGs. 4A-4D. DEFINITIONS

[0060] As used herein and in the claims, the singular forms“a,”“an,” and“the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to“an agent” includes a single agent and a plurality of such agents.

Adenosine deaminase

[0061] As used herein, an“adenosine deaminase” is an enzyme that catalyzes the deamination of adenosine, converting it to the nucleoside hypoxanthine. Under standard Watson-Crick hydrogen bond pairing, an adenosine base hydrogen bonds to a thymine base (or a uracil in case of RNA). When adenine is converted to hypoxanthine, the hypoxanthine undergoes hydrogen bond pairing with cytosine. Thus, a conversion of“A” to hypoxanthine by adenosine deaminase will cause the insertion of“C” instead of a“T” during cellular repair and/or replication processes. Since the cytosine“C” pairs with guanine“G”, the adenosine deaminase in coordination with DNA replication causes the conversion of an A T pairing to a C-G pairing in the double- stranded DNA molecule.

Ancestral sequence reconstruction (ASR)

[0062] Ancestral sequence reconstruction (ASR) is the process of analyzing modern sequences within an evolutionary/phylogenetic context to infer the ancestral sequences at particular nodes of a tree using an ASR algorithm. ASR algorithms are known in the art.

Base editing

[0063] Base editing is a genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain aspects, this can be achieved without requiring double- stranded DNA breaks (DSB). Since many genetic diseases arise from point mutations, this technology has important implications in the study of human health and disease.

[0064] To date, other genome editing techniques, including CRISPR-based systems, begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low (e.g., typically 0.1% to 5%), with the major genome editing products being indels. In order to increase the efficiency of gene correction without simultaneously introducing random indels, the present inventors previously modified the CRISPR/Cas9 system to directly convert one DNA base into another without DSB formation.

Base editors

[0065] The term“base editors (BEs)” or“nucleobase editors (NBEs)” or as used herein, refers to the improved Cas-fusion proteins described herein. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase which still binds DNA in a guide RNA-programmed manner via the formation of an R-loop, but does not cleave the DNA backbone. For example, the dCas9 of the fusion protein can comprise a D10A and a H840A mutation (which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex) as described in PCT/US2016/058344 (published as WO 2017/070632), which is incorporated herein by reference in its entirety. In some

embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase, e.g., a cytidine deaminase (rAPOBECl) which converts a DNA base cytosine to uracil. One such base editor is referred to as“BE1” in the literature. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 fused to a deaminase and further fused to a UGI domain (uracil DNA glycosylase inhibitor, which prevents the subsequent U:G mismatch from being repaired back to a C:G base pair). One such base editor is referred to as“BE2” in the literature. In other embodiments, to improve base editing efficiency, the catalytic His residue at position 840 in the Cas9 HNH domain of BE2 can be restore (resulting in“BE3” as described in the literature), which nicks only the non-edited strand, simulating newly synthesized DNA and leading to the desired U:A product. In other embodiments, the dCas9 is any dCas9 disclosed or described in PCT/US2017/045381 (published as WO

2018/027078), which is incorporated herein by reference in its entirety. The terms “nucleobase editors (NBEs)” and“base editors (BEs)” may be used interchangeably. The term“base editors” encompasses any base editor known or described in the art at the time of this filing, but also the improved base editors described herein. The base editors known in the state of the art which may be modified by the methods and strategies described herein to improve editing efficiency include, for example, BE1, BE2, BE3, or BE4.

Cas9 or Cas9 moiety

[0066] The term“Cas9” or“Cas9 nuclease” or“Cas9 moiety” refers to a CRISPR associated protein 9, or functional fragment thereof, and embraces any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. More broadly, a Cas9 is a type of “RNA-programmable nuclease” or“RNA-guided nuclease” or more broadly a type of “nucleic acid programmable DNA binding protein (napDNAbp)”. The term Cas9 is not meant to be particularly limiting and may be referred to as a“Cas9 or equivalent.”

Exemplary Cas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the improved base editors of the invention.

dCas9

[0067] As used herein, the term“dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a“dCas9 or equivalent.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference.

Cytidine deaminase

[0068] As used herein, a“cytidine deaminase” encoded by the CDA gene is an enzyme that catalyzes the removal of an amine group from cytidine (i.e., the base cytosine when attached to a ribose ring) to uridine (C to U) and deoxycytidine to deoxyuridine (C to U). A non-limiting example of a cytidine deaminase is APOBEC1. Under standard Watson- Crick hydrogen bond pairing, a cytosine base hydrogen bonds to a guanine base. When cytidine is converted to uridine (or deoxycytidine is converted to deoxyuridine), the uridine (or the uracil base of uridine) undergoes hydrogen bond pairing with the base adenine. Thus, a conversion of“C” to uridine (“U”) by cytidine deaminase will cause the insertion of“A” instead of a“G” during cellular repair and/or replication processes. Since the adenine“A” pairs with thymine“T”, the cytidine deaminase in coordination with DNA replication causes the conversion of an C-G pairing to a T· A pairing in the double-stranded DNA molecule.

CRISPR [0069] CRISPR is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that have invaded the prokaryote. The snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively compose, along with an array of CRISPR-associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (me) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently,

Cas9/crRNA/tracrRNA endonucleolytic ally cleaves linear or circular dsDNA target complementary to the RNA. Specifically, the target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 "-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species - the guide RNA. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al, J.J., McShan W.M., Ajdic D.J.,

Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S.,

Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White L, Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001);

“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.”

Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel L, Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA- guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus . Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

Deaminase

[0070] As used herein, the term“deaminase” or“deaminase domain” or“deaminase moiety” refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine ( e.g an engineered adenosine deaminase that deaminates adenosine in DNA). In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is a naturally-occuring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occuring deaminase from an organism that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is 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%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occuring deaminase from an organism. The term deaminase also embraces any genetically engineered deaminase that may comprise genetic modifications (e.g., one or more mutations) that results in a variant deaminase having an amino acid sequence comprising one or more changes relative to a wildtype counterpart deaminase. Examples of deaminases are given herein, and the term is not meant to be limiting.

Effective amount

[0071] The term“effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a base editor may refer to the amount of the base editor that is sufficient to edit a target site nucleotide sequence, e.g., a genome. In some embodiments, an effective amount of a base editor provided herein, e.g., of a fusion protein comprising a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a deaminase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used.

Inhibitor of base repair

[0072] The term“inhibitor of base repair” or“IBR” refers to a protein that is capable of inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair.

Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or a catalytically inactive hAAG.

Isolated

[0073] As used herein, the term“isolated protein” or“isolated nucleic acid” refers to a protein or nucleic acid that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins or nucleic acids from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide or nucleic acid that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein or nucleic acid may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

Linker

[0074] The term“linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of an RNA- programmable nuclease and the catalytic domain of a recombinase. In some embodiments, a linker joins a dCas9 and base editor moiety (e.g., a cytidine or adenosine deaminase).

Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

Mutation

[0075] The term“mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include“loss-of-function” mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. There are some exceptions where a loss-of-function mutation is dominant, one example being haploinsufficiency, where the organism is unable to tolerate the approximately 50% reduction in protein activity suffered by the heterozygote. This is the explanation for a few genetic diseases in humans, including Marfan syndrome which results from a mutation in the gene for the connective tissue protein called fibrillin. Mutations also embrace“gain-of- function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Alternatively the mutation could lead to overexpression of one or more genes involved in control of the cell cycle, thus leading to uncontrolled cell division and hence to cancer. Because of their nature, gain-of-function mutations are usually dominant.

Non-naturally occurring or engineered

[0076] The terms“non-naturally occurring” or“engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides (e.g., Cas9 or deaminases) mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and/or as found in nature (e.g., an amino acid sequence not found in nature).

Nucleic acid/nucleic acid molecule

[0077] The terms“nucleic acid” and“nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments,“nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues.

Nucleic acid programmable R/DNA binding protein (napR/DNAbp)

[0078] The term“nucleic acid programmable D/RNA binding protein

(napR/DNAbp)” refers to any protein that may associate (e.g., form a complex) with one or more nucleic acid molecules (i.e., which may broadly be referred to as a“napR/DNAbp- programming nucleic acid molecule” and includes, for example, guide RNA in the case of Cas systems) which direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the protein to bind to the nucleotide sequence at the specific target site. This term napR/DNAbp embraces CRISPR Cas 9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR- Cas system). Further Cas-equivalents are described in Makarova et al.,“C2c2 is a single component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference. However, the nucleic acid programmable DNA binding protein (napDNAbp) that may be used in connection with this invention are not limited to CRISPR-Cas systems. The invention embraces any such programmable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo) which may also be used for DNA-guided genome editing. NgAgo-guide DNA system does not require a PAM sequence or guide RNA molecules, which means genome editing can be performed simply by the expression of generic NgAgo protein and

introduction of synthetic oligonucleotides on any genomic sequence. See Gao F, Shen XZ, Jiang F, Wu Y, Han C. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat Biotechnol 2016; 34(7):768-73, which is incorporated herein by reference.

napR/DNAbp-programming nucleic acid molecule or guide sequence

[0079] The term“napR/DNAbp-programming nucleic acid molecule” or equivalently “guide sequence” refers the one or more nucleic acid molecules which associate with and direct or otherwise program a napR/DNAbp protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the napR/DNAbp protein to bind to the nucleotide sequence at the specific target site. A non limiting example is a guide RNA of a Cas protein of a CRISPR-Cas genome editing system.

Nuclear localization signal (NLS)

[0080] A nuclear localization signal or sequence (NLS) is an amino acid sequence that tags, designates, or otherwise marks a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus. Thus, a single nuclear localization signal can direct the entity with which it is associated to the nucleus of a cell. Such sequences can be of any size and composition, for example more than 25, 25, 15, 12, 10, 8, 7, 6, 5 or 4 amino acids, but will preferably comprise at least a four to eight amino acid sequence known to function as a nuclear localization signal (NLS).

Nucleobase modification moiety or nucleic acid effector domain

[0081] The term, as used herein,“nucleobase modification moiety” or equivalently a “nucleic acid effector domain” embraces any protein, enzyme, or polypeptide (or functional fragment thereof) which is capable of modifying a DNA or RNA molecule. Nucleobase modification moieties can be naturally occurring, or can be recombinant. For example, a nucleobase modification moiety can include one or more DNA repair enzymes, for example, and an enzyme or protein involved in base excision repair (BER), nucleotide excision repair (NER), homology-dependnent recombinational repair (HR), non-homologous end-joining repair (NHEJ), microhomology end-joining repair (MMEJ), mismatch repair (MMR), direct reversal repair, or other known DNA repair pathway. A nucleobase modification moiety can have one or more types of enzymatic activities, including, but not limited to endonuclease activity, polymerase activity, ligase activity, replication activity, proofreading activity.

Nucleobase modification moieties can also include DNA or RNA-modifying enzymes and/or mutagenic enzymes, such as, DNA methylases and deaminating enzymes (i.e., deaminases, including cytidine deaminases and adenosine deaminases, all defined above), which deaminate nucleobases leading in some cases to mutagenic corrections by way of normal cellular DNA repair and replication processes. The“nucleic acid effector domain” (e.g., a DNA effector domain or an RNA effector domain) as used herein may also refer to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA). Exemplary nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments the nucleic acid editing domain is a deaminase (e.g., a cytidine deaminase, such as an APOBEC or an AID deaminase).

Oligonucleotide/polynucleotide

[0082] As used herein, the terms“oligonucleotide” and“polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments,“nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms“nucleic acid,”“DNA,”“RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

Protein/peptide/polypeptide

[0083] The terms“protein,”“peptide,” and“polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term“fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy- terminal (C-terminal) protein thus forming an“amino-terminal fusion protein” or a“carboxy- terminal fusion protein,” respectively. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain or a catalytic domain of a recombinase. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. It should be appreciated that any of the disclosure provides any of the polypeptide sequences provided herein without an N-terminal methionine (M) residue.

Recombinant

[0084] The term“recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For exampe, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence.

RNA-programmable nuclease/RNA-guided nuclease [0085] The term“RNA-programmable nuclease,” and“RNA-guided nuclease” are used interchangeably herein and refer to a nuclease that forms a complex with ( e.g ., binds or associates with) one or more RNA that is not a target for cleavage (e.g., a Cas9 or homolog or variant thereof). In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though“gRNA” is used interchangeabley to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules.

Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 (or equivalent) complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is homologous to a tracrRNA as depicted in Figure 1E of Jinek et al, Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S.S.N. 61/874,682, filed September 6, 2013, entitled“Switchable Cas9 Nucleases And Uses Thereof,” and U.S. Provisional Patent Application, U.S.S.N. 61/874,746, filed September 6, 2013, entitled“Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an“extended gRNA.” For example, an extended gRNA will, e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example Cas9 (Csnl) from Streptococcus pyogenes (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes .” Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White L, Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference.

[0086] Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA

hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e0047l (2013); Dicarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR- Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

Subject

[0087] The term“subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some

embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.

Target site [0088] The term“target site” refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a fusion protein comprising a deaminase ( e.g ., a dCas9- deaminase fusion protein provided herein).

Uracil glycosylase inhibitor or UGI

[0089] The term“uracil glycosylase inhibitor” or“UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme.

In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 10 or 108. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 10 or 108. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 10 or 108. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 10 or 108, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 10 or 108. In some

embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as“UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 10 or 108. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the

corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 10 or 108. In some embodiments, the UGI comprises the following amino acid sequence:

MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTD EN VMLLT S D APE YKPW ALVIQDS N GENKIKML (SEQ ID NO: 10)

(P 14739 IUN GI_B PPB 2 Uracil-DNA glycosylase inhibitor). Treatment

[0090] The terms“treatment,”“treat,” and“treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms“treatment,” “treat,” and“treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

Variant

[0091] As used herein the term“variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant Cas9 is a Cas9 comprising one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence.

Wild type

[0092] As used herein the term“wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0093] The specification relates to improved base editors that achieve a significant increase in editing efficiencies by making various modifications which address certain underlying biological restrictions (e.g., restricted expression and nuclear localization) in known base editor systems surprisingly found to significantly reduce the efficiency of genome editing that is achievable by base editing. In particular, the inventors surprisingly found that base editor modifications (e.g., base editors comprising improved codon-usage and at least two NLSs) resulting in improved expression and nuclear localization and thereby improved editing efficiencies. [0094] Despite these recent advances in the design of base editors, the efficiency of base editing varies widely. To increase base editing efficiency, the inventors sought to identify the factors that limit base editing efficiency in cells. It was surprisingly found by the inventors that expression and nuclear localization in human cells imposed key bottlenecks on editing efficiency. The inventors discovered that optimizing codon usage, using improved nuclear localization sequences (NLSs) (e.g., at least two NLS moieties), and performing ancestral reconstruction of deaminases resulted in base editors with greatly increased editing efficiency, often more than doubling target nucleotide conversion yields as compared to the unmodified counterpart editors. The resulting base editors were shown, as demonstrated in the Examples, to install point mutations relevant to human disease in a variety of mammalian cell types much more efficiently than previously described base editors. These approaches can be used to provide improved base editors that can be used to efficiently edit a nucleic acid molecule in a manner that is dramatically improved as compared to base editors known in the art. The improved base editors may be used to efficiently edit nucleic acid molecules, e.g., a genome, for example, by correcting a disease-causing point mutation.

[0095] Thus, the instant specification provides improved base editors that comprise one or modifications that result in increased expression (e.g., by way of optimizing codon usage and/or conducting ancestral reconstruction of deaminases) and/or nuclear localization. Further, the specification in certain aspects describes nucleic acid molecules encoding and/or expressing the improved base editors disclosed herein, as well as cloning and/or expression vectors for cloning and/or expressing the improved base editors described herein, host cells comprising said nucleic acid molecules and cloning and/or expression vectors, and compositions for delivering and/or administering nucleic acid-based embodiments described herein. In addition, the disclosure provides for improved base editors, as well as

compositions comprising said improved base editors. Still further, the present disclosure provides for methods of making the improved base editors, as well as methods of using the improved base editors or nucleic acid molecules encoding the improved base editors in applications including editing a nucleic acid molecule, e.g., a genome, with improved efficiency as compared to base editor that forms the state of the art. The specification also provides methods for efficiently editing a target nucleic acid molecule, e.g., a single nucleobase of a genome, with a base editing system described herein (e.g., in the form of an improved base editor protein or a vector encoding same) and conducting based editing. Still further, the specification provides therapeutic methods for treating a genetic disease and/or for altering or changing a genetic trait or condition by contacting a target nucleic acid molecule, e.g., a genome, with a base editing system (e.g., in the form of an improved base editor protein or a vector encoding same) and conducting based editing to treat the genetic disease and/or change the genetic trait (e.g., eye color).

I. IMPROVED BASE-EDITORS

[0096] In various aspects, the instant specification provides improved base editors that comprising one or modifications that result in increased expression (e.g., by way of optimizing codon usage and/or conducting ancestral reconstruction of deaminases) and/or nuclear localization (e.g., by incorporating at least two NLSs). The improved base editors described herein achieve a significant increase in editing efficiencies as compared to unmodified counterparts by making various modifications which address certain fundamental underlying biological restrictions in known base editor systems surprisingly found to significantly reduce the efficiency of genome editing that is achievable by base editing. In particular, the inventors surprising found that base editor modifications resulting in improved expression and nuclear localization specifically resulted in improved editing efficiencies.

[0097] In certain aspects, the methods described herein for modifying and improving base editors begins with a base editor known in the art upon which one or improvements are imparted. The state of the art has described numerous base editors as of this filing. The methods and approaches herein described for improving base editors may be applied to any previously known base editor, or to base editors that may be developed in the further but which lack the beneficial characteristics imparted by the instant methods and modification approaches. Examplary base editors that may be modified by the methods described herein to achieve the improved base editors of the invention can include, for example, those described in the following references and/or patent publications, each of which are incorporated by reference in their entireties: (a) PCT/US2014/070038 (published as

WO2015/089406, June 18, 2015) and its equivalents in the US or around the world; (b) PCT/US2016/058344 (published as W02017/070632, April 27, 2017) and its equivalents in the US or around the world; (c) PCT/US2016/058345 (published as W02017/070633, April 27. 20l7)and its equivalent in the US or around the world; (d) PCT/US2017/045381

(published as WO2018/027078, February 8, 2018) and its equivalents in the US or around the world; (e) PCT/US2017/056671 (published as WO2018/071868, April 19, 2018) and its equivalents in the US or around the world; PCT/2017/048390 (W02017/048390, March 23, 2017) and its equivalents in the US or around the world; (f) PCT/US2017/068114 (not published) and its equivalents in the US or around the world; (g) PCT/US2017/068105 (not published)and its equivalents in the US or around the world; (h) PCT/US2017/046144 (WO2018/031683, February 15, 2018) and its equivalents in the US or around the world; (i) PCT/US2018/024208 (not published) and its equivalents in the US or around the world; (j) PCT/2018/021878 (WO2018/021878, February 1, 2018) and its equivalents in the US and around the world; (k) Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. & Liu, D.R.

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420- (2016); (1) Gaudelli, N.M. et al. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 551, 464- (2017); (m) any of the references listed in this specification entitled“References” and which reports or describes a base editor known in the art.

[0001] In various aspects, the improved or modified base editors described herein have the following generalized structure:

[0002] A - B - C,

[0003] wherein“A” is a Cas moiety or napDNAbp,“B” is nucleic acid effector domain (e.g., a deaminase, such as a cytidine or adenosine deaminase), and“C” is as least two nuclear localization signals (NLS). In addition, the represents a linker that covalently joins moieties A, B, and C. The linkers can be any suitable type (e.g., amino acid sequences or other biopolymers, or synthetic chemical linkages in the case where the moieties are bioconjugated to one another) or length. In addition, a functional improved base editor of the invention would also include one or more“R” or guide sequences (e.g., guide RNA in the case of a Cas9 or Cas9 equivalent) in order to carry out the R/DNA-programmable functionality of base editors for targeting specific sites to be corrected.

[0004] The order of linkage of the moieties is not meant to be particularly limiting so long as the particular arrangement of the elements of moieties produces a functional base editor. That is, the improved base editors of the invention may also include editors represented by the following structures:

[0005] B - A - C;

[0006] B - C - A;

[0007] C - B - A; [0008] C - A - B; and

[0009] A - C - B.

[0010] In some embodiments, the improved base editors provided herein can be made a recombinant fusion protein comprising one or more protein domains, thereby generating an improved base editor. In certain embodiments, the base editors provided herein comprise one or more features that improve the base editing activity (e.g., efficiency, selectivity, and/or specificity) of the base editor proteins. For example, the base editor proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, the base editor proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a T opposite the targeted A.

Mutation of the catalytic residue (e.g., D10 to A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants are able to generate a single strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non- edited strand.

[0011] In some embodiments, any of the base editor proteins provided herein may further comprise one or more additional nucleic acid effector moieties, such as, for example, an inhibitor of inosine base excision repair (e.g., a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease (dISN)). Without wishing to be bound by any particular theory, the UGI domain or dISN may inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which may improve the activity or efficiency of the base editor.

The Cas9 moiety or equivalent protein

[0012] The improved base editors provided by the instant specification include any suitable Cas9 moiety or equivalent protein, such as a CRISPR associated protein 9, or functional fragment thereof, and embraces any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally- occurring or engineered. More broadly, a Cas9 is a type of“RNA-programmable nuclease” or“RNA-guided nuclease” or“nucleic acid programmable DNA-binding protein.” The terms napR/DNAbp or Cas9 are not meant to be particularly limiting. The present disclosure is unlimited with regard to the particular napR/DNAbp, Cas9 or Cas9 equivalent that is employed in the improved base editors of the invention.

[0013] In some embodiments, the napR/DNAbp is a Cas moiety.

[0014] In various embodiment, the Cas moiety is a S. pyogenes Cas9, which has been mostly widely used as a tool for genome engineering. This Cas9 protein is a large, multi- domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, dCas9 can target that protein to virtually any DNA sequence simply by co expression with an appropriate sgRNA.

[0015] In other embodiments, the Cas moiety is a Cas9 from: Corynebacterium ulcerans (NCBI Refs: NC_0l5683.l, NC_0l73l7.l); Corynebacterium diphtheria (NCBI Refs: NC 016782.1, NC_0l6786.l); Spiroplasma syrphidicola (NCBI Ref: NC_02l284.l); Prevotella intermedia (NCBI Ref: NC_0l786l.l); Spiroplasma taiwanense (NCBI Ref: NC_02l846.l); Streptococcus iniae (NCBI Ref: NC_02l3l4.l); Belliella baltica (NCBI Ref: NC_0l80l0.l); Psychroflexus torquisl (NCBI Ref: NC_0l872l.l); Streptococcus

thermophilus (NCBI Ref: YP_820832.l); Listeria innocua (NCBI Ref: NP_472073.l);

Campylobacter jejuni (NCBI Ref: YP_002344900.l); ox Neisseria meningitidis (NCBI Ref: YP_002342100.1 ) .

[0016] In still other embodiments, the Cas moiety may include any CRISPR associated protein, including but not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5,

Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some

embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S.

pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.

[0017] A Cas moiety may also be referred to as a casnl nuclease or a CRISPR

(clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans- encoded small RNA (tracrRNA), endogenous ribonuclease 3 (me) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytic ally cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 "-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference.

[0018] Cas9 and equivalents recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. As noted herein, Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti el al, J J., McShan W.M., Ajdic D J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., Vogel J., Charpentier E., Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara L, Hauer M., Doudna J.A., Charpentier E. Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference).

[0019] The Cas moiety may include any suitable homologs and/or orthologs. Cas9 homologs and/or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus . Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier,“The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase.

[0020] In various embodiments, the improved base editors may comprise a nuclease- inactivated Cas protein may interchangeably be referred to as a“dCas” or“dCas9” protein (for nuclease-“dead” Cas9). Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al, Science.

337:816-821(2012); Qi et al,“Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al, Science. 337:816-821(2012); Qi et al, Cell. 28; 152(5): 1173-83 (2013)). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9.

[0021] In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as“Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 ( e.g ., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is is 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%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

[0022] In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_0l7053.l). In other embodiments, wild type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference

Sequence: NC_002737.2). In still other embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. [0023] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 relative to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCB I Reference Sequence: NC_0l7053.l).

[0024] Without wishing to be bound by any particular theory, the presence of the catalytic residue H840 restores the acvitity of the Cas9 to cleave the non-edited (e.g., non- deaminated) strand containing a G opposite the targeted C. Restoration of H840 (e.g., from A840) does not result in the cleavage of the target strand containing the C. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a G to A change on the non-edited strand. Briefly, the C of a C-G basepair can be deaminated to a U by a deaminase, e.g., an APOBEC deaminase. Nicking the non-edited strand, having the G, facilitates removal of the G via mismatch repair mechanisms. UGI inhibits UDG, which prevents removal of the U.

[0025] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC 1 subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_0l7053.l). In some embodiments, variants or homologues of dCas9 (e.g., variants of Cas9 from

Streptococcus pyogenes (NCBI Reference Sequence: NC_0l7053.l)) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to NCBI Reference Sequence:

NC_0l7053.l. In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_0l7053.l) are provided having amino acid sequences which are shorter, or longer than NC_0l7053.l by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

[0026] In some embodiments, the base editors as provided herein comprise the full- length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art.

[0027] It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 comprises the amino acid sequence (SEQ ID NO: 53). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the nCas9 comprises the amino acid sequence (SEQ ID NO:

54). In some embodiments, the Cas9 protein is a nuclease active Cas9. In some

embodiments, the nuclease active Cas9 comprises the amino acid sequence (SEQ ID NO:

55).

[0028] Exemplary catalytically inactive Cas9 (dCas9) (SEQ ID NO: 53):

[0029]

VDELVKVMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV LTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYK VREINN YHH AHD A YLN A V V GT ALIKK YPKLES EF V Y GD YKV YD VRKMIAKS E QEIGKAT AKYFFYSNIMNFFKTEITLAN GEIRKRPLIETN GETGEIVWDKGRDFATVR KVLS MPQ VNIVKKTE V QT GGF S KES ILPKRN S DKLIARKKD WDPKKY GGFD SPT V AY S VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLP KY S LFELEN GRKRML AS AGELQKGNELALPS KY VNFLYLAS H YEKLKGS PEDNEQK QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKE VLD ATLIHQS ITGLYETRIDLS QLGGD (SEQ ID NO: 53)

[0030] Exemplary Cas9 nickase (nCas9) (SEQ ID NO: 54):

[0031] DKKY S IGLAIGTN S V GW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV EEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDN GS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTY AHLFDDKVMKQLKRRRYTGW GRLSRKLIN GIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYK VREINN YHH AHD A YLN A V V GT ALIKK YPKLES EF V Y GD YKV YD VRKMIAKS E QEIGKAT AKYFFYSNIMNFFKTEITLAN GEIRKRPLIETN GETGEIVWDKGRDFATVR KVLS MPQ VNIVKKTE V QT GGF S KES ILPKRN S DKLIARKKD WDPKKY GGFD SPT V AY S VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLP KY S LFELEN GRKRML AS AGELQKGNELALPS KY VNFLYLAS H YEKLKGS PEDNEQK QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKE VLD ATLIHQS ITGLYETRIDLS QLGGD (SEQ ID NO: 54)

[0032] Exemplary catalytically active Cas9 (SEQ ID NO: 55):

[0033] DKKY S IGLDIGTN S V GW A VETDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV EEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDN GS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTY AHLFDDKVMKQLKRRRYTGW GRLSRKLIN GIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYK VREINN YHH AHD A YLN A V V GT ALIKK YPKLES EF V Y GD YKV YD VRKMIAKS E QEIGKAT AKYFFYSNIMNFFKTEITLAN GEIRKRPLIETN GETGEIVWDKGRDFATVR KVLS MPQ VNIVKKTE V QT GGF S KES ILPKRN S DKLIARKKD WDPKKY GGFD SPT V AY S VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLP KY S LFELEN GRKRML AS AGELQKGNELALPS KY VNFLYLAS H YEKLKGS PEDNEQK QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKE VLD ATLIHQS ITGLYETRIDLS QLGGD (SEQ ID NO: 55). [0034] In some embodiments, a Cas moiety refers to a Cas9 or Cas9 homolog from archaea (e.g. nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et ah,“New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb 21. doi: l0.l038/cr.20l7.2l, the entire contents of which is hereby incorporated by reference. Using genome -resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure.

[0035] In some embodiments, the Cas9 moiety is a nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some

embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein. In some embodiments, the napDNAbp comprises an amino acid sequence of any one of SEQ ID NOs: 56-58. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

These sequences are shown below.

[0036] CasX (uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53)

[0037] >trlF0NN 87 IFONN 87_SULIH CRISPR-associated Casx protein

OS=Sulfolobus islandicus (strain HVE10/4) GN=SiH_0402 PE=4 SV=l [0038] ME VPLYNIF GDN YIIQ V ATE AEN S TIYNNKVEIDDEELRN VLNLA YKIA KNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALS E VFKNF S Q VKECEE V S APS F VKPEF YEF GRS PGM VERTRR VKLE VEPH YLIIA A AGW VLTRLGKAKV S EGD Y V G VN VFTPTRGILY S LIQN VN GIVPGIKPET AF GLWIARKV V S S VTNPN V S V VRIYTIS D A V GQNPTTIN GGF S IDLTKLLEKR YLLS ERLE AIARN ALS IS S NMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNG ELIRGEG (SEQ ID NO: 56)

[0039] CasX OS=Sulfolobus islandicus (strain REY15A)

[0040] >trlF0NH53IF0NH53_SULIR CRISPR associated protein, CasX

OS=Sulfolobus islandicus (strain REY15A) GN=SiRe_077 l PE=4 SV=l

[0041] ME VPLYNIF GDN YIIQ V ATE AEN S TIYNNKVEIDDEELRN VLNLA YKIA KNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALS E VFKNF S Q VKECEE V S APS F VKPEF YKF GRS PGM VERTRRVKLE VEPH YLIM AA AG W VLTRLGKAKV S EGD Y V GVN VFTPTRGILY S LIQN VN GIVPGIKPET AF GLWIARKV VS S VTNPN V S V V S IYTIS D A V GQNPTTIN GGF S IDLTKLLEKRDLLS ERLE AIARN ALS I SSNMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYV NGELIRGEG (SEQ ID NO: 57)

[0042] CasY (ncbi.nlm.nih.gov/protein/APG80656.1)

[0043] >APG80656. l CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]

[0044] MS KRHPRIS G VKG YRLH AQRLE YT GKS G AMRTIKYPLY S S PS GGRT VP REIVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAP GLLKN V AE VRGGS YELTKTLKGS HLYDELQIDKVIKFLNKKEIS RAN GS LDKLKKDII DCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQKKLFRDFFGISEQSENDKP SFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKLKEYAQKLDKNEGSLEMWEYIGIGNS GTAFSNFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQEEELEKRLRILAALTIKL REPKFDNHW GGYRSDIN GKLS S WLQNYIN QT VKIKEDLKGHKKDLKKAKEMINRFG ESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDV QEALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNFYGDSK RELYKKYKN A AIYTD ALWKA VEKIYKS AFS S S LKN S FFDTDFDKDFFIKRLQKIF S V Y RRFNTDKWKPIVKN S F AP Y CDIVS LAENE VL YKPKQS RS RKS A AIDKNR VRLPS TENI AKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALD

[0045] In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 ( e.g dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, and Argonaute. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpfl). Similar to Cas9, Cpfl is also a class 2 CRISPR effector. It has been shown that Cpflmediates robust DNA interference with features distinct from Cas9. Cpfl is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN).

Moreover, Cpfl cleaves DNA via a staggered DNA double- stranded break. Out of 16 Cpfl- family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpfl proteins are known in the art and have been described previously, for example Yamano et al,“Crystal structure of Cpfl in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.

[0046] Also useful in the present compositions and methods are nuclease-inactive Cpfl (dCpfl) variants that may be used as a guide nucleotide sequence-programmable DNA- binding protein domain. The Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al, Cell , 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpfl is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpfl nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in Francisella novicida Cpfl (SEQ ID NO:

65) inactivates Cpfl nuclease activity. In some embodiments, the dCpfl of the present disclosure comprises mutations corresponding to D917A, E1006A, D1255A,

D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/ E1006A/D1255A in SEQ ID NO: 59. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions that inactivate the RuvC domain of Cpfl, may be used in accordance with the present disclosure.

[0047] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cpfl protein. In some embodiments, the Cpfl protein is a Cpfl nickase (nCpfl). In some embodiments, the Cpfl protein is a nuclease inactive Cpfl (dCpfl). In some embodiments, the Cpfl, the nCpfl, or the dCpfl comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 59-66. In some embodiments, the dCpfl comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 59-66, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A,

D917A/D1255A, E1006A/D1255A, or D917A/ E1006A/D1255A in SEQ ID NO: 59. In some embodiments, the dCpfl comprises an amino acid sequence of any one SEQ ID NOs: 59-66. It should be appreciated that Cpfl from other bacterial species may also be used in accordance with the present disclosure.

[0048] Wild type Francisella novicida Cpfl (SEQ ID NO: 59) (D917, E1006, and D1255 are bolded and underlined)

[0049]

EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA

EDDVKAIKDFFDQTNNFFHKFKIFHISQSEDKANIFDKDEHFYFVFEECYFEFANIV P

FYNKIRNYITQKPYSDEKFKFNFENSTFANGWDKNKEPDNTAIFFIKDDKYYFGVMN

KKNNKIFDDKAIKENKGEGYKKIVYKFFPGANKMFPKVFFSAKSIKFYNPSEDIFRI R

NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I

DEF YRE VEN QG YKFTFENIS ES YIDS V VN QGKFYFF QIYNKDF S AY S KGRPNFHTFY

WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE

YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IDRGERHL

AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN

YLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVN

QLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR

LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKL T

S VLNTILQMRN S KTGTELD YLIS P V AD VN GNFFDS RQ APKNMPQD AD AN G A YHIGL

KGLMLLGRIKNN QEGKKLNLVIKNEEYFEFV QNRNN (SEQ ID NO: 59)

[0050] Francisella novicida Cpfl D917A (SEQ ID NO: 60) (A917, E1006, and D1255 are bolded and underlined)

[0051] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S D V YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN V Y S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLED DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVP LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIR NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I DEF YRE VEN QG YKLTFENIS ES YIDS V VN QGKLYLF QIYNKDF S AY S KGRPNLHTLY WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IARGERHL AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVN QFYPKYESVSKSQEFFSKFDKICYNFDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR FINFRNSDKNHNWDTREVYPTKEFEKFFKDYSIEYGHGECIKAAICGESDKKFFAKFT S VFNTIFQMRN S KTGTEFD YFIS P V AD VN GNFFDS RQ APKNMPQD AD AN G A YHIGF KGFMFFGRIKNN QEGKKFNFVIKNEEYFEFV QNRNN (SEQ ID NO: 60)

[0052] Francisella novicida Cpfl E1006A (SEQ ID NO: 61) (D917, A1006, and D1255 are bolded and underlined)

[0053] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S D V YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN V Y S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLED DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVP LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIR NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I DEF YRE VEN QG YKLTFENIS ES YIDS V VN QGKLYLF QIYNKDF S AY S KGRPNLHTLY WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IDRGERHL AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKL NYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFV NQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGS RLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAK LTSVLNTILQMRNSKTGTELD YLISPV AD VNGNFFDSRQ APKNMPQD AD AN GAYHIG LKGLMLLGRIKNN QEGKKLNLVIKNEE YFEFV QNRNN (SEQ ID NO: 61)

[0054] Francisella novicida Cpfl D1255A (SEQ ID NO: 62) (D917, E1006, and A 1255 are bolded and underlined)

[0055] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S D V YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN V Y S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLED DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVP LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIR NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I DEF YRE VEN QG YKLTFENIS ES YIDS V VN QGKLYLF QIYNKDF S AY S KGRPNLHTLY WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IDRGERHL AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVN QLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLT S VLNTILQMRN S KTGTELD YLIS P V AD VN GNFFDS RQ APKNMPQD A A AN G A YHIGL KGLMLLGRIKNN QEGKKLNLVIKNEEYFEFV QNRNN (SEQ ID NO: 62)

[0056] Francisella novicida Cpfl D917A/E1006A (SEQ ID NO: 63) (A917, A1006, and D1255 are bolded and underlined)

[0057] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S DV YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN VY S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG

GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLE D

DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL

S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE

EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQAS A

EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIV P

LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN

KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRI R

NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I

DEF YRE VEN QG YKLTFENIS ES YIDS V VN QGKLYLF QIYNKDF S AY S KGRPNLHTLY

WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE

YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IARGERHL

AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE

MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKL

NYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFV

NQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGS

RLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAK

LTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIG

LKGLMLLGRIKNN QEGKKLNLVIKNEE YFEFV QNRNN (SEQ ID NO: 63)

[0058] Francisella novicida Cpfl D917A/D1255A (SEQ ID NO: 64) (A917, E1006, and A 1255 are bolded and underlined)

[0059] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S D V YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN V Y S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLED DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVP LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIR NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I DEF YRE VEN QG YKLTFENIS ES YIDS V VN QGKLYLF QIYNKDF S AY S KGRPNLHTLY WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IARGERHL AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE MKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLN YLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVN QLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLT S VLNTILQMRN S KTGTELD YLIS P V AD VN GNFFDS RQ APKNMPQD A A AN G A YHIGL KGLMLLGRIKNN QEGKKLNLVIKNEEYFEFV QNRNN (SEQ ID NO: 64)

[0060] Francisella novicida Cpfl E1006A/D1255A (SEQ ID NO: 65) (D917, A1006, and A 1255 are bolded and underlined)

[0061] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S D V YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN V Y S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLED DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVP LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIR NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I DEF YRE VEN QG YKLTFENIS ES YIDS VVN QGKLYLF QIYNKDF S AY S KGRPNLHTLY WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IDRGERHL AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKL

NYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFV

NQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGS

RLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAK

LTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAAANGAYHIG

LKGLMLLGRIKNN QEGKKLNLVIKNEE YFEFV QNRNN (SEQ ID NO: 65)

[0062] Francisella novicida Cpfl D917A/E1006A/D1255A (SEQ ID NO: 66) (A917, A1006, and A1255 are bolded and underlined)

[0063] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKK AKQIIDKYHQFFIEEILS S VCIS EDLLQN Y S D V YFKLKKS DDDNLQKDFKS AKDTIKKQ ISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEI IKS FKGWTT YFKGFHENRKN V Y S S NDIPT S IIYRIVDDNLPKFLENKAKYES LKDKAP E AIN YEQIKKDLAEELTFDID YKT S E VN QR VF S LDE VFEIANFNN YLN QS GITKFNTIIG GKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLED DSD VVTTMQSFYEQIAAFKT VEEKS IKETLS LLFDDLKAQKLDLS KIYFKNDKS LTDL S QQVFDD YS VIGT A VLE YITQQIAPKNLDNPS KKEQELIAKKTEKAKYLS LETIKLALE EFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASA EDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVP LYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMN KKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIR NHS THTKN GS PQKG YEKFEFNIEDCRKFIDF YKQS IS KHPE WKDF GFRF S DTQR YN S I DEF YRE VEN QG YKLTFENIS ES YIDS V VN QGKLYLF QIYNKDF S AY S KGRPNLHTLY WKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFE YDLIKDKRFTEDKFFFHCPITINFKS S GANKFNDEINLLLKEKAND VHILS IARGERHL AYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKE MKEGYLSQVVHEIAKLVIEYNAIVVFADLNFGFKRGRFKVEKQVYQKLEKMLIEKL NYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFV NQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGS RLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAK LTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDAAANGAYHIG LKGLMLLGRIKNN QEGKKLNLVIKNEE YFEFV QNRNN (SEQ ID NO: 66)

[0064] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5' phosphorylated ssDNA of -24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al, Nat Biotechnol., 2016 Jul;34(7):768-73. PubMed PMID: 27136078; Swarts et al, Nature. 507(7491) (20l4):258-6l ; and Swarts et al., Nucleic Acids Res. 43(10) (20l5):5120-9, each of which is incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided in SEQ ID NO: 67.

[0065] Wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 67)

[0066]

[0067] In some embodiments, the napDNAbp is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and have been described, for example, in Makarova K., et ah,“Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements”, Biol Direct. 2009 Aug 25;4:29. doi: 10.1186/1745-6150-4-29, the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp is a Marinitoga piezophila Argunaute (MpAgo) protein. The CRISPR-associated Marinitoga piezophila Argunaute (MpAgo) protein cleaves single- stranded target sequences using 5’- phosphorylated guides. The 5’ guides are used by all known Argonautes. The crystal structure of an MpAgo-RNA complex shows a guide strand binding site comprising residues that block 5’ phosphate interactions. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for a 5’-hydroxylated guide. See, e.g., Kaya et ah,“A bacterial Argonaute with noncanonical guide RNA specificity”, Proc Natl Acad Sci U SA. 2016 Apr 12;113(15):4057-62, the entire contents of which are hereby incorporated by reference). It should be appreciated that other argonaute proteins may be used, and are within the scope of this disclosure.

[0068] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpfl, C2cl, C2c2, and C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpfl are Class 2 effectors. In addition to Cas9 and Cpfl, three distinct Class 2 CRISPR-Cas systems (C2cl, C2c2, and C2c3) have been described by Shmakov et ah,“Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, C2cl and C2c3, contain RuvC-like endonuclease domains related to Cpfl. A third system, C2c2 contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2cl. C2cl depends on both CRISPR RNA and tracrRNA for DNA cleavage. Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single- stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpfl. See, e.g., East-Seletsky, et ah,“Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection”, Nature, 2016 Oct l3;538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2 is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage. Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al.,“C2c2 is a single-component

programmable RNA-guided RNA-targeting CRISPR effector”, Science, 2016 Aug 5;

353(6299), the entire contents of which are hereby incorporated by reference.

[0069] The crystal structure of Alicyclobaccillus acidoterrastris C2cl (AacC2cl) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al.,“C2cl-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage

Mechanism”, Mol. Cell, 2017 Jan 19;65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris C2cl bound to target DNAs as ternary complexes. See e.g., Yang et al.,

“P AM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas

endonuclease”, Cell, 2016 Dec 15; 167(7): 1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2cl, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2cl -mediated cleavage resulting in a staggered seven- nucleotide break of target DNA. Structural comparisons between C2cl ternary complexes and previously identified Cas9 and Cpfl counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.

[0070] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a C2cl, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2cl protein. In some embodiments, the napDNAbp is a C2c2 protein. In some embodiments, the napDNAbp is a C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2cl, C2c2, or C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring C2cl, C2c2, or C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 68 or 69. In some embodiments, the napDNAbp comprises an amino acid sequence of any one SEQ ID NOs: 68 or 69. It should be appreciated that C2cl, C2c2, or C2c3 from other bacterial species may also be used in accordance with the present disclosure.

[0071] C2cl (uniprot.org/uniprot/T0D7 A2#)

[0072] splT0D7 A2IC2C l_ALIAG CRISPR-associated endonuclease C2cl

OS=Alicyclobacillus acidoterrestris (strain ATCC 49025 / DSM 3922 / CIP 106132 /

NCIMB 13137 / GD3B) GN=c2cl PE=l SV=l

[0073] MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQE NLYRRSPNGDGEQECDKTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQL YELLVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPG WEEEKEKAETRKS ADRT AD VLR ALADF GLKPLMR V YTDS EMS S VE WKPLRKGQ A V RTWDRDMFQQAIERMMS WES WN QRV GQE Y AKLVEQKNRFEQKNFVGQEHLVHLV NQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPFDLYDAEI KN V QRRNTRRF GS HDLF AKLAEPE Y Q ALWRED AS FLTRY A V YN S ILRKLNH AKMF A TFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREVD DVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMH RRRG ARD V YLN V S VR V QS QS E ARGERRPP Y A A VFRLV GDNHRAFVHFDKLS D YLA EHPDDGKLGS EGLLS GLRVMS VDLGLRT S AS IS VFRV ARKDELKPN S KGRVPFFFPIK GNDNLVAVHERSQLLKLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSED VGRRERSWAKLIEQPVDAANHMTPDWREAFENELQKLKSLHGICSDKEWMDAVYE S VRR VWRHMGKQ VRD WRKD VRS GERPKIRG Y AKD V V GGN S IEQIE YLERQ YKFLK SWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKEDRLKKLADRIIMEALGYVY ALDER GKGKW V AKYPPC QLILLEELS E Y QFNNDRPPS ENN QLMQW S HRG VF QELIN Q AQ VH DLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPWWLNKFVVEHTLD ACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDFDISQIRLRC DWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQEKLS EEE AELLVE ADE AREKS V VLMRDPS GIINRGNWTRQKEFW S M VN QRIEG YL VKQIRS RVPLQDSACENTGDI (SEQ ID NO: 68) [0074] C2c2 (uniprot.org/uniprot/PODOC6)

[0075] >splPODOC6IC2C2_LEPSD CRISPR-associated endoribonuclease C2c2 OS=Leptotrichia shahii (strain DSM 19757 / CCUG 47503 / CIP 107916 / JCM 16776 / LB37) GN=c2c2 PE=l SV=l

[0076]

G G G

[0077] Cas9 Domains with Reduced PAM Exclusivity

[0078] Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example where a target base is placed within a 4 base region ( e.g ., a“deamination window”), which is approximately 15 bases upstream of the PAM. See Komor, A.C., et al,“Programmable editing of a target base in genomic DNA without double- stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al.,“Engineered CRISPR-Cas9 nucleases with altered PAM

specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al.,“Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference.

[0079] In some embodiments, the Cas9 domain is a Cas9 domain from

Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises the amino acid sequence SEQ ID NO: 70. In some embodiments, the SaCas9 comprises a N579X mutation of SEQ ID NO: 70, wherein X is any amino acid except for N. In some embodiments, the SaCas9 comprises a N579A mutation of SEQ ID NO: 70. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid seuqnce having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of a E781X, a N967X, and a R1014X mutation of SEQ ID NO: 70, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation of SEQ ID NO: 70. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation of SEQ ID NO: 70. [0080] In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 70. In some

embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of SEQ ID NOs: 70. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of SEQ ID NOs: 70.

Exemplary SaCas9 sequence

[0081

[0082] Residue N579 of SEQ ID NO: 70, which is underlined and in bold, may be mutated ( e.g to a A579) to yield a SaCas9 nickase.

Nucleobase modification moiety/ nucleic acid effector domain / nucleic acid editing domain [0083] In various embodiments, the improved base editors provided herein comprise one or more nucleic acid effector domains. In various embodiments, the nucleic acid effector domain may be any protein, enzyme, or polypeptide (or functional fragment thereof) which is capable of modifying a DNA or RNA molecule. Nucleobase modification moieties can be naturally occurring, or can be recombinant. For example, a nucleobase modification moiety can include one or more DNA repair enzymes, for example, and an enzyme or protein involved in base excision repair (BER), nucleotide excision repair (NER), homology- dependnent recombinational repair (HR), non-homologous end-joining repair (NHEJ), microhomology end-joining repair (MMEJ), mismatch repair (MMR), direct reversal repair, or other known DNA repair pathway. A nucleobase modification moiety can have one or more types of enzymatic activities, including, but not limited to endonuclease activity, polymerase activity, ligase activity, replication activity, proofreading activity. Nucleobase modification moieties can also include DNA or RNA-modifying enzymes and/or mutagenic enzymes, such as, DNA methylases and deaminating enzymes (i.e., deaminases, including cytidine deaminases and adenosine deaminases, all defined above), which deaminate nucleobases leading in some cases to mutagenic corrections by way of normal cellular DNA repair and replication processes. The“nucleic acid effector domain” (e.g., a DNA effector domain or an RNA effector domain) as used herein may also refer to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA). Exemplary nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments the nucleic acid editing domain is a deaminase (e.g., a cytidine deaminase, such as an APOBEC or an AID deaminase).

[0084] In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine deaminase. In other embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC 1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an

APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a Lamprey CDA1 (pmCDAl) deaminase. In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat. In some embodiments, the deaminase is a rat APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 98). In some embodiments, the deaminase is a human APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 96). In some embodiments, the deaminase is pmCDAl (SEQ ID NO: 103). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 82). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 104-106). In some embodiments, the deaminase is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 4-9 or 72-106.

[0085] Some exemplary suitable nucleic-acid editing domains, e.g., deaminases and deaminase domains, that can be fused to Cas9 domains according to aspects of this disclosure are provided below. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).

[0086] Human AID:

[0087] MDS LLMNRRKFLY OFKN VRW AKGRRET YLC Y V VKRRDS AT S FS LDF GYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGN PNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTF KAWEGEHENSVRESROERRTEEPEYEVDDERDAERTEGE (SEQ ID NO: 72)

(underline: nuclear localization sequence; double underline: nuclear export signal)

[0088] Mouse AID:

[0089] MDS LLMKO KKFLYHFKN VRW AKGRHET YLC YV VKRRDS AT S CS LDF GHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWN PNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTF KAWEGLHENSVRLTROLRRILLPLYEVDDLRDAFRMLGF (SEQ ID NO: 73)

(underline: nuclear localization sequence; double underline: nuclear export signal) [0090] Dog AID:

[0091]

(underline: nuclear localization sequence; double underline: nuclear export signal) [0092] Bovine AID:

[0093]

(underline: nuclear localization sequence; double underline: nuclear export signal) [0094]

(underline: nuclear localization sequence; double underline: nuclear export signal) [0095] Mouse APOBEC-3:

[0096]

(italic: nucleic acid editing domain)

[0097] Rat APOBEC-3:

[0098

(italic: nucleic acid editing domain)

[0099] Rhesus macaque APOBEC-3G:

[0100]

(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0101] Chimpanzee APOBEC-3G:

[0102]

ID NO: 80)

(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0103] Green monkey APOBEC-3G:

[0104]

(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0105] Human APOBEC-3G:

[0106]

(italic: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0107] Human APOBEC-3F: [0108]

(italic: nucleic acid editing domain)

[0109] Human APOBEC-3B:

[0110]

(italic: nucleic acid editing domain) [0111]

[0112]

[0113] Bovine APOBEC-3B:

[0114]

[0115] Chimpanzee APOBEC-3B:

[0116]

[0117] Human APOBEC-3C:

[0118]

(italic: nucleic acid editing domain) [0119] Gorilla APOBEC3C:

(italic: nucleic acid editing domain)

[0120] Human APOBEC-3A:

[0121]

(italic: nucleic acid editing domain)

[0122] Rhesus macaque APOBEC-3A:

(italic: nucleic acid editing domain)

[0123] Bovine APOBEC-3A:

(italic: nucleic acid editing domain)

[0124] Human APOBEC-3H:

[0125]

(italic: nucleic acid editing domain)

[0126] Rhesus macaque APOBEC-3H: [0127]

[0128] Human APOBEC-3D:

[0129]

(italic: nucleic acid editing domain)

[0130] Human APOBEC-l:

[0131]

[0132] Mouse APOBEC-l:

[0133]

ATGLK (SEQ ID NO: 97)

[0134] Rat APOBEC-l:

[0135]

WPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWA TGFK (SEQ ID NO: 98)

[0136] Human APOBEC-2:

[0137] M AQKEE A A V ATE A AS QN GEDFENFDDPEKFKEFIEFPPFEIVT GERFP ANFFKFQFRN VEY S S GRNKTFLC YVVE AQGKGGQV Q ASRGYLEDEHAAAHAEE AFF NTIFPAFDPAFRYNVTW YV S S SPC AAC ADRIIKTFS KTKNFRFFIFV GRFFMWEEPEIQ AAFKKFKEAGCKFRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFFYYEEKF ADIFK (SEQ ID NO: 99)

[0138] Mouse APOBEC-2:

[0139] MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLP VNFFKFQFRN VEY S S GRNKTFLC YVVE V QS KGGQAQ ATQGYLEDEHAGAHAEEAFF NTILP AFDP ALK YN VT W Y V S S S PC A AC ADRILKTLS KTKNLRLLILV S RLFMWEEPE V QAALKKLKE AGCKLRIMKPQDFE YIW QNFVEQEEGES KAFEPWEDIQENFLYYEEKL ADILK (SEQ ID NO: 100)

[0140] Rat APOBEC-2:

[0141] MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLP VNFFKFQFRN VEY S S GRNKTFLC YVVE AQS KGGQV Q ATQGYLEDEHAGAHAEEAFF NTILP AFDP ALK YNVTWYV S S S PC A AC ADRILKTLS KTKNLRLLILV S RLFMWEEPE V QAALKKLKE AGCKLRIMKPQDFE YLW QNFVEQEEGES KAFEPWEDIQENFLYYEEK LADILK (SEQ ID NO: 101)

[0142] Bovine APOBEC-2:

[0143] MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPA H YFKFQFRN VE Y S S GRNKTFLC YVVE AQS KGGQV Q AS RG YLEDEH ATNH AEE AFFN S IMPTFDP ALRYM VT W Y V S S S PC A AC ADRIVKTLNKTKNLRLLIL V GRLFM WEEPEI QAALRKLKE AGCRLRIMKPQDFEYIW QNFVEQEEGES KAFEPWEDIQENFLYYEEKL ADILK (SEQ ID NO: 102)

[0144] Petromyzon marinus CDA1 (pmCDAl)

[0145] MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRA CFW GY A VNKPQS GTERGIHAEIFS IRKVEE YLRDNPGQFTINWY S S WSPC ADC AEKIL EW YN QELRGN GHTLKIW AC KLY YEKN ARN QIGLWNLRDN G V GLN VM V S EH Y QCC RKIFIQS S HN QLNENRWLEKTLKR AEKRRS ELS IMIQ VKILHTTKS P A V (SEQ ID NO: 103) [0146] Human APOBEC3G D3 l6R_D3 l7R

[0147]

[0148] Human APOBEC3G chain A

[0149]

[0150] Human APOBEC3G chain A Dl20R_Dl2lR

[0151

Cytidine deaminases

[0152] Some aspects of the disclosure provide cytidine deaminases.

[0153] In some embodiments, second protein comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC 1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an

APOBEC3A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBECl. In some embodiments, the deaminase is a Petromyzon marinus cytidine deaminase 1 (pmCDAl). In some embodiments, the deminase is a human APOBEC3G (SEQ ID NO: 82). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 105). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D3l6R_D3l7R mutation (SEQ ID NO: 104). In some embodiments, the deaminase is a frantment of the human APOBEC3G and comprising mutations corresponding to the D3l6R_D3l7R mutations in SEQ ID NO: 82 (SEQ ID NO: 106).

[0154] In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the deaminase domain of any one of SEQ ID NOs: 4-9, or 72-106. In some embodiments, the nucleic acid editing domain comprises the amino acid sequence of any one of SEQ ID NOs: 4-9, or 72-106.

[0155] Deaminase Domains that Modulate the Editing Window of Base Editors

[0156] Some aspects of the disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins provided herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors). For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deaminataion window may prevent unwanted deamination of residues adjacent of specific target residues, which may decrease or prevent off-target effects. [0157] In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain ( e.g ., a cytidine deaminase domain) that has reduced catalytic deaminase activity. In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has a reduced catalytic deaminase activity as compared to an appropriate control. For example, the appropriate control may be the deaminase activity of the deaminase prior to introducing one or more mutations into the deaminase. In other embodiments, the appropriate control may be a wild-type deaminase. In some embodiments, the appropriate control is a wild-type apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the appropriate control is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an

APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an

APOBEC3F deaminase, an APOBEC3G deaminase, or an APOBEC3H deaminase. In some embodiments, the appropriate control is an activation induced deaminase (AID). In some embodiments, the appropriate control is a cytidine deaminase 1 from Petromyzon marinus, (pmCDAl). In some embodiments, the deaminse domain may be a deaminase domain that has at least 1%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at lest 70%, at least 80%, at least 90%, or at least 95% less catalytic deaminase activity as compared to an appropriate control.

[0158] In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase, wherin X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase.

[0159] In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase, wherin X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase.

[0160] In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a H121R and a Hl22Rmutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126A mutation of rAPOBECl (SEQ ID NO: 98), or one or more

corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126E mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R118A mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90A mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R132E mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y and a R126E mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some

embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R126E and a R132E mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y and a R132E mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W90Y, R126E, and R132E mutation of rAPOBECl (SEQ ID NO: 98), or one or more corresponding mutations in another APOBEC deaminase.

[0161] In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a D316R and a D317R mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320A mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320E mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R313A mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285A mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R326E mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y and a R320E mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320E and a R326E mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y and a R326E mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a W285Y, R320E, and R326E mutation of hAPOBEC3G (SEQ ID NO: 82), or one or more corresponding mutations in another APOBEC deaminase.

[0162] Some aspects of this disclosure provide fusion proteins comprising (i) a nuclease-inactive Cas9 domain; and (ii) a nucleic acid editing domain. In some

embodiments, a nuclease-inactive Cas9 domain (dCas9), comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of a Cas9 as provided by any one of the Cas9 moiteies provided herein, and comprises mutations that inactivate the nuclease activity of Cas9. Mutations that render the nuclease domains of Cas9 inactive are well-known in the art. For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek el al, Science. 337:816-821(2012); Qi et al, Cell. 28; 152(5): 1173-83 (2013)). In some embodiments, the dCas9 of this disclosure comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 107, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the dCas9 of this disclosure comprises a H840A mutation of the amino acid sequence provided in SEQ ID NO: 107, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the dCas9 of this disclosure comprises both D10A and H840A mutations of the amino acid sequence provided in SEQ ID NO: 107, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the Cas9 further comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 107, or a corresponding mutation in any of the amino acid sequences provided herein. The presence of the catalytic residue H840 restores the acvitity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C. In some embodiments, the dCas9 comprises an amino acid sequence of SEQ ID NO: 53. It is to be understood that other mutations that inactivate the nuclease domains of Cas9 may also be included in the dCas9 of this disclosure. [0163] Wild type Cas9 corresponding to Cas9 from Streptococcus pyogenes

[0164] MDKKY S IGLDIGTN S V GW A VITDE YK VPS KKFKVLGNTDRHS IKKNLI GALLFDS GET AEATRLKRT ARRRYTRRKNRIC YLQEIFSNEM AKVDDSFFHRLEES FL VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AK AILS ARLS KS RR LENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNL LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK ALVRQQLPEKYKEIFFDQS KN GY AG YIDGGAS QEEFYKFIKPILEKMDGTEELLVKLN REDLLRKQRTFDN GS IPHQIHLGELH AILRRQEDF YPFLKDNREKIEKILTFRIP Y Y V GP LARGN S RF A WMTRKS EETITPWNFEE V VDKG AS AQS FIERMTNFDKNLPNE KVLPK HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL KSDGFANRNFMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT V KVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLS ELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVY GD YKVYDVRKMI AKS EQEIGKAT AKYFFY S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF A TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPT VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLII KLPKY S LFELEN GRKRMLAS AGELQKGNEL ALPS KY VNFLYLAS H YEKLKGS PEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

(SEQ ID NO: 107)

[0165] The Cas9 or dCas9 domains comprising the mutations disclosed herein, may be a full-length Cas9, or a fragment thereof. In some embodiments, proteins comprising Cas9, or fragments thereof, are referred to as“Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 ( e.g ., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9, e.g., a Cas9 comprising the amino acid sequence of SEQ ID NO: 107.

[0166] Any of the Cas9 fusion proteins of this disclosure may further comprise a nucleic acid editing domain (e.g., an enzyme that is capable of modifying nucleic acid, such as a deaminase). In some embodiments, the nucleic acid editing domain is a DNA-editing domain. In some embodiments, the nucleic acid editing domain has deaminase activity. In some embodiments, the nucleic acid editing domain comprises or consists of a deaminase or deaminase domain. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC 1 family deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). Some nucleic-acid editing domains as well as Cas9 fusion proteins including such domains are described in detail herein. Additional suitable nucleic acid editing domains will be apparent to the skilled artisan based on this disclosure and knowledge in the field.

[0167] Some aspects of the disclosure provide a fusion protein comprising a Cas9 domain fused to a nucleic acid editing domain, wherein the nucleic acid editing domain is fused to the N-terminus of the Cas9 domain. In some embodiments, the Cas9 domain and the nucleic acid editing-editing domain are fused via a linker. In some embodiments, the linker comprises a (GGGS)„ (SEQ ID NO: 109), a (GGGGS)„ (SEQ ID NO: 110), a (G)„ (SEQ ID NO: 118), an (EAAAK)„ (SEQ ID NO: 111), a (GGS)„ (SEQ ID NO: 112), (SGGS)„ (SEQ ID NO: 113), an SGSETPGTSESATPES (SEQ ID NO: 114) motif (see, e.g., Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or an (XP) _n motif (SEQ ID NO: 120), or a combination of any of these, wherein n is independently an integer between 1 and 30. In some

embodiments, n is independently 1, 2, 3, 4, 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, or 30, or, if more than one linker or more than one linker motif is present, any combination thereof. In some embodiments, the linker comprises a (GGS) _n motif (SEQ ID NO: 112), wherein n is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the linker comprises a (GGS) _n motif (SEQ ID NO: 112), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence

SGSETPGTSESATPES (SEQ ID NO: 114). Additional suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al, Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69, the entire contents of which are incorporated herein by reference. Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure. In some embodiments, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure:

[NH ₂ ]- [nucleic acid editing domain]-[Cas9]-[COOH] or

[NH ₂ ]- [nucleic acid editing domain]-[linker]-[Cas9]-[COOH],

wherein NH ₂ is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.

[0168] The fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS of the fusion protein is localized between the nucleic acid editing domain and the Cas9 domain. In some

embodiments, the NLS of the fusion protein is localized C-terminal to the Cas9 domain.

[0169] In some embodiments, the nucleic acid editing domain is a deaminase. For example, in some embodiments, the general architecture of exemplary Cas9 fusion proteins with a deaminase domain comprises the structure:

[NH ₂ ] - [NLS ] - [deaminase] - [Cas9] - [COOH] ,

[NH ₂ ] - [Cas9] - [deaminase] - [COOH] ,

[NH ₂ ] - [deaminase] - [Cas9] - [COOH] , or

[NH ₂ ] - [deaminase] - [Cas9] - [NLS ] - [COOH]

wherein NLS is a nuclear localization sequence, NH ₂ is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al, PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 115) or MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO:

116). In some embodiments, a linker is inserted between the Cas9 and the deaminase. In some embodiments, the NLS is located C-terminal of the Cas9 domain. In some

embodiments, the NLS is located N-terminal of the Cas9 domain. In some embodiments, the NLS is located between the deaminase and the Cas9 domain. In some embodiments, the NLS is located N-terminal of the deaminase domain. In some embodiments, the NLS is located C- terminal of the deaminase domain.

[0170] One exemplary suitable type of nucleic acid editing domain is a cytidine deaminase, for example, of the APOBEC family. The apolipoprotein B mRNA-editing complex (APOBEC) family of cytidine deaminase enzymes encompasses eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner. ²⁹ One family member, activation-induced cytidine deaminase (AID), is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion. ³⁰ The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-l strain via the deamination of cytosines in reverse- transcribed viral ssDNA. These proteins all require a Zn ²⁺ -coordinating motif (His-X-Glu- X23-26-Pro-Cys-X _2-4 -Cys; SEQ ID NO: 117) and bound water molecule for catalytic activity. The Glu residue acts to activate the water molecule to a zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own particular“hotspot”, ranging from WRC (W is A or T, R is A or G) for hAID, to TTC for hAPOBEC3F. ³² A recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure comprised of a five-stranded b-sheet core flanked by six a-helices, which is believed to be conserved across the entire family. ³³ The active center loops have been shown to be responsible for both ssDNA binding and in determining“hotspot” identity. ³⁴ Overexpression of these enzymes has been linked to genomic instability and cancer, thus highlighting the importance of sequence-specific targeting.

[0171] Some aspects of this disclosure relate to the recognition that the activity of cytidine deaminase enzymes such as APOBEC enzymes can be directed to a specific site in genomic DNA. Without wishing to be bound by any particular theory, advantages of using Cas9 as a recognition agent include (1) the sequence specificity of Cas9 can be easily altered by simply changing the sgRNA sequence; and (2) Cas9 binds to its target sequence by denaturing the dsDNA, resulting in a stretch of DNA that is single- stranded and therefore a viable substrate for the deaminase. It should be understood that other catalytic domains, or catalytic domains from other deaminases, can also be used to generate fusion proteins with Cas9, and that the disclosure is not limited in this regard.

[0172] Some aspects of this disclosure are based on the recognition that

Cas9:deaminase fusion proteins can efficiently deaminate nucleotides. In view of the results provided herein regarding the nucleotides that can be targeted by Cas9:deaminase fusion proteins, a person of skill in the art will be able to design suitable guide RNAs to target the fusion proteins to a target sequence that comprises a nucleotide to be deaminated.

[0173] In some embodiments, the deaminase domain and the Cas9 domain are fused to each other via a linker. Various linker lengths and flexibilities between the deaminase domain ( e.g ., AID) and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGGS)„ (SEQ ID NO: 110), (GGS)„, (SEQ ID NO: 112) and (G)„

(SEQ ID NO: 118) to more rigid linkers of the form (EAAAK) _n (SEQ ID NO: 111), (SGGS) _n (SEQ ID NO: 113), SGSETPGTSESATPES (SEQ ID NO: 114) (see, e.g., Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to Fokl nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP) _n (SEQ ID NO: 120)) ³⁶ in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, the linker comprises a (GGS) _n motif (SEQ ID NO: 112), wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (an SGSETPGTSESATPES (SEQ ID NO: 114) motif.

[0174] Some exemplary suitable nucleic-acid editing domains, e.g., deaminases and deaminase domains, that can be fused to Cas9 domains according to aspects of this disclosure are provided below. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal).

Adenosine deaminases

[0175] Some aspects of the disclosure provide adenosine deaminases. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenosine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenosine in a deoxyadenosine residue of DNA. The adenosine deaminase may be derived from any suitable organism ( e.g ., E. coli). In some embodiments, the adenosine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.

[0176] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs: 15, or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identiy plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30,

31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NO: 15 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 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 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NO: 15 or any of the adenosine deaminases provided herein.

[0177] In some embodiments, the adenosine deaminase comprises a D108X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase. It should be appreciated, however, that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein.

[0178] In some embodiments, the adenosine deaminse comprises an A106X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0179] In some embodiments, the adenosine deaminase comprises a E155X mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild- type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a E155D, E155G, or E155V mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0180] In some embodiments, the adenosine deaminase comprises a D147X mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild- type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y, mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0181] It should be appreciated that any of the mutations provided herein ( e.g ., based on the ecTadA amino acid sequence of SEQ ID NO: 15) may be introduced into other adenosine deaminases, such as S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It would be apparent to the skilled artisan how to identify amino acid residues from other adenosine deaminases that are homologous to the mutated residues in ecTadA. Thus, any of the mutations identified in ecTadA may be made in other adenosine deaminases that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein may be made individually or in any combination in ecTadA or another adenosine deaminase. For example, an adenosine deaminase may contain a D108N, a A106V, a E155V, and/or a D147Y mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase. In some embodiments, an adenosine deaminase comprises the following group of mutations (groups of mutations are separated by a in ecTadA SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase:

[0182] D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V, and E55V; D108N, A106V, and D147Y; D108N, E55V, and D147Y; A106V, E55V, and D147Y; and D108N, A106V, E55V, and D147Y. It should be appreciated, however, that any combination of corresponding mutations provided herein may be made in an adenosine deaminase ( e.g ecTadA).

[0183] In some embodiments, the adenosine deaminase comprises one or more of a H8X, T17X, L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X,

V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and/or K157X mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L, 1951, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N, or D108V, or D108A, or D108Y, Kl 101, Ml 18K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D, and/or K157R mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase.

[0184] In some embodiments, the adenosine deaminase comprises one or more of a H8X, D108X, and/or N127X mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of a H8Y, D108N, and/or N127S mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase.

[0185] In some embodiments, the adenosine deaminase comprises one or more of H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and/or T166X mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and/or T166P mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase.

[0186] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, D108X, N127X, E155X, and K161X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R126X, L68X, D108X, N127X, D147X, and E155X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X in SEQ ID NO: 15, or a

corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0187] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G and Q163H in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. In some

embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R126W, L68Q, D108N, N127S, D147Y, and E155V in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase.

[0188] In some embodiments, the adenosine deaminase comprises one or more of the mutations provided in Figure 16 corresponding to SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D108G, or D108V mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106V and D108N mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises R107C and D108N mutations in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, N127S, D147Y, and Q154H mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, R24W, D108N, N127S, D147Y, and E155V mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108N, D147Y, and E155V mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a H8Y, D108N, and S127S mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A106V, D108N, D147Y and E155V mutation in SEQ ID NO: 15, or corresponding mutations in another adenosine deaminase.

[0189] In some embodiments, the adenosine deaminase comprises one or more of a, S2X, H8X, I49X, L84X, H123X, N127X, I156X and/or K160X mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of S2A, H8Y, I49F, L84F, H123Y, N127S, I156F and/or K160S mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase.

[0190] In some embodiments, the adenosine deaminse comprises an L84X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0191] In some embodiments, the adenosine deaminse comprises an H123X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0192] In some embodiments, the adenosine deaminse comprises an I157X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I157F mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0193] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, A106X, D108X, N127X, and K160X in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase.

[0194] In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. In some

embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S in SEQ ID NO: 15, or a corresponding mutation or mutations in another adenosine deaminase. [0195] In some embodiments, the adenosine deaminase comprises one or more of a, E25X, R26X, R107X, A142X, and/or A143X mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R07K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase.

[0196] In some embodiments, the adenosine deaminse comprises an E25X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0197] In some embodiments, the adenosine deaminse comprises an R26X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an, R26G, R26N, R26Q, R26C, R26L, or R26K mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0198] In some embodiments, the adenosine deaminse comprises an R107X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R107P, R07K, R107A, R107N, R107W, R107H, or R107S mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0199] In some embodiments, the adenosine deaminse comprises an A142X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G, mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0200] In some embodiments, the adenosine deaminse comprises an A143X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0201] In some embodiments, the adenosine deaminase comprises one or more of a, H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S146X, Q154X, K157X, and/or K161X mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and/or K161T mutation in SEQ ID NO: 15, or one or more corresponding mutations in another adenosine deaminase.

[0202] In some embodiments, the adenosine deaminse comprises an H36X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0203] In some embodiments, the adenosine deaminse comprises an N37X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T, or N37S mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0204] In some embodiments, the adenosine deaminse comprises an P48X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an P48T, or P48L mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0205] In some embodiments, the adenosine deaminse comprises an R51X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H, or R51L mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0206] In some embodiments, the adenosine deaminse comprises an S146X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S146R, or S146C mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0207] In some embodiments, the adenosine deaminse comprises an K157X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0208] In some embodiments, the adenosine deaminse comprises an P48X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0209] In some embodiments, the adenosine deaminse comprises an A142X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A142N mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0210] In some embodiments, the adenosine deaminse comprises an W23X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R, or W23L mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0211] In some embodiments, the adenosine deaminse comprises an R152X mutation in ecTadA SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P, or R52H mutation in SEQ ID NO: 15, or a corresponding mutation in another adenosine deaminase.

[0212] In some embodiments, the adenosine deaminase comprises the combination of mutations of any of the adenosine deaminases ( e.g ecTadA adenosine deaminases) described herein. For example, the adenosine deaminase may comprise the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S 146C, D147Y, E155V, I156F, and K157N (relative to SEQ ID NO: 15) of clone pNMG-477. In some embodiments, the adenosine deaminase comprises the following combination of mutations relative to SEQ ID NO: 15, where each mutation of a combination is separated by a and each combination of mutations is between parentheses: (Al06V_Dl08N), (Rl07C_Dl08N), (H8Y_Dl08N_S l27S_Dl47Y_Ql54H),

(H8Y_R24W_Dl08N_Nl27S_Dl47Y_El55V), (Dl08N_Dl47Y_El55V),

(H8Y_Dl08N_S l27S), (H8Y_Dl08N_Nl27S_Dl47Y_Ql54H),

(Al06V_Dl08N_Dl47Y_El55V), (Dl08Q_Dl47Y_El55V), (Dl08M_Dl47Y_El55V), (Dl08L_Dl47Y_El55V), (Dl08K_Dl47Y_El55V), (Dl08I_Dl47Y_El55V),

(Dl08F_Dl47Y_El55V), (Al06V_Dl08N_Dl47Y), (Al06V_Dl08M_Dl47Y_El55V), (E59A_Al06V_Dl08N_Dl47Y_El55V), (E59A cat

dead_A 106 V_D 108N_D 147 Y_E 155 V) ,

(L84F_Al06V_Dl08N_Hl23Y_Dl47Y_El55V_Il56Y),

(L84F_Al06V_Dl08N_Hl23Y_Dl47Y_El55V_Il56F), (Dl03A_D0l4N),

(G22P_D 103 A_D 104N) , (G22P_D 103 A_D 104N_S 138 A) , (D 103 A_D l04N_S 138A), (R26G_L84F_Al06V_Rl07H_Dl08N_Hl23Y_Al42N_Al43D_Dl47Y_El55V_I l56F), (E25G_R26G_L84F_Al06V_Rl07H_Dl08N_Hl23Y_Al42N_Al43D_Dl47Y_El 55V_Il5 6F) ,(E25D_R26G_L84F_A 106 V_R 107K_D 108N_H 123 Y_A 142N_A 143 G_D 147 Y_E 155 V _Il56), (R26Q_L84F_A 106 V_D 108N_H 123 Y_A 142N_D 147 Y_E 155 V_1156F) ,

[0213] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95, 98%, 99%, or 99.5% identical to any one of SEQ ID NOs: 15-23, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NO:

15 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 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 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 166, identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NO: 15 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 15 or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase consists of the amino acid sequence of SEQ ID NO: 15 or any of the adenosine deaminases provided herein.

Nuclear localization signals

[0214] In various embodiments, the base editors disclosed herein further comprise one or more, preferably at least two nuclear localization signals. In a preferred embodiment, the base editors comprise at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLSs or they can be different NLSs. In addition, the NLSs may be expressed as part of a fusion protein with the remaining portions of the base editors. The location of the NLS fusion can be at the N-terminus, the C-terminus, or within a sequence of a base editor (e.g., inserted between the encoded napR/DNAbp component (e.g., Cas9) and a DNA effector moiety (e.g., a deaminase)).

[0215] The NLSs may be any known NLS sequence in the art. The NLSs may also be any future-discovered NLSs for nuclear localization. The NLSs also may be any naturally-occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations).

[0216] A nuclear localization signal or sequence (NLS) is an amino acid sequence that tags, designates, or otherwise marks a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus. A nuclear localization signal can also target the exterior surface of a cell. Thus, a single nuclear localization signal can direct the entity with which it is associated to the exterior of a cell and to the nucleus of a cell. Such sequences can be of any size and composition, for example more than 25, 25, 15, 12, 10, 8, 7, 6, 5 or 4 amino acids, but will preferably comprise at least a four to eight amino acid sequence known to function as a nuclear localization signal (NLS).

[0217] The term“nuclear localization sequence” or“NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al, international PCT application, PCT/EP2000/011690, filed November 23, 2000, published asWO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 115),

MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 116),

KRT ADGS EFES PKKKRKV (SEQ ID NO: 1), or KRT ADGS EFEPKKKRKV (SEQ ID NO:

2).

[0218] In one aspect of the invention, a base editor (e.g., a known base editor, such as BE1, BE2, BE3, or BE4) may be modified with one or more nuclear localization signals (NLS), preferably at least two NLSs. In preferred embodiments, the base editors are modified with two or more NLSs. The invention contemplates the use of any nuclear localization signal known in the art at the time of the invention, or any nuclear localization signal that is identified or otherwise made available in the state of the art after the time of the instant filing. A representative nuclear localization signal is a peptide sequence that directs the protein to the nucleus of the cell in which the sequence is expressed. A nuclear localization signal is predominantly basic, can be positioned almost anywhere in a protein's amino acid sequence, generally comprises a short sequence of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem. 273: 14731-37, incorporated herein by reference) to eight amino acids, and is typically rich in lysine and arginine residues (Magin et ah, (2000) Virology 274: 11-16, incorporated herein by reference). Nuclear localization signals often comprise proline residues. A variety of nuclear localization signals have been identified and have been used to effect transport of biological molecules from the cytoplasm to the nucleus of a cell. See, e.g., Tinland et ah, (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moede et ah, (1999) FEBS Leff. 461:229-34, which is incorporated by reference. Translocation is currently thought to involve nuclear pore proteins. [0219] Most NLSs can be classified in three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV SEQ ID NO: 115); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL SEQ ID NO: 119); and (iii) noncanonical sequences such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991).

[0220] Nuclear localization signals appear at various points in the amino acid sequences of proteins. NLS's have been identified at the N-terminus, the C-terminus and in the central region of proteins. Thus, the specification provides base editors that may be modified with one or more NLSs at the C-terminus, the N-terminus, as well as at in internal regaion of the base editor. The residues of a longer sequence that do not function as component NLS residues should be selected so as not to interfere, for example tonically or sterically, with the nuclear localization signal itself. Therefore, although there are no strict limits on the composition of an NLS -comprising sequence, in practice, such a sequence can be functionally limited in length and composition.

[0221] The present disclosure contemplates any suitable means by which to modify a base editor to include one or more NLSs. In one aspect, the base editors can be engineered to express a base editor protein that is translationally fused at its N-terminus or its C-terminus (or both) to one or more NLSs, i.e., to form a base editor-NLS fusion construct. In other embodiments, the base editor-encoding nucleotide sequence can be genetically modified to incorporate a reading frame that encodes one or more NLSs in an internal region of the encoded base editor. In addition, the NLSs may include various amino acid linkers or spacer regions encoded between the base editor and the N-terminally, C-terminally, or internally- attached NLS amino acid sequence, e.g, and in the central region of proteins. Thus, the present disclosure also provides for nucleotide constructs, vectors, and host cells for expressing fusion proteins that comprise a base editor and one or more NLSs.

[0222] The improved base editors described herein may also comprise nuclear localization signals which are linked to a base editor through one or more linkers, e.g., and polymeric, amino acid, nucleic acid, polysaccharide, chemical, or nucleic acid linker element. The linkers within the contemplated scope of the disclosure are not intented to have any limitations and can be any suitable type of molecule (e.g., polymer, amino acid, polysaccharide, nucleic acid, lipid, or any synthetic chemical linker moiety) and be joined to the base editor by any suitable strategy that effectuates forming a bond (e.g., covalent linkage, hydrogen bonding) between the base editor and the one or more NLSs.

Additional functionalities

[0223] The improved base editors described herein also may include one or more additional functionalities. In certain embodiments, the additional functionalities may include an effector of base repair.

[0224] In certain embodiments, the base editors described herein may comprise an inhibitor of base repair. The term“inhibitor of base repair” or“IBR” refers to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or a catalytically inactive hAAG.

[0225] In other embodiments, the base editors described herein may comprise a uracil glycosylase inhibitor. The term“uracil glycosylase inhibitor” or“UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 10. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as“UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 10. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 10. In some embodiments, the UGI comprises the following amino acid sequence:

[0226] >splPl4739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor

MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVML LT S D APE YKPW ALVIQDS N GENKIKML (SEQ ID NO: 10).

[0227] In some embodiments, the base editor described herein may comprise one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the base editor components). A base editor may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a base editor or component thereof (e.g., the napR/DNAbp moiety, the nucleic acid effector moiety, or the NLS moeity) include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta- glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A base editor may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a fusion protein comprising a base editor are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged base editor is used to identify the location of a target sequence.

[0228] In an aspect of the invention, a reporter gene which includes but is not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), may be introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In a further embodiment of the invention, the DNA molecule encoding the gene product may be introduced into the cell via a vector. In a preferred embodiment of the invention the gene product is luciferase. In a further embodiment of the invention the expression of the gene product is decreased.

[0229] Other exemplary features that may be present are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione- S- transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags,

Softags ( e.g ., Softag 1, Softag 3), strep-tags , biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags.

The guide sequence (e.g., a guide RNA)

[0230] In various embodiments, the improved base editors can be complexed, bound, or otherwise associated with (e.g., via any type of covalent or non-covalent bond) one or more guide sequences, i.e., the sequence which becomes associated or bound to the base editor and directs its localization to a specific target sequence having complementarity to the guide sequence or a portion thereof. The particular design aspects of a guide sequence will depend upon the nucleotide sequence of a genomic target site of interest (i.e., the desired site to be edited) and the type of napR/DNAbp (e.g., type of Cas protein) present in the base editor, among other factors, such as PAM sequence locations, percent G/C content in the target sequence, the degree of microhomology regions, secondary structures, etc.

[0231] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napR/DNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%,

85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 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, 75, or more nucleotides in length.

[0232] In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, the components of a base editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. [0233] A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S.

pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 121) where

NNNNNNNNNNNNXGG (SEQ ID NO: 122) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ ID NO: 123) where NNNNNNNNNNNXGG (SEQ ID NO: 124) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. For the S. thermophilus

CRISPRlCas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 125) where

NNNNNNNNNNNNXXAGAAW (SEQ ID NO: 126) (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR 1 Cas9 target site of the form

MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 127) where

NNNNNNNNNNNXXAGAAW (SEQ ID NO: 128) (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form

MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 129) where

NNNNNNNNNNNNXGGXG (SEQ ID NO: 130) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form

MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 131) where

NNNNNNNNNNNXGGXG (SEQ ID NO: 132) (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. In each of these sequences“M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique.

[0234] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online Webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et ah, 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature

Biotechnology 27(12): 1151-62). Further algorithms may be found in U.S. application Ser.

No. 61/836,080; Broad Reference B 1-2013/004 A); incorporated herein by reference.

[0235] In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a complex at a target sequence, wherein the complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5' to 3'), where“N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1)

NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaag ataaggctt

catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTT TT (SEQ ID NO: 133); (2) NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggc ttcatgccgaaatca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 134); (3)

NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagat aaggcttcatgccgaa atca acaccctgtcattttatggcagggtgtTTTTT (SEQ ID NO: 135); (4)

NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtc cgttatcaacttga aaa agtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 136); (5)

NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtc cgttatcaactt gaa aaagtgTTTTTTT (SEQ ID NO: 137); and (6)

NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtc cgttatcaTTT TT TTT (SEQ ID NO: 138). In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.

[0236] It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and an adenosine deaminase, as disclosed herein, to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein.

[0237] In some embodiments, the guide RNA comprises a structure 5’-[guide sequence] - guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaag uggcaccgagucggugcuu uuu-3’ (SEQ ID NO: 139), wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50

nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. Additional guide sequences are are well known in the art and can be used with the base editors described herein.

Linkers

[0238] In certain embodiments, linkers may be used to link any of the peptides or peptide domains or moieties of the invention (e.g., moiety A covalently linked to moiety B which is covalently linked to moiety C).

[0239] As defined above, the term“linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., a binding domain and a cleavage domain of a nuclease. In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease and the catalytic domain of a recombinase. In some

embodiments, a linker joins a dCas9 and base editor moiety (e.g., a cytidine or adenosine deaminase). Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 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, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated

[0240] The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or hetero aliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5- pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain

embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0241] In some other embodiments, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 110), (G)n (SEQ ID NO: 118), (EAAAK)n (SEQ ID NO: 111), (GGS)n (SEQ ID NO: 112), (SGGS)n (SEQ ID NO: 113), SGSETPGTSESATPES SEQ ID NO: 114), (XP)n (SEQ ID NO: 120), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n (SEQ ID NO: 112), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 114).

[0242] In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain] -[optional linker sequence]-[dCas9 or Cas9 nickase]- [optional linker sequence] -[UGI]. In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain] -[optional linker sequence] -[UGI] -[optional linker sequence]-[dCas9 or Cas9 nickase]; [UGI] -[optional linker sequence] -[nucleic acid editing domain] -[optional linker sequence]-[dCas9 or Cas9 nickase]; [UGI] -[optional linker sequence]-[dCas9 or Cas9 nickase] -[optional linker sequence] -[nucleic acid editing domain]; [dCas9 or Cas9 nickase]- [optional linker sequence] -[UGI] -[optional linker sequence] -[nucleic acid editing domain]; or [dCas9 or Cas9 nickase] -[optional linker sequence] -[nucleic acid editing domain] -[optional linker sequence] -[UGI]. Improved editing efficiencies

[0243] As exemplified in the Examples, the efficiency of base editing may be increased by the various approaches described herein for improving base editors in cells. In one aspect, base editing efficiency may be increased by optimizing base editor codon usage which increases base editor mRNA expression levels thereby increase base editing efficiencies. In another aspect, base editing efficiency may be increased by optimizing base editor amino acid sequences through ancestral sequence reconstruction. In still other aspects, base editing efficiency may be increased by modifying base editors to include at least two NLSs, e.g., wherein one is located at the N-terminus and another (same or difference NLS) is located as the C-terminus of a base editor fusion protein. The level or degree of increase in efficiency may be measured or expressed in any suitable manner such as the percentage of nucleotides correctly edited from the total number of nucleotides attempted to be edited by a base editor described herein.

[0244] The base editors in various embodiment may be characterized with an improved editing capability that is at least 0.5-fold, or at least 0.6-fold, or at least 0.7-fold, or at least 0.8-fold, or at least 0.9-fold, or at least 1.0-fold, or at least 1.5-fold, or at least 2.0- fold, or at least 3.0-fold, or at least 4.0-fold, or at least 5.0-fold, or at least 6.0-fold, or at least 7.0-fold, or at least 8.0-fold, or at least 9.0-fold, or at least 10.0-fold, or at least 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or lOO-fold or more higher than the base editing efficiency of a base editor that has not been modified by at least one of the modification approached described herein (e.g., codon optimization, at least 2 NLSs, or by ancestral sequence reconstruction. Examples of specific increases in base editing efficiencies are exemplified in the Examples.

II. METHODS FOR MAKING THE IMPROVED BASE-EDITORS

[0245] Despite recent advances in the design of base editors, the efficiency of base editing varies widely. To increase base editing efficiency, the inventors sought to identify the factors that limit base editing efficiency in cells. It was surprisingly found by the inventors that expression and nuclear localization in human cells imposed key bottlenecks on editing efficiency. The inventors discovered that by optimizing codon usage, using improved nuclear localization sequences (NLSs), and performing ancestral reconstruction of deaminases resulted in base editors with greatly increased editing efficiency, often more than doubling target nucleotide conversion yields as compared to the unmodified counterpart editors. The resulting base editors were shown, as demonstrated in the Examples, to install point mutations relevant to human disease in a variety of mammalian cell types much more efficiently than previously described base editors. These methods can be used to provide improved base editors that can be used to efficiently edit a nucleic acid molecule in a manner that is dramatically improved as compared to base editors known in the art. The improved base editors may be used to efficiently edit nucleic acid molecules, e.g., a genome, for example, by correcting a disease-causing point mutation.

[0246] Thus, the invention relates in various aspects to methods of making the disclosed improved base editors by various modes of manipulation that include but are not limited to codon optimization and performance of ancestral reconstruction of components of the base editors (e.g., of a deaminase) to achieve greater expression levels in a cell, and the use of nuclear localization sequences (NLS)s, preferably at least two NLSs to increase the localization of the expressed base editors into a cell nucleus.

Increasing expression

[0247] The base editors contemplated herein can include modifications that result in increased expression through codon optimization and ancestral reconstruction analysis.

[0248] In some embodiments, the base editors (or a component thereof) is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The

predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the“Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).

Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0249] In other embodiments, the base editors of the invention have improved expression (as compared to non-modified or state of the art counterpart editors) as a result of ancestral sequence reconstruction analysis. Ancestral sequence reconstruction (ASR) is the process of analyzing modern sequences within an evolutionary/phylogenetic context to infer the ancestral sequences at particular nodes of a tree. These ancient sequences are most often then synthesized, recombinantly expressed in laboratory microorganisms or cell lines, and then characterized to reveal the ancient properties of the extinct biomolecules 2, 3, 4, 5, 6. This process has produced tremendous insights into the mechanisms of molecular adaptation and functional divergence7. Despite such insights, a major criticism of ASR is the general inability to benchmark accuracy of the implemented algorithms. It is difficult to benchmark ASR for many reasons. Notably, genetic material is not preserved in fossils on a long enough time scale to satisfy most ASR studies (many millions to billions of years ago), and it is not yet physically possible to travel back in time to collect samples. Reference can be made to Cai et ah,“Reconstruction of ancestral protein sequences and its applications,” BMC

Evolutionary Biology 2004, 4:33 and Zakas et ah,“Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction,” Nature Biotechnology, 35, pp. 35-37 (2017), each of which are incorporated herein by reference.

[0250] There are many software packages available which can perform ancestral state reconstruction. Generally, these software packages have been developed and maintained through the efforts of scientists in related fields and released under free software licenses.

The following list is not meant to be a comprehensive itemization of all available packages, but provides a representative sample of the extensive variety of packages that implement methods of ancestral reconstruction with different strengths and features: PAML

(Phylogenetic Analysis by Maximum Likelihood, available at

//abacus. gene.ucl.ac.uk/software/paml.html ), BEAST (Bayesian evolutionary analysis by sampling trees, available at //www.beast2.org/wiki/index.php/Main_Page), and Diversitree (FitzJohn RG, 2012. Diversitree: comparative phylogenetic analyses of diversification in R. Methods in Ecology and Evolution), and HyPHy (Hypothesis testing using phylogenies, available at //hyphy.org/w/index.php/Main_Page).

[0251] The Examples demonstrate one embodiment for using ASR to increase overall expression of base editors disclosed herein.

[0252] The above description is meant to be non-limiting with regard to making base editors having increased expression, and thereby increase editing efficiencies.

Increasing nuclear localization

[0253] In one aspect, the specification provides a strategy for improving a base editor by incorporating one or more nuclear localization signals (NLS) therein, e.g., as a N-terminal or C-terminal fusion protein. Preferably, at least two NLSs are incorporated into a base editor. In the Examples, the inventors explored whether sub-optimal nuclear localization could be a basis or poor editing efficiency. The inventors test six combinations of the base editor“BE4” as N- and/or C-terminal fusions to either the SV40 NLS or the bipartite NLS (bpNLS). As shown in the Examples, all the variants using one or two bpNLSs showed improvements in editing efficiency. The presence of a bpNLS at both the N- and C-terminus (referred to hereafter as“bis-bpNLS”) performed best, resulting in a 1.3-fold average improvement in BE4-mediated OG-to-T*A editing efficiency at five exemplary tested genomic loci (48+8.0% average editing compared to 37+5.6% for the C-terminal SV40 NLS used in BE4). These results together suggest that modifying base editors with one or more nuclear localization signals, e.g., a bis-bpNLS, can significantly improve the editing efficiency of previously described for known base editors, such as, BE3 and BE4 (6, 7).

[0254] However, the Examples are not intended to be limiting, but only

demonstrative of wider strategy for improving base editor efficiency through the modification of the base editor with one or more nuclear localization signals, preferably at least two NLSs. The invention is not intended to be limiting with regard to which NLS is employed, and the manner by which the NLS is attached to or otherwise coupled to a base editor. NLS sequences are known in the art and examples are disclosed herein.

Vectors

[0255] Several aspects of the making and using the base editors of the invention relate to vector systems comprising one or more vectors, or vectors as such. Vectors can be designed to clone and/or express the improved base editors of the disclosure. Vectors can also be designed to transfect the improved base editors of the disclosure into one or more cells, e.g., a target diseased eukaryotic cell for treatment with the base editor systems and methods disclosed herein.

[0256] Vectors can be designed for expression of base editor transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, base editor transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press. San Diego, Calif. (1990). Alternatively, expression vectors encoding one or more improved base editors described herein can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0257] Vectors may be introduced and propagated in a prokaryotic cells. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.

[0258] Fusion expression vectors also may be used to express the improved base editors of the disclosure. Such vectors generally add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0259] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET l ld (Studier et al., GENE

EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,

San Diego, Calif. (1990) 60-89).

[0260] In some embodiments, a vector is a yeast expression vector for expressing the improved base editors described herein. Examples of vectors for expression in yeast

Saccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

[0261] In some embodiments, a vector drives protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0262] In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2,

cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0263] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver- specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid- specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235- 275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and

Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas- specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland- specific promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a- fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

Increasing base editor efficiencies

[0264] Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An“indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions ( i.e ., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended point mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method, for example the methods used in the below Examples. In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two lO-bp sequences that flank both sides of a window in which indels might occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively.

[0265] In some embodiments, the base editors provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, an number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor.

[0266] Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g. a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, a intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation associated with a disease or disorder. In some embodiments, the intended mutation is a adenine (A) to guanine (G) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a thymine (T) to cytosine (C) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a adenine (A) to guanine (G) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a thymine (T) to cytosine (C) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. In some embodiments, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene ( e.g ., a gene promotor or gene repressor). In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point m utati o n s : u n i n t c n dcd point mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point m utati o n s : u n i n t c n dcd point mutations) that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more. It should be appreciated that the characteristics of the base editors described in the“ Base Editor Efficiency" section, herein, may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein.

III. METHODS OF USING IMPROVED BASE-EDITORS

[0267] Some aspects of this disclosure provide methods of using the improved base editors disclosed herein, or base editor complexes comprising one or more napR/DNAbp- programming nucleic acid molecules (e.g., Cas9 guide RNAs) and a nucleobase editor provided herein.

Editing DNA or RNA

[0268] Some aspects of the disclosure provide methods for editing a nucleic acid using the base editors described herein. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to an adenosine deaminase) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the first nucleobase is an adenine. In some embodiments, the second nucleobase is a deaminated adenine, or inosine. In some embodiments, the third nucleobase is a thymine. In some embodiments, the fourth nucleobase is a cytosine. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair ( e.g ., A:T to G:C). In some embodiments, the fifth nucleobase is a guanine. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base paires are edited.

[0269] In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100: 1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is adenine, and the second base is not a G, C, A, or T. In some embodiments, the second base is inosine. In some embodiments, the first base is adenine. In some embodiments, the second base is not a G, C, A, or T. In some embodiments, the second base is inosine. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some

embodiments, the base editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical ( e.g ., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length.

In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1- 7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window.

[0270] In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double- stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1,

5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the first base is adenine. In some embodiments, the second nucleobase is not G, C, A, or T. In some embodiments, the second base is inosine. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects (e.g., form base excision repair) or binds the non-edited strand. In some embodiments, the nucleobase editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some embodiments, the nucleobase editor comprises nickase activity. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some

embodiments, the intended edited basepair is downstream of a PAM site. In some

embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some

embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1- 5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein. [0271] In another embodiment, the disclosure provides editing methods comprising contacting a DNA, or RNA molecule with any of the base editors provided herein, and with at least one guide nucleic acid ( e.g ., guide RNA), wherein the guide nucleic acid, (e.g., guide RNA) is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3’ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3’ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3’ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

[0272] In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a point mutation associated with a disease or disorder. In some embodiments, the activity of the fusion protein (e.g., comprising an adenosine deaminase and a Cas9 domain), or the complex, results in a correction of the point mutation. In some embodiments, the target DNA sequence comprises a G A point mutation associated with a disease or disorder, and wherein the deamination of the mutant A base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein, and the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant A results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant A results in the codon encoding the wild-type amino acid. In some embodiments, the contacting is in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease or disorder. In some embodiments, the disease or disorder is phenylketonuria, von Willebrand disease (vWD), a neoplastic disease associated with a mutant PTEN or BRCA1, or Li-Fraumeni syndrome. A list of exemplary diseases and disorders that may be treated using the base editors described herein is shown in Table 1. Table 1 includes the target gene, the mutation to be corrected, the related disease and the nucleotide sequence of the associated protospacer and PAM.

[0273] Table 1 - List of exemplary diseases that may be treated using the base editors described herein. The A to be edited in the protospacer is indicated by underlining and the PAM is indicated in bold.

[0274] Some embodiments provide methods for using the improved base editors provided herein. In some embodiments, the base editors are used to introduce a point mutation into a nucleic acid by deaminating a target nucleobase, e.g., an A residue. In some embodiments, the deamination of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease or disorder, e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes. In some embodiments, the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder. For example, in some embodiments, methods are provided herein that employ a DNA editing fusion protein to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease). A deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein.

[0275] In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional gene via genome editing. The nucleobase editing proteins provided herein can be validated for gene editing-based human therapeutics in vitro, e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins comprising a nucleic acid programmable DNA binding protein (e.g., Cas9) and an adenosine deaminase domain can be used to correct any single point G to A or C to T mutation. In the first case, deamination of the mutant A to I corrects the mutation, and in the latter case, deamination of the A that is base-paired with the mutant T, followed by a round of replication, corrects the mutation. Exemplary point mutations that can be corrected are listed in Tables 1.

[0276] The successful correction of point mutations in disease-associated genes and alleles opens up new strategies for gene correction with applications in therapeutics and basic research. Site-specific single-base modification systems like the disclosed fusions of a nucleic acid programmable DNA binding protein and an adenosine deaminase domain also have applications in“reverse” gene therapy, where certain gene functions are purposely suppressed or abolished. In these cases, site-specifically mutating residues that lead to inactivating mutations in a protein, or mutations that inhibit function of the protein can be used to abolish or inhibit protein function

Methods of treatment

[0277] The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a DNA editing fusion protein provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a point mutation as described above, an effective amount of an adenosine deaminase fusion protein that corrects the point mutation or introduces a deactivating mutation into a disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.

[0278] The instant disclosure provides methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by deaminase-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Exemplary suitable diseases and disorders are listed below. It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Exemplary suitable diseases and disorders include, without limitation: 2-methyl-3-hydroxybutyric aciduria; 3 beta- Hydroxysteroid dehydrogenase deficiency; 3-Methylglutaconic aciduria; 3-Oxo-5 alpha- steroid delta 4-dehydrogenase deficiency; 46, XY sex reversal, type 1, 3, and 5; 5- Oxoprolinase deficiency; 6-pyruvoyl-tetrahydropterin synthase deficiency; Aarskog syndrome; Aase syndrome; Achondrogenesis type 2; Achromatopsia 2 and 7; Acquired long QT syndrome; Acrocallosal syndrome, Schinzel type; Acrocapitofemoral dysplasia;

Acrodysostosis 2, with or without hormone resistance; Acroerythrokeratoderma; Acromicric dysplasia; Acth-independent macronodular adrenal hyperplasia 2; Activated PI3K-delta syndrome; Acute intermittent porphyria; deficiency of Acyl-CoA dehydrogenase family, member 9; Adams-Oliver syndrome 5 and 6; Adenine phosphoribosyltransferase deficiency; Adenylate kinase deficiency; hemolytic anemia due to Adenylosuccinate lyase deficiency; Adolescent nephronophthisis; Renal-hepatic-pancreatic dysplasia; Meckel syndrome type 7; Adrenoleukodystrophy; Adult junctional epidermolysis bullosa; Epidermolysis bullosa, junctional, localisata variant; Adult neuronal ceroid lipofuscinosis; Adult neuronal ceroid lipofuscinosis; Adult onset ataxia with oculomotor apraxia; ADULT syndrome;

Afibrinogenemia and congenital Afibrinogenemia; autosomal recessive

Agammaglobulinemia 2; Age-related macular degeneration 3, 6, 11, and 12; Aicardi

Goutieres syndromes 1, 4, and 5; Chilbain lupus 1; Alagille syndromes 1 and 2; Alexander disease; Alkaptonuria; Allan-Herndon-Dudley syndrome; Alopecia universalis congenital; Alpers encephalopathy; Alpha- 1 -antitrypsin deficiency; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndromes; Alzheimer disease, familial, 3, with spastic paraparesis and apraxia; Alzheimer disease, types, 1, 3, and 4; hypocalcification type and hypomaturation type, IIA1 Amelogenesis imperfecta; Aminoacylase 1 deficiency; Amish infantile epilepsy syndrome; Amyloidogenic transthyretin amyloidosis; Amyloid

Cardiomyopathy, Transthyretin-related; Cardiomyopathy; Amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia), 22 (with or without frontotemporal dementia), and 10; Frontotemporal dementia with TDP43 inclusions, TARDBP-related; Andermann syndrome; Andersen Tawil syndrome; Congenital long QT syndrome; Anemia, nonspherocytic hemolytic, due to G6PD deficiency; Angelman syndrome; Severe neonatal- onset encephalopathy with microcephaly; susceptibility to Autism, X-linked 3; Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps; Angiotensin i-converting enzyme, benign serum increase; Aniridia, cerebellar ataxia, and mental retardation;

Anonychia; Antithrombin III deficiency; Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis; Aortic aneurysm, familial thoracic 4, 6, and 9; Thoracic aortic aneurysms and aortic dissections; Multisystemic smooth muscle dysfunction syndrome; Moyamoya disease 5; Aplastic anemia; Apparent mineralocorticoid excess; Arginase deficiency; Argininosuccinate lyase deficiency; Aromatase deficiency; Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10; Primary familial hypertrophic

cardiomyopathy; Arthrogryposis multiplex congenita, distal, X-linked; Arthrogryposis renal dysfunction cholestasis syndrome; Arthrogryposis, renal dysfunction, and cholestasis 2; Asparagine synthetase deficiency; Abnormality of neuronal migration; Ataxia with vitamin E deficiency; Ataxia, sensory, autosomal dominant; Ataxia-telangiectasia syndrome; Hereditary cancer-predisposing syndrome; Atransferrinemia; Atrial fibrillation, familial, 11, 12, 13, and 16; Atrial septal defects 2, 4, and 7 (with or without atrioventricular conduction defects); Atrial standstill 2; Atrioventricular septal defect 4; Atrophia bulborum hereditaria; ATR-X syndrome; Auriculocondylar syndrome 2; Autoimmune disease, multisystem, infantile-onset; Autoimmune lymphoproliferative syndrome, type la; Autosomal dominant hypohidrotic ectodermal dysplasia; Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions 1 and 3; Autosomal dominant torsion dystonia 4; Autosomal recessive centronuclear myopathy; Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; Autosomal recessive cutis laxa type IA and 1B; Autosomal recessive hypohidrotic ectodermal dysplasia syndrome; Ectodermal dysplasia l lb; hypohidrotic/hair/tooth type, autosomal recessive; Autosomal recessive hypophosphatemic bone disease; Axenfeld-Rieger syndrome type 3; Bainbridge-Ropers syndrome; Bannayan-Riley-Ruvalcaba syndrome;

PTEN hamartoma tumor syndrome; Baraitser-Winter syndromes 1 and 2; Barakat syndrome; Bardet-Biedl syndromes 1, 11, 16, and 19; Bare lymphocyte syndrome type 2,

complementation group E; Bartter syndrome antenatal type 2; Bartter syndrome types 3, 3 with hypocalciuria , and 4; Basal ganglia calcification, idiopathic, 4; Beaded hair; Benign familial hematuria; Benign familial neonatal seizures 1 and 2; Seizures, benign familial neonatal, 1, and/or myokymia; Seizures, Early infantile epileptic encephalopathy 7; Benign familial neonatal-infantile seizures; Benign hereditary chorea; Benign scapuloperoneal muscular dystrophy with cardiomyopathy; Bemard-Soulier syndrome, types Al and A2 (autosomal dominant); Bestrophinopathy, autosomal recessive; beta Thalassemia; Bethlem myopathy and Bethlem myopathy 2; Bietti crystalline corneoretinal dystrophy; Bile acid synthesis defect, congenital, 2; Biotinidase deficiency; Birk Barel mental retardation dysmorphism syndrome; Blepharophimosis, ptosis, and epicanthus inversus; Bloom syndrome; Borjeson-Forssman-Lehmann syndrome; Boucher Neuhauser syndrome;

Brachydactyly types Al and A2; Brachydactyly with hypertension; Brain small vessel disease with hemorrhage; Branched-chain ketoacid dehydrogenase kinase deficiency;

Branchiootic syndromes 2 and 3; Breast cancer, early-onset; Breast-ovarian cancer, familial 1, 2, and 4; Brittle cornea syndrome 2; Brody myopathy; Bronchiectasis with or without elevated sweat chloride 3; Brown- Vialetto- Van laere syndrome and Brown- Vialetto- Van Laere syndrome 2; Brugada syndrome; Brugada syndrome 1; Ventricular fibrillation;

Paroxysmal familial ventricular fibrillation; Brugada syndrome and Brugada syndrome 4; Long QT syndrome; Sudden cardiac death; Bull eye macular dystrophy; Stargardt disease 4; Cone-rod dystrophy 12; Bullous ichthyosiform erythroderma; Burn-Mckeown syndrome; Candidiasis, familial, 2, 5, 6, and 8; Carbohydrate-deficient glycoprotein syndrome type I and II; Carbonic anhydrase VA deficiency, hyperammonemia due to; Carcinoma of colon;

Cardiac arrhythmia; Long QT syndrome, LQT1 subtype; Cardioencephalo myopathy, fatal infantile, due to cytochrome c oxidase deficiency; Cardiofaciocutaneous syndrome;

Cardiomyopathy; Danon disease; Hypertrophic cardiomyopathy; Left ventricular

noncompaction cardiomyopathy; Camevale syndrome; Carney complex, type 1; Carnitine acylcarnitine translocase deficiency; Carnitine palmitoyltransferase I , II, II (late onset), and II (infantile) deficiency; Cataract 1, 4, autosomal dominant, autosomal dominant, multiple types, with microcornea, coppock-like, juvenile, with microcornea and glucosuria, and nuclear diffuse nonprogressive; Catecholaminergic polymorphic ventricular tachycardia; Caudal regression syndrome; Cd8 deficiency, familial; Central core disease; Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency; Cerebellar ataxia infantile with progressive external ophthalmoplegi and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome 2; Cerebral amyloid angiopathy, APP-related; Cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations 2; Cerebrooculofacioskeletal syndrome 2; Cerebro-oculo- facio- skeletal syndrome; Cerebroretinal microangiopathy with calcifications and cysts;

Ceroid lipofuscinosis neuronal 2, 6, 7, and 10; Ch\xc3\xa9diak-Higashi syndrome , Chediak- Higashi syndrome, adult type; Charcot-Marie-Tooth disease types 1B, 2B2, 2C, 2F, 21, 2U (axonal), 1C (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF, and X; Scapuloperoneal spinal muscular atrophy; Distal spinal muscular atrophy, congenital nonprogressive; Spinal muscular atrophy, distal, autosomal recessive, 5; CHARGE association; Childhood hypophosphatasia; Adult hypophosphatasia; Cholecystitis; Progressive familial intrahepatic cholestasis 3; Cholestasis, intrahepatic, of pregnancy 3; Cholestanol storage disease; Cholesterol monooxygenase (side-chain cleaving) deficiency; Chondrodysplasia Blomstrand type; Chondrodysplasia punctata 1, X-linked recessive and 2 X-linked dominant; CHOPS syndrome; Chronic granulomatous disease, autosomal recessive cytochrome b-positive, types 1 and 2; Chudley-McCullough syndrome; Ciliary dyskinesia, primary, 7, 11, 15, 20 and 22; Citrullinemia type I; Citrullinemia type I and II; Cleidocranial dysostosis; C-like syndrome; Cockayne syndrome type A, ; Coenzyme Q10 deficiency, primary 1, 4, and 7; Coffin Siris/Intellectual Disability; Coffin-Lowry syndrome; Cohen syndrome, ; Cold-induced sweating syndrome 1; COLE-CARPENTER SYNDROME 2; Combined cellular and humoral immune defects with granulomas; Combined d-2- and 1-2- hydroxyglutaric aciduria; Combined malonic and methylmalonic aciduria; Combined oxidative phosphorylation deficiencies 1, 3, 4, 12, 15, and 25; Combined partial and complete l7-alpha-hydroxylase/ 17, 20-lyase deficiency; Common variable immunodeficiency 9;

Complement component 4, partial deficiency of, due to dysfunctional cl inhibitor;

Complement factor B deficiency; Cone monochromatism; Cone-rod dystrophy 2 and 6;

Cone-rod dystrophy amelogenesis imperfecta; Congenital adrenal hyperplasia and Congenital adrenal hypoplasia, X-linked; Congenital amegakaryocytic thrombocytopenia; Congenital aniridia; Congenital central hypoventilation; Hirschsprung disease 3; Congenital contractural arachnodactyly; Congenital contractures of the limbs and face, hypotonia, and developmental delay; Congenital disorder of glycosylation types 1B, 1D, 1G, 1H, 1J, 1K, 1N, 1P, 2C, 2J,

2K, Ilm; Congenital dyserythropoietic anemia, type I and II; Congenital ectodermal dysplasia of face; Congenital erythropoietic porphyria; Congenital generalized lipodystrophy type 2; Congenital heart disease, multiple types, 2; Congenital heart disease; Interrupted aortic arch; Congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; Non-small cell lung cancer; Neoplasm of ovary; Cardiac conduction defect, nonspecific; Congenital microvillous atrophy; Congenital muscular dystrophy; Congenital muscular dystrophy due to partial LAMA2 deficiency; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, types A2, A7, A8, Al l, and A14; Congenital muscular dystrophy- dystroglycanopathy with mental retardation, types B2, B3, B5, and B15; Congenital muscular dystrophy-dystroglycanopathy without mental retardation, type B5; Congenital muscular hypertrophy-cerebral syndrome; Congenital myasthenic syndrome, acetazolamide- responsive; Congenital myopathy with fiber type disproportion; Congenital ocular coloboma; Congenital stationary night blindness, type 1A, 1B, 1C, 1E, 1F, and 2A; Coproporphyria; Cornea plana 2; Comeal dystrophy, Fuchs endothelial, 4; Comeal endothelial dystrophy type 2; Comeal fragility keratoglobus, blue sclerae and joint hypermobility; Cornelia de Fange syndromes 1 and 5; Coronary artery disease, autosomal dominant 2; Coronary heart disease; Hyperalphalipoproteinemia 2; Cortical dysplasia, complex, with other brain malformations 5 and 6; Cortical malformations, occipital; Corticosteroid-binding globulin deficiency;

Corticosterone methyloxidase type 2 deficiency; Costello syndrome; Cowden syndrome 1; Coxa plana; Craniodiaphyseal dysplasia, autosomal dominant; Craniosynostosis 1 and 4; Craniosynostosis and dental anomalies; Creatine deficiency, X-linked; Crouzon syndrome; Cryptophthalmos syndrome; Cryptorchidism, unilateral or bilateral; Cushing symphalangism; Cutaneous malignant melanoma 1; Cutis laxa with osteodystrophy and with severe pulmonary, gastrointestinal, and urinary abnormalities; Cyanosis, transient neonatal and atypical nephropathic; Cystic fibrosis; Cystinuria; Cytochrome c oxidase i deficiency;

Cytochrome-c oxidase deficiency ; D-2-hydroxyglutaric aciduria 2; Darier disease, segmental; Deafness with labyrinthine aplasia microtia and microdontia (FAMM); Deafness, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17, 20, and 65; Deafness, autosomal recessive 1A, 2, 3, 6, 8, 9, 12, 15, 16, l8b, 22, 28, 31, 44,

49, 63, 77, 86, and 89; Deafness, cochlear, with myopia and intellectual impairment, without vestibular involvement, autosomal dominant, X-linked 2; Deficiency of 2-methylbutyryl-CoA dehydrogenase; Deficiency of 3-hydroxyacyl-CoA dehydrogenase; Deficiency of alpha- mannosidase; Deficiency of aromatic-F-amino-acid decarboxylase; Deficiency of

bisphosphoglycerate mutase; Deficiency of butyryl-CoA dehydrogenase; Deficiency of ferroxidase; Deficiency of galactokinase; Deficiency of guanidinoacetate methyltransferase; Deficiency of hyaluronoglucosaminidase; Deficiency of ribose-5-phosphate isomerase;

Deficiency of steroid 11 -beta-monooxygenase; Deficiency of UDPglucose-hexose-l- phosphate uridylyltransferase; Deficiency of xanthine oxidase; Dejerine-Sottas disease;

Charcot-Marie-Tooth disease, types ID and IVF; Dejerine-Sottas syndrome, autosomal dominant; Dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiency; Desbuquois dysplasia 2; Desbuquois syndrome; DFNA 2 Nonsyndromic Hearing Loss;

Diabetes mellitus and insipidus with optic atrophy and deafness; Diabetes mellitus, type 2, and insulin-dependent, 20; Diamond-Blackfan anemia 1, 5, 8, and 10; Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tufting enteropathy, congenital); Dicarboxylic aminoaciduria; Diffuse palmoplantar keratoderma, Bothnian type; Digitorenocerebral syndrome; Dihydropteridine reductase deficiency; Dilated cardiomyopathy 1A, 1AA, 1C, 1G, 1BB, 1DD, 1FF, 1HH, II, 1KK, 1N, 1S, 1Y, and 3B; Left ventricular noncompaction 3; Disordered steroidogenesis due to cytochrome p450 oxidoreductase deficiency; Distal arthrogryposis type 2B; Distal hereditary motor neuronopathy type 2B; Distal myopathy Markesbery-Griggs type; Distal spinal muscular atrophy, X-linked 3; Distichiasis- lymphedema syndrome; Dominant dystrophic epidermolysis bullosa with absence of skin; Dominant hereditary optic atrophy; Donnai Barrow syndrome; Dopamine beta hydroxylase deficiency; Dopamine receptor d2, reduced brain density of; Dowling-degos disease 4; Doyne honeycomb retinal dystrophy; Malattia leventinese; Duane syndrome type 2; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Becker muscular dystrophy; Dysfibrinogenemia; Dyskeratosis congenita autosomal dominant and autosomal dominant, 3; Dyskeratosis congenita, autosomal recessive, 1, 3, 4, and 5; Dyskeratosis congenita X-linked; Dyskinesia, familial, with facial myokymia; Dysplasminogenemia; Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (Dopa-responsive type ), 10, 12, 16, 25, 26 (Myoclonic); Seizures, benign familial infantile, 2; Early infantile epileptic encephalopathy 2, 4, 7, 9, 10,

11, 13, and 14; Atypical Rett syndrome; Early T cell progenitor acute lymphoblastic leukemia; Ectodermal dysplasia skin fragility syndrome; Ectodermal dysplasia-syndactyly syndrome 1; Ectopia lentis, isolated autosomal recessive and dominant; Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3; Ehlers-Danlos syndrome type 7 (autosomal recessive), classic type, type 2 (progeroid ), hydroxylysine-deficient, type 4, type 4 variant, and due to tenascin-X deficiency; Eichsfeld type congenital muscular dystrophy; Endocrine-cerebroosteodysplasia; Enhanced s-cone syndrome; Enlarged vestibular aqueduct syndrome; Enterokinase deficiency; Epidermodysplasia verruciformis; Epidermolysa bullosa simplex and limb girdle muscular dystrophy, simplex with mottled pigmentation, simplex with pyloric atresia, simplex, autosomal recessive, and with pyloric atresia; Epidermolytic palmoplantar keratoderma; Familial febrile seizures 8; Epilepsy, childhood absence 2, 12 (idiopathic generalized, susceptibility to) 5 (nocturnal frontal lobe), nocturnal frontal lobe type 1, partial, with variable foci, progressive myoclonic 3, and X-linked, with variable learning disabilities and behavior disorders; Epileptic encephalopathy, childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Epiphyseal dysplasia, multiple, with myopia and conductive deafness; Episodic ataxia type 2; Episodic pain syndrome, familial, 3; Epstein syndrome; Fechtner syndrome; Erythropoietic protoporphyria; Estrogen resistance; Exudative vitreoretinopathy 6; Fabry disease and Fabry disease, cardiac variant; Factor H, VII, X, v and factor viii, combined deficiency of 2, xiii, a subunit, deficiency; Familial adenomatous polyposis 1 and 3; Familial amyloid nephropathy with urticaria and deafness; Familial cold urticarial; Familial aplasia of the vermis; Familial benign pemphigus; Familial cancer of breast; Breast cancer, susceptibility to; Osteosarcoma; Pancreatic cancer 3; Familial cardiomyopathy; Familial cold autoinflammatory syndrome 2; Familial colorectal cancer; Familial exudative vitreoretinopathy, X-linked; Familial hemiplegic migraine types 1 and 2; Familial hypercholesterolemia; Familial hypertrophic cardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24; Familial hypokalemia-hypomagnesemia; Familial hypoplastic, glomerulocystic kidney; Familial infantile myasthenia; Familial juvenile gout; Familial Mediterranean fever and Familial mediterranean fever, autosomal dominant; Familial porencephaly; Familial porphyria cutanea tarda; Familial pulmonary capillary hemangiomatosis; Familial renal glucosuria; Familial renal hypouricemia; Familial restrictive cardiomyopathy 1; Familial type 1 and 3 hyperlipoproteinemia; Fanconi anemia, complementation group E, I, N, and O; Fanconi-Bickel syndrome; Favism, susceptibility to; Febrile seizures, familial, 11; Feingold syndrome 1; Fetal hemoglobin quantitative trait locus 1; FG syndrome and FG syndrome 4; Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with or without extraocular

involvement), 3b; Fish-eye disease; Fleck comeal dystrophy; Floating-Harbor syndrome; Focal epilepsy with speech disorder with or without mental retardation; Focal segmental glomerulosclerosis 5; Forebrain defects; Frank Ter Haar syndrome; Borrone Di Rocco Crovato syndrome; Frasier syndrome; Wilms tumor 1; Freeman-Sheldon syndrome;

Frontometaphyseal dysplasia land 3; Frontotemporal dementia; Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4; Frontotemporal Dementia Chromosome 3- Linked and Frontotemporal dementia ubiquitin-positive; Fructose-biphosphatase deficiency; Fuhrmann syndrome; Gamma-aminobutyric acid transaminase deficiency; Gamstorp- Wohlfart syndrome; Gaucher disease type 1 and Subacute neuronopathic; Gaze palsy, familial horizontal, with progressive scoliosis; Generalized dominant dystrophic

epidermolysis bullosa; Generalized epilepsy with febrile seizures plus 3, type 1, type 2; Epileptic encephalopathy Lennox-Gastaut type; Giant axonal neuropathy; Glanzmann thrombasthenia; Glaucoma 1, open angle, e, F, and G; Glaucoma 3, primary congenital, d; Glaucoma, congenital and Glaucoma, congenital, Coloboma; Glaucoma, primary open angle, juvenile-onset; Glioma susceptibility 1; Glucose transporter type 1 deficiency syndrome; Glucose-6-phosphate transport defect; GLUT1 deficiency syndrome 2; Epilepsy, idiopathic generalized, susceptibility to, 12; Glutamate formiminotransferase deficiency; Glutaric acidemia IIA and IIB; Glutaric aciduria, type 1; Gluthathione synthetase deficiency;

Glycogen storage disease 0 ( muscle), II (adult form), IXa2, IXc, type 1A; type II, type IV,

IV (combined hepatic and myopathic), type V, and type VI; Goldmann-Favre syndrome; Gordon syndrome; Gorlin syndrome; Holoprosencephaly sequence; Holoprosencephaly 7; Granulomatous disease, chronic, X-linked, variant; Granulosa cell tumor of the ovary; Gray platelet syndrome; Griscelli syndrome type 3; Groenouw comeal dystrophy type I; Growth and mental retardation, mandibulofacial dysostosis, microcephaly, and cleft palate; Growth hormone deficiency with pituitary anomalies; Growth hormone insensitivity with

immunodeficiency; GTP cyclohydrolase I deficiency; Hajdu-Cheney syndrome; Hand foot uterus syndrome; Hearing impairment; Hemangioma, capillary infantile; Hematologic neoplasm; Hemochromatosis type 1, 2B, and 3; Microvascular complications of diabetes 7; Transferrin serum level quantitative trait locus 2; Hemoglobin H disease, nondeletional; Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency;

Hemophagocytic lymphohistiocytosis, familial, 2; Hemophagocytic lymphohistiocytosis, familial, 3; Heparin cofactor II deficiency; Hereditary acrodermatitis enteropathica;

Hereditary breast and ovarian cancer syndrome; Ataxia-telangiectasia-like disorder;

Hereditary diffuse gastric cancer; Hereditary diffuse leukoencephalopathy with spheroids; Hereditary factors II, IX, VIII deficiency disease; Hereditary hemorrhagic telangiectasia type 2; Hereditary insensitivity to pain with anhidrosis; Hereditary lymphedema type I; Hereditary motor and sensory neuropathy with optic atrophy; Hereditary myopathy with early respiratory failure; Hereditary neuralgic amyotrophy; Hereditary Nonpolyposis Colorectal Neoplasms; Lynch syndrome I and II; Hereditary pancreatitis; Pancreatitis, chronic, susceptibility to; Hereditary sensory and autonomic neuropathy type IIB amd IIA; Hereditary sideroblastic anemia; Hermansky-Pudlak syndrome 1, 3, 4, and 6; Heterotaxy, visceral, 2, 4, and 6, autosomal; Heterotaxy, visceral, X-linked; Heterotopia; Histiocytic medullary reticulosis; Histiocytosis-lymphadenopathy plus syndrome; Holocarboxylase synthetase deficiency; Holoprosencephaly 2, 3,7, and 9; Holt-Oram syndrome; Homocysteinemia due to MTHFR deficiency, CBS deficiency, and Homocystinuria, pyridoxine-responsive;

Homocystinuria-Megaloblastic anemia due to defect in cobalamin metabolism, cblE complementation type; Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome; Hydrocephalus; Hyperammonemia, type III; Hypercholesterolaemia and

Hypercholesterolemia, autosomal recessive; Hyperekplexia 2 and Hyperekplexia hereditary; Hyperferritinemia cataract syndrome; Hyperglycinuria; Hyperimmunoglobulin D with periodic fever; Mevalonic aciduria; Hyperimmunoglobulin E syndrome; Hyperinsulinemic hypoglycemia familial 3, 4, and 5; Hyperinsulinism-hyperammonemia syndrome;

Hyperlysinemia; Hypermanganesemia with dystonia, polycythemia and cirrhosis;

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; Hyperparathyroidism 1 and 2; Hyperparathyroidism, neonatal severe; Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency, BH4-deficient, D, and non-pku; Hyperphosphatasia with mental retardation syndrome 2, 3, and 4; Hypertrichotic osteochondrodysplasia;

Hypobetalipoproteinemia, familial, associated with apob32; Hypocalcemia, autosomal dominant 1; Hypocalciuric hypercalcemia, familial, types 1 and 3; Hypochondrogenesis; Hypochromic microcytic anemia with iron overload; Hypoglycemia with deficiency of glycogen synthetase in the liver; Hypogonadotropic hypogonadism 11 with or without anosmia; Hypohidrotic ectodermal dysplasia with immune deficiency; Hypohidrotic X-linked ectodermal dysplasia; Hypokalemic periodic paralysis 1 and 2; Hypomagnesemia 1, intestinal; Hypomagnesemia, seizures, and mental retardation; Hypomyelinating

leukodystrophy 7; Hypoplastic left heart syndrome; Atrioventricular septal defect and common atrioventricular junction; Hypospadias 1 and 2, X-linked; Hypothyroidism, congenital, nongoitrous, 1; Hypotrichosis 8 and 12; Hypotrichosis-lymphedema- telangiectasia syndrome; I blood group system; Ichthyosis bullosa of Siemens; Ichthyosis exfoliativa; Ichthyosis prematurity syndrome; Idiopathic basal ganglia calcification 5; Idiopathic fibrosing alveolitis, chronic form; Dyskeratosis congenita, autosomal dominant, 2 and 5; Idiopathic hypercalcemia of infancy; Immune dysfunction with T-cell inactivation due to calcium entry defect 2; Immunodeficiency 15, 16, 19, 30, 31C, 38, 40, 8, due to defect in cd3-zeta, with hyper IgM type 1 and 2, and X-Linked, with magnesium defect, Epstein-Barr virus infection, and neoplasia; Immunodeficiency-centromeric instability-facial anomalies syndrome 2; Inclusion body myopathy 2 and 3; Nonaka myopathy; Infantile convulsions and paroxysmal choreoathetosis, familial; Infantile cortical hyperostosis; Infantile GM1 gangliosidosis; Infantile hypophosphatasia; Infantile nephronophthisis; Infantile nystagmus, X-linked; Infantile Parkinsonism-dystonia; Infertility associated with multi-tailed

spermatozoa and excessive DNA; Insulin resistance; Insulin-resistant diabetes mellitus and acanthosis nigricans; Insulin-dependent diabetes mellitus secretory diarrhea syndrome;

Interstitial nephritis, karyomegalic; Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies; Iodotyrosyl coupling defect; IRAK4 deficiency; Iridogoniodysgenesis dominant type and type 1; Iron accumulation in brain; Ischiopatellar dysplasia; Islet cell hyperplasia; Isolated 17, 20-lyase deficiency; Isolated lutropin deficiency; Isovaleryl-CoA dehydrogenase deficiency; Jankovic Rivera syndrome; Jervell and Lange-Nielsen syndrome 2; Joubert syndrome 1, 6, 7, 9/15 (digenic), 14, 16, and 17, and Orofaciodigital syndrome xiv; Junctional epidermolysis bullosa gravis of Herlitz; Juvenile GM>l< gangliosidosis; Juvenile polyposis syndrome; Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome; Juvenile retinoschisis; Kabuki make-up syndrome; Kallmann syndrome 1, 2, and 6; Delayed puberty; Kanzaki disease; Karak syndrome;

Kartagener syndrome; Kenny-Caffey syndrome type 2; Keppen-Lubinsky syndrome;

Keratoconus 1; Keratosis follicularis; Keratosis palmoplantaris striata 1; Kindler syndrome; L-2-hydroxyglutaric aciduria; Larsen syndrome, dominant type; Lattice corneal dystrophy Type III; Leber amaurosis; Zellweger syndrome; Peroxisome biogenesis disorders; Zellweger syndrome spectrum; Leber congenital amaurosis 11, 12, 13, 16, 4, 7, and 9; I ber optic atrophy; Aminoglycoside-induced deafness; Deafness, nonsyndromic sensorineural, mitochondrial; Left ventricular noncompaction 5; Left-right axis malformations; Leigh disease; Mitochondrial short-chain Enoyl-CoA Hydratase 1 deficiency; Leigh syndrome due to mitochondrial complex I deficiency; Leiner disease; Leri Weill dyschondrosteosis; Lethal congenital contracture syndrome 6; Leukocyte adhesion deficiency type I and III;

Leukodystrophy, Hypomyelinating, 11 and 6; Leukoencephalopathy with ataxia, with Brainstem and Spinal Cord Involvement and Lactate Elevation, with vanishing white matter, and progressive, with ovarian failure; Leukonychia totalis; Lewy body dementia;

Lichtenstein-Knorr Syndrome; Li-Fraumeni syndrome 1; Lig4 syndrome; Limb-girdle muscular dystrophy, type 1B, 2A, 2B, 2D, Cl, C5, C9, C14; Congenital muscular dystrophy- dystroglycanopathy with brain and eye anomalies, type A14 and B14; Lipase deficiency combined; Lipid proteinosis; Lipodystrophy, familial partial, type 2 and 3; Lissencephaly 1, 2 (X-linked), 3, 6 (with microcephaly), X-linked; Subcortical laminar heterotopia, X-linked; Liver failure acute infantile; Loeys-Dietz syndrome 1, 2, 3; Long QT syndrome 1, 2, 2/9, 2/5, (digenic), 3, 5 and 5, acquired, susceptibility to; Lung cancer; Lymphedema, hereditary, id; Lymphedema, primary, with myelodysplasia; Lymphoproliferative syndrome 1, 1 (X-linked), and 2; Lysosomal acid lipase deficiency; Macrocephaly, macrosomia, facial dysmorphism syndrome; Macular dystrophy, vitelliform, adult-onset; Malignant hyperthermia susceptibility type 1; Malignant lymphoma, non-Hodgkin; Malignant melanoma; Malignant tumor of prostate; Mandibuloacral dysostosis; Mandibuloacral dysplasia with type A or B

lipodystrophy, atypical; Mandibulofacial dysostosis, Treacher Collins type, autosomal recessive; Mannose-binding protein deficiency; Maple syrup urine disease type 1A and type 3; Marden Walker like syndrome; Marfan syndrome; Marinesco-Sj\xc3\xb6gren syndrome; Martsolf syndrome; Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3, and type 9; May-Hegglin anomaly; MYH9 related disorders; Sebastian syndrome; McCune- Albright syndrome; Somatotroph adenoma; Sex cord-stromal tumor; Cushing syndrome; McKusick Kaufman syndrome; McLeod neuroacanthocytosis syndrome; Meckel-Gruber syndrome; Medium-chain acyl-coenzyme A dehydrogenase deficiency; Medulloblastoma; Megalencephalic leukoencephalopathy with subcortical cysts land 2a; Megalencephaly cutis marmorata telangiectatica congenital; PIK3CA Related Overgrowth Spectrum;

Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2; Megaloblastic anemia, thiamine-responsive, with diabetes mellitus and sensorineural deafness; Meier-Gorlin syndromes land 4; Melnick-Needles syndrome; Meningioma; Mental retardation, X-linked, 3, 21, 30, and 72; Mental retardation and microcephaly with pontine and cerebellar hypoplasia; Mental retardation X-linked syndromic 5; Mental retardation, anterior maxillary protrusion, and strabismus; Mental retardation, autosomal dominant 12, 13, 15, 24, 3, 30, 4,

5, 6, and 9; Mental retardation, autosomal recessive 15, 44, 46, and 5; Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations; Mental retardation, syndromic, Claes-Jensen type, X-linked; Mental retardation, X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type; Merosin deficient congenital muscular dystrophy; Metachromatic leukodystrophy juvenile, late infantile, and adult types;

Metachromatic leukodystrophy; Metatrophic dysplasia; Methemoglobinemia types I and 2; Methionine adenosyltransferase deficiency, autosomal dominant; Methylmalonic acidemia with homocystinuria, ; Methylmalonic aciduria cblB type, ; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency; METHYLMALONIC ACIDURIA, mut(0) TYPE; Microcephalic osteodysplastic primordial dwarfism type 2; Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation; Microcephaly, hiatal hernia and nephrotic syndrome; Microcephaly; Hypoplasia of the corpus callosum; Spastic paraplegia 50, autosomal recessive; Global developmental delay; CNS hypomyelination; Brain atrophy; Microcephaly, normal intelligence and immunodeficiency; Microcephaly-capillary malformation syndrome; Microcytic anemia; Microphthalmia syndromic 5, 7, and 9;

Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6; Microspherophakia; Migraine, familial basilar; Miller syndrome; Minicore myopathy with external ophthalmoplegia;

Myopathy, congenital with cores; Mitchell-Riley syndrome; mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase deficiency; Mitochondrial complex I, II, III, III (nuclear type 2, 4, or 8) deficiency; Mitochondrial DNA depletion syndrome 11, 12 (cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type); Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and 13 (encephalomyopathic type); Mitochondrial phosphate carrier and pyruvate carrier deficiency; Mitochondrial trifunctional protein deficiency; Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Miyoshi muscular dystrophy 1; Myopathy, distal, with anterior tibial onset; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency, complementation group A; Mowat-Wilson syndrome; Mucolipidosis III Gamma; Mucopolysaccharidosis type VI, type VI (severe), and type VII; Mucopolysaccharidosis, MPS-I-H/S, MPS-II, MPS-III-A, MPS-III-B, MPS-III-C, MPS-IV-A, MPS-IV-B; Retinitis Pigmentosa 73; Gangliosidosis GM1 typel (with cardiac involvenment) 3; Multicentric osteolysis nephropathy; Multicentric osteolysis, nodulosis and arthropathy; Multiple congenital anomalies; Atrial septal defect 2; Multiple congenital anomalies -hypotonia- seizures syndrome 3; Multiple Cutaneous and Mucosal Venous Malformations; Multiple endocrine neoplasia, types land 4; Multiple epiphyseal dysplasia 5 or Dominant; Multiple gastrointestinal atresias; Multiple pterygium syndrome Escobar type; Multiple sulfatase deficiency; Multiple synostoses syndrome 3; Muscle AMP deaminase deficiency; Muscle eye brain disease; Muscular dystrophy, congenital, megaconial type; Myasthenia, familial infantile, 1; Myasthenic Syndrome, Congenital, 11, associated with acetylcholine receptor deficiency; Myasthenic Syndrome, Congenital, 17, 2A (slow-channel), 4B (fast-channel), and without tubular aggregates; Myeloperoxidase deficiency; MYH-associated polyposis;

Endometrial carcinoma; Myocardial infarction 1; Myoclonic dystonia; Myoclonic-Atonic Epilepsy; Myoclonus with epilepsy with ragged red fibers; Myofibrillar myopathy 1 and ZASP-related; Myoglobinuria, acute recurrent, autosomal recessive; Myoneural

gastrointestinal encephalopathy syndrome; Cerebellar ataxia infantile with progressive external ophthalmoplegia; Mitochondrial DNA depletion syndrome 4B, MNGIE type;

Myopathy, centronuclear, 1, congenital, with excess of muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1, mitochondrial progressive with congenital cataract, hearing loss, and developmental delay, and tubular aggregate, 2; Myopia 6; Myosclerosis, autosomal recessive; Myotonia congenital; Congenital myotonia, autosomal dominant and recessive forms; Nail-patella syndrome; Nance-Horan syndrome; Nanophthalmos 2; Navajo neurohepatopathy; Nemaline myopathy 3 and 9; Neonatal hypotonia; Intellectual disability; Seizures; Delayed speech and language development; Mental retardation, autosomal dominant 31; Neonatal intrahepatic cholestasis caused by citrin deficiency; Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus, X-linked; Nephrolithiasis/osteoporosis, hypophosphatemic, 2; Nephronophthisis 13, 15 and 4; Infertility; Cerebello-oculo-renal syndrome (nephronophthisis, oculomotor apraxia and cerebellar abnormalities); Nephrotic syndrome, type 3, type 5, with or without ocular abnormalities, type 7, and type 9; Nestor- Guillermo progeria syndrome; Neu-Laxova syndrome 1; Neurodegeneration with brain iron accumulation 4 and 6; Neuroferritinopathy; Neurofibromatosis, type land type 2;

Neurofibrosarcoma; Neurohypophyseal diabetes insipidus; Neuropathy, Hereditary Sensory, Type IC; Neutral 1 amino acid transport defect; Neutral lipid storage disease with myopathy; Neutrophil immunodeficiency syndrome; Nicolaides-Baraitser syndrome; Niemann-Pick disease type Cl, C2, type A, and type Cl, adult form; Non-ketotic hyperglycinemia; Noonan syndrome 1 and 4, LEOPARD syndrome 1; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia; Normokalemic periodic paralysis, potassium-sensitive; Norum disease; Epilepsy, Hearing Loss, And Mental Retardation Syndrome; Mental Retardation, X-Linked 102 and syndromic 13; Obesity; Ocular albinism, type I; Oculocutaneous albinism type 1B, type 3, and type 4; Oculodentodigital dysplasia;

Odontohypophosphatasia; Odontotrichomelic syndrome; Oguchi disease; Oligodontia- colorectal cancer syndrome; Opitz G/BBB syndrome; Optic atrophy 9; Oral-facial-digital syndrome; Ornithine aminotransferase deficiency; Orofacial cleft 11 and 7, Cleft lip/palate- ectodermal dysplasia syndrome; Orstavik Lindemann Solberg syndrome; Osteoarthritis with mild chondrodysplasia; Osteochondritis dissecans; Osteogenesis imperfecta type 12, type 5, type 7, type 8, type I, type III, with normal sclerae, dominant form, recessive perinatal lethal; Osteopathia striata with cranial sclerosis; Osteopetrosis autosomal dominant type 1 and 2, recessive 4, recessive 1, recessive 6; Osteoporosis with pseudoglioma; Oto-palato-digital syndrome, types I and II; Ovarian dysgenesis 1; Ovarioleukodystrophy; Pachyonychia congenita 4 and type 2; Paget disease of bone, familial; Pallister-Hall syndrome;

Palmoplantar keratoderma, nonepidermolytic, focal or diffuse; Pancreatic agenesis and congenital heart disease; Papillon-Lef\xc3\xa8vre syndrome; Paragangliomas 3;

Paramyotonia congenita of von Eulenburg; Parathyroid carcinoma; Parkinson disease 14, 15, 19 (juvenile-onset), 2, 20 (early-onset), 6, (autosomal recessive early-onset, and 9; Partial albinism; Partial hypoxanthine-guanine phosphoribosyltransferase deficiency; Patterned dystrophy of retinal pigment epithelium; PC-K6a; Pelizaeus-Merzbacher disease; Pendred syndrome; Peripheral demyelinating neuropathy, central dysmyelination; Hirschsprung disease; Permanent neonatal diabetes mellitus; Diabetes mellitus, permanent neonatal, with neurologic features; Neonatal insulin-dependent diabetes mellitus; Maturity-onset diabetes of the young, type 2; Peroxisome biogenesis disorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B;

Perrault syndrome 4; Perry syndrome; Persistent hyperinsulinemic hypoglycemia of infancy; familial hyperinsulinism; Phenotypes; Phenylketonuria; Pheochromocytoma; Hereditary Paraganglioma- Pheochromocytoma Syndromes; Paragangliomas 1; Carcinoid tumor of intestine; Cowden syndrome 3; Phosphoglycerate dehydrogenase deficiency;

Phosphoglycerate kinase 1 deficiency; Photosensitive trichothiodystrophy; Phytanic acid storage disease; Pick disease; Pierson syndrome; Pigmentary retinal dystrophy; Pigmented nodular adrenocortical disease, primary, 1; Pilomatrixoma; Pitt- Hopkins syndrome; Pituitary dependent hypercortisolism; Pituitary hormone deficiency, combined 1, 2, 3, and 4;

Plasminogen activator inhibitor type 1 deficiency; Plasminogen deficiency, type I; Platelet- type bleeding disorder 15 and 8; Poikiloderma, hereditary fibrosing, with tendon contractures, myopathy, and pulmonary fibrosis; Polycystic kidney disease 2, adult type, and infantile type; Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy;

Polyglucosan body myopathy 1 with or without immunodeficiency; Polymicrogyria, asymmetric, bilateral frontoparietal; Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract; Pontocerebellar hypoplasia type 4; Popliteal pterygium syndrome; Porencephaly 2; Porokeratosis 8, disseminated superficial actinic type; Porphobilinogen synthase deficiency; Porphyria cutanea tarda; Posterior column ataxia with retinitis pigmentosa; Posterior polar cataract type 2; Prader-Willi-like syndrome; Premature ovarian failure 4, 5, 7, and 9; Primary autosomal recessive microcephaly 10, 2, 3, and 5; Primary ciliary dyskinesia 24; Primary dilated cardiomyopathy; Left ventricular noncompaction 6; 4, Left ventricular noncompaction 10; Paroxysmal atrial fibrillation; Primary hyperoxaluria, type I, type, and type III; Primary hypertrophic osteoarthropathy, autosomal recessive 2; Primary hypomagnesemia; Primary open angle glaucoma juvenile onset 1; Primary pulmonary hypertension; Primrose syndrome; Progressive familial heart block type 1B; Progressive familial intrahepatic cholestasis 2 and 3; Progressive intrahepatic cholestasis; Progressive myoclonus epilepsy with ataxia; Progressive pseudorheumatoid dysplasia;

Progressive sclerosing poliodystrophy; Prolidase deficiency; Proline dehydrogenase deficiency; Schizophrenia 4; Properdin deficiency, X-linked; Propionic academia; Proprotein convertase 1/3 deficiency; Prostate cancer, hereditary, 2; Protan defect; Proteinuria; Finnish congenital nephrotic syndrome; Proteus syndrome; Breast adenocarcinoma;

Pseudoachondroplastic spondyloepiphyseal dysplasia syndrome; Pseudohypoaldosteronism type 1 autosomal dominant and recessive and type 2; Pseudohypoparathyroidism type 1A, Pseudopseudohypoparathyroidism; Pseudoneonatal adrenoleukodystrophy; Pseudoprimary hyperaldosteronism; Pseudoxanthoma elasticum; Generalized arterial calcification of infancy 2; Pseudoxanthoma elasticum-like disorder with multiple coagulation factor deficiency; Psoriasis susceptibility 2; PTEN hamartoma tumor syndrome; Pulmonary arterial hypertension related to hereditary hemorrhagic telangiectasia; Pulmonary Fibrosis And/Or Bone Marrow Failure, Telomere-Related, 1 and 3; Pulmonary hypertension, primary, 1, with hereditary hemorrhagic telangiectasia; Purine-nucleoside phosphorylase deficiency; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase El -alpha deficiency; Pyruvate kinase deficiency of red cells; Raine syndrome; Rasopathy; Recessive dystrophic epidermolysis bullosa; Nail disorder, nonsyndromic congenital, 8; Reifenstein syndrome; Renal adysplasia; Renal carnitine transport defect; Renal coloboma syndrome; Renal dysplasia; Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia; Renal tubular acidosis, distal, autosomal recessive, with late-onset sensorineural hearing loss, or with hemolytic anemia; Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; Retinal cone dystrophy 3B; Retinitis pigmentosa; Retinitis pigmentosa 10, 11, 12, 14, 15, 17, and 19; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48, 66, 7, 70, 72; Retinoblastoma; Rett disorder; Rhabdoid tumor predisposition syndrome 2;

Rhegmatogenous retinal detachment, autosomal dominant; Rhizomelic chondrodysplasia punctata type 2 and type 3; Roberts-SC phocomelia syndrome; Robinow Sorauf syndrome; Robinow syndrome, autosomal recessive, autosomal recessive, with brachy-syn-polydactyly; Rothmund-Thomson syndrome; Rapadilino syndrome; RRM2B -related mitochondrial disease; Rubinstein-Taybi syndrome; Salla disease; Sandhoff disease, adult and infantil types; Sarcoidosis, early-onset; Blau syndrome; Schindler disease, type 1; Schizencephaly;

Schizophrenia 15; Schneckenbecken dysplasia; Schwannomatosis 2; Schwartz Jampel syndrome type 1; Sclerocornea, autosomal recessive; Sclerosteosis; Secondary

hypothyroidism; Segawa syndrome, autosomal recessive; Senior-Loken syndrome 4 and 5, ; Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis; Sepiapterin reductase deficiency; SeSAME syndrome; Severe combined immunodeficiency due to ADA

deficiency, with microcephaly, growth retardation, and sensitivity to ionizing radiation, atypical, autosomal recessive, T cell-negative, B cell-positive, NK cell-negative of NK- positive; Partial adenosine deaminase deficiency; Severe congenital neutropenia; Severe congenital neutropenia 3, autosomal recessive or dominant; Severe congenital neutropenia and 6, autosomal recessive; Severe myoclonic epilepsy in infancy; Generalized epilepsy with febrile seizures plus, types 1 and 2; Severe X-linked myotubular myopathy; Short QT syndrome 3; Short stature with nonspecific skeletal abnormalities; Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities; Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis; Primordial dwarfism; Short-rib thoracic dysplasia 11 or 3 with or without polydactyly; Sialidosis type I and II; Silver spastic paraplegia syndrome; Slowed nerve conduction velocity, autosomal dominant; Smith-Lemli-Opitz syndrome;

Snyder Robinson syndrome; Somatotroph adenoma; Prolactinoma; familial, Pituitary adenoma predisposition; Sotos syndrome 1 or 2; Spastic ataxia 5, autosomal recessive, Charlevoix- Saguenay type, 1,10, or 11, autosomal recessive; Amyotrophic lateral sclerosis type 5; Spastic paraplegia 15, 2, 3, 35, 39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Bile acid synthesis defect, congenital, 3; Spermatogenic failure 11, 3, and 8;

Spherocytosis types 4 and 5; Spheroid body myopathy; Spinal muscular atrophy, lower extremity predominant 2, autosomal dominant; Spinal muscular atrophy, type II;

Spinocerebellar ataxia 14, 21, 35, 40, and 6; Spinocerebellar ataxia autosomal recessive 1 and 16; Splenic hypoplasia; Spondylocarpotarsal synostosis syndrome; Spondylocheirodysplasia, Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type, with congenital joint dislocations, short limb-hand type, Sedaghatian type, with cone-rod dystrophy, and Kozlowski type; Parastremmatic dwarfism; Stargardt disease 1; Cone-rod dystrophy 3;

Stickler syndrome type 1; Kniest dysplasia; Stickler syndrome, types l(nonsyndromic ocular) and 4; Sting-associated vasculopathy, infantile-onset; Stormorken syndrome; Sturge -Weber syndrome, Capillary malformations, congenital, 1 ; Succinyl-CoA acetoacetate transferase deficiency; Sucrase-isomaltase deficiency; Sudden infant death syndrome; Sulfite oxidase deficiency, isolated; Supravalvar aortic stenosis; Surfactant metabolism dysfunction, pulmonary, 2 and 3; Symphalangism, proximal, lb; Syndactyly Cenani Lenz type;

Syndactyly type 3; Syndromic X-linked mental retardation 16; Talipes equinovarus; Tangier disease; TARP syndrome; Tay-Sachs disease, Bl variant, Gm2-gangliosidosis (adult), Gm2- gangliosidosis (adult-onset); Temtamy syndrome; Tenorio Syndrome; Terminal osseous dysplasia; Testosterone l7-beta-dehydrogenase deficiency; Tetraamelia, autosomal recessive; Tetralogy of Fallot; Hypoplastic left heart syndrome 2; Truncus arteriosus; Malformation of the heart and great vessels; Ventricular septal defect 1; Thiel-Behnke corneal dystrophy; Thoracic aortic aneurysms and aortic dissections; Marfanoid habitus; Three M syndrome 2; Thrombocytopenia, platelet dysfunction, hemolysis, and imbalanced globin synthesis;

Thrombocytopenia, X-linked; Thrombophilia, hereditary, due to protein C deficiency, autosomal dominant and recessive; Thyroid agenesis; Thyroid cancer, follicular; Thyroid hormone metabolism, abnormal; Thyroid hormone resistance, generalized, autosomal dominant; Thyrotoxic periodic paralysis and Thyrotoxic periodic paralysis 2; Thyrotropin releasing hormone resistance, generalized; Timothy syndrome; TNF receptor-associated periodic fever syndrome (TRAPS); Tooth agenesis, selective, 3 and 4; Torsades de pointes; Townes-Brocks-branchiootorenal-like syndrome; Transient bullous dermolysis of the newborn; Treacher collins syndrome 1; Trichomegaly with mental retardation, dwarfism and pigmentary degeneration of retina; Trichorhinophalangeal dysplasia type I;

Trichorhinophalangeal syndrome type 3; Trimethylaminuria; Tuberous sclerosis syndrome; Lymphangiomyomatosis; Tuberous sclerosis 1 and 2; Tyrosinase-negative oculocutaneous albinism; Tyrosinase-positive oculocutaneous albinism; Tyrosinemia type I; UDPglucose-4- epimerase deficiency; Ullrich congenital muscular dystrophy; Ulna and fibula absence of with severe limb deficiency; Upshaw-Schulman syndrome; Urocanate hydratase deficiency; Usher syndrome, types 1, 1B, 1D, 1G, 2A, 2C, and 2D; Retinitis pigmentosa 39; UV- sensitive syndrome; Van der Woude syndrome; Van Maldergem syndrome 2; Hennekam lymphangiectasia-lymphedema syndrome 2; Variegate porphyria; Ventriculomegaly with cystic kidney disease; Verheij syndrome; Very long chain acyl-CoA dehydrogenase deficiency; Vesicoureteral reflux 8; Visceral heterotaxy 5, autosomal; Visceral myopathy; Vitamin D-dependent rickets, types land 2; Vi tel 1i form dystrophy ; von Willebrand disease type 2M and type 3; Waardenburg syndrome type 1, 4C, and 2E (with neurologic

involvement); Klein-Waardenberg syndrome; Walker-Warburg congenital muscular dystrophy; Warburg micro syndrome 2 and 4; Warts, hypogammaglobulinemia, infections, and myelokathexis; Weaver syndrome; Weill-Marchesani syndrome 1 and 3; Weill- Marchesani-like syndrome; Weissenbacher-Zweymuller syndrome; Werdnig-Hoffmann disease; Charcot-Marie-Tooth disease; Wemer syndrome; WFSl-Related Disorders;

Wiedemann- Steiner syndrome; Wilson disease; Wolfram-like syndrome, autosomal dominant; Worth disease; Van Buchem disease type 2; Xeroderma pigmentosum, complementation group b, group D, group E, and group G; X-linked agammaglobulinemia; X-linked hereditary motor and sensory neuropathy; X-linked ichthyosis with steryl-sulfatase deficiency; X-linked periventricular heterotopia; Oto-palato-digital syndrome, type I; X- linked severe combined immunodeficiency; Zimmermann-Laband syndrome and

Zimmermann-Laband syndrome 2; and Zonular pulverulent cataract 3.

[0279] The instant disclosure provides lists of genes comprising pathogenic G to A or C to T mutations. Such pathogenic G to A or C to T mutations may be corrected using the methods and compositions provided herein, for example by mutating the A to a G, and/or the T to a C, thereby restoring gene function. Table 2 includes exemplary mutations that can be corrected using base editors described herein. Table 2 includes the gene symbol, the associated phenotype, the mutation to be corrected and exemplary gRNA sequences which may be used to correct the mutations. The gRNA sequences provided in Table 2 are sequences that encode RNA that can direct Cas9, or any of the base editors provided herin, to a target site. For example, the gRNA sequences provided in Table 2 may be cloned into a gRNA expression vector, such as pFYF to encode a gRNA that targets Cas9, or any of the base editors provided herein, to a target site in order to correct a disease-related mutation. It should be appreciated, however, that additional mutations may be corrected to treat additional diseases associated with a G to A or C to T mutation. Furthermore, additional gRNAs may be designed based on the disclosure and the knowledge in the art, which would be

appreciated by the skilled artisan.

Pharmaceutical compositions

[0280] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the adenosine deaminases, fusion proteins, or the fusion protein-gRNA complexes described herein. The term“pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the

pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).

[0281] As used here, the term“pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose,

methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as“excipient”,“carrier”,“pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0282] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

[0283] In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

[0284] In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989 , Ann. Neurol. 25:351; Howard et al, 1989, /. Neurosurg. 71:105.) Other controlled release systems are discussed, for example, in Langer, supra.

[0285] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or

subcutaneous administration to a subject, e.g., a human. In some embodiments,

pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0286] A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer’s or Hank’s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

[0287] The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral

administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid

dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al, Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N- trimethyl-amoniummethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference. [0288] The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term“unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0289] Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0290] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution.

It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Delivery methods

[0291] In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a base editor to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIB TECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1 ):31-44 (1995);

Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0292] Methods of non-viral delivery of nucleic acids include lipofection,

nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid ucleic acid conjugates, naked DNA, artificial virions, and agent- enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,

4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g.,

Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).

[0293] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0294] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0295] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.

4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0296] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and y2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle.

The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.

Kits, vectors, cells

[0297] Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding an adenosine deaminase capable of deaminating an adenosine in a deoxyribonucleic acid (DNA) molecule. In some

embodiments, the nucleotide sequence encodes any of the adenosine deaminases provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the adenosine deaminase. [0298] Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to an adenosine deaminase, or a fusion protein comprising a napDNAbp (e.g., Cas9 domain) and an adenosine deaminase as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide nucleic acid backbone, (e.g., a guide RNA backbone), wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide nucleic acid (e.g., guide RNA backbone).

[0299] Some aspects of this disclosure provide cells comprising any of the adenosine deaminases, fusion proteins, or complexes provided herein. In some embodiments, the cells comprise a nucleotide that encodes any of the adenosine deaminases or fusion proteins provided herein. In some embodiments, the cells comprise any of the nucleotides or vectors provided herein.

[0300] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa- S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-l, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2,

HeLa B, HeLa T4, COS, COS-l, COS-6, COS-M6A, BS-C-l monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, l32-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-l cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV- 434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL- 60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku8l2, KCL22, KG1, KY01, LNCap, Ma- Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI- H69/FX10, NCI-H69/FX20, NCI-H69/FX4, NIH-3T3, NAFM-l, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-l, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.

[0301] The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure.

[0302] It should be appreciated however, that additional fusion proteins would be apparent to the skilled artisan based on the present disclosure and knowledge in the art.

[0303] The function and advantage of these and other embodiments of the present invention will be more fully understood from the Examples below. The following Examples are intended to illustrate the benefits of the present invention and to describe particular embodiments, but are not intended to exemplify the full scope of the invention. Accordingly, it will be understood that the Examples are not meant to limit the scope of the invention. EXAMPLES

[0304] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

Example 1

[0305] Base editors, catalytically impaired Cas9 proteins fused to nucleobase modification enzymes, enable targeted single-nucleotide conversion in the genomes of a wide range of cells and organisms without inducing double- stranded DNA breaks. Previously, both C*G-to-T»A base editors (BE3 and BE4) and A»T-to-G*C base editors (ABE) were developed, and their targeting scope, product purity, and DNA specificity were enhanced.

The usefulness of base editors for research and therapeutic applications is also strongly dependent on the efficiency with which they can modify target nucleotides. Here, factors that limit base editing efficiency in mammalian cells were examined and it was determined that the expression level and nuclear localization of current-generation BE4 and ABE base editors limit their editing efficiency. By improving the nuclear localization signal (NLS), optimizing the codon usage of base editor constructs, and performing ancestral reconstruction of the component deaminase domains, the efficiency of BE4- and ABE-mediated genome editing was greatly improved, in most tested cases to -80% target base conversion in HEK293T cells. It is shown that these optimized“BE4max”,“AncBE4max”, and“ABEmax” base editors are especially enabling under unfavorable conditions such as when delivery of constructs is limiting. These optimized editors corrected several pathogenic SNPs in a variety of mammalian cell types with substantially higher efficiencies than BE4 and ABE.

AncBE4max, BE4max, and ABEmax substantially expand the capabilities of both cytidine and adenosine base editing and represent current state-of-the-art mammalian cell base editors.

[0306] Point mutations represent the majority of known human genetic variants associated with disease ¹ . Developing robust methods to introduce and correct point mutations is therefore an important challenge to understand and treat diseases with a genetic

component. Base editors, fusions of catalytically disabled Cas9, natural or evolved

nucleobase deaminases, and, in some cases, proteins have been recently developed, that alter cellular DNA repair processes to increase the efficiency and stability of the resulting single nucleotide change ^{2, 3} . Two classes of base editors have been described to date: cytidine base editors convert target OG base pairs to T·A, and adenosine base editors convert A·T to G*C. Collectively, these two classes of base editors enable the targeted installation of all four transition mutations (C-to-T, G-to-A, A-to-G, and T-to-C), which collectively account for 61% of known human pathogenic SNPs in the ClinVar database (FIGs. 1A, 1B). Base editors have been used widely in organisms ranging from prokaryotes to plants to amphibians to mammals, and have even been used to correct pathogenic mutations in human embryos ^{4 18} .

[0307] The utility of base editing is limited by several constraints, including the PAM requirement imposed by the Cas9 moiety, off-target base editing,“bystander editing” of non target Cs or As very close to the target nucleotides, the production of undesired byproducts, and overall editing efficiency. Next-generation base editors have been developed that address some of these limitations, including base editors with different or expanded PAM

compatibilities ^{19 21} , high-fidelity base editors with reduced off-target activity ^{20, 22-25} , base editors with narrowed editing windows (normally ~5 nucleotides wide) ¹⁹ , and a current- generation cytidine base editor (BE4) with greatly reduced byproducts ⁶ .

[0308] Despite these recent advances, the efficiency of base editing by BE4 and current adenine base editor (ABE) variants varies widely by cell type and target locus. To broadly increase base editing efficiency, it was sought to identify the factors that limit base editing efficiency in mammalian cells. In this study it was found that for both BE4 and ABE, expression and nuclear localization in human cells impose key bottlenecks on editing efficiency. Optimizing codon usage, using improved nuclear localization sequences (NLSs), and performing ancestral reconstruction of cytidine deaminases result in base editors with greatly increased editing efficiency, often more than doubling target nucleotide conversion yields. The resulting AncBE4max, BE4max, and ABEmax base editors install point mutations relevant to human disease in a variety of mammalian cell types much more efficiently than previously described base editors. AncBE4max, BE4max, and ABEmax substantially advance the utility of both classes of base editors, and their use is recommended for general base editing applications in mammalian cells.

Results

[0309] Several factors could limit the ability of a base editor to achieve conversion of the target nucleotide. If the base editor is delivered as encoded DNA or RNA, cellular uptake of the nucleic acid can be limiting. Among cells that have acquired a DNA construct encoding a base editor, transcription may be limited by the choice of promoter or regulatory sequences or by the editor’s coding sequence. Once transcribed, translation efficiency may also be limiting depending on factors including codon usage and mRNA processing. In protein form, a base editor protein may be inefficiently trafficked to the nucleus or may be degraded too quickly. Given that APOBEC1, the cytidine deaminase component of BE4, expresses poorly in bacteria ²⁶ it was speculated that altering the coding sequence or codon usage of this component might augment base editing efficiency.

[0310] These possibilities were dissected using three fluorescent protein expression experiments. To establish a baseline editing efficiency level among viable cells capable of taking up plasmid DNA during transfection, a three-plasmid mixture consisting of a plasmid expressing BE4, a plasmid expressing an sgRNA, and a separate plasmid expressing mCherry, were co-transfected into human HEK293T cells to mark viable cells that received plasmid. Base editing was measured by high-throughput DNA sequencing (HTS) from mCherry-positive cells isolated by FACS, revealing an average of 45+7.1% OG-to-T*A editing across the base editing activity window (positions 4-8, counting the PAM as positions 21-23) at five test genomic loci (FIG. 1C, 1D). To examine editing efficiency only among cells that contain the DNA plasmid encoding the base editor, HEK293T cells were transfected with a two-plasmid mixture consisting of a plasmid encoding an sgRNA and a separate plasmid encoding both BE4 and GFP, expressed from separate promoters. Isolation by FACS of GFP-positive cells followed by HTS resulted in an average of 35+7.3% editing at the same five test sites (FIG. 1D). The lack of improvement in editing efficiencies among GFP-positive cells containing the base editor plasmid in the second experiment versus transfected (mCherry-positive) cells in the first experiment suggests that transfection of HEK293T cells was not limiting base editing outcomes.

[0311] In a third experiment, editing efficiencies in HEK293T cells were evaluated following transfection of a BE4-P2A-GFP construct, where P2A is a self-cleaving peptide ²⁷ that enables co-expression of the GFP protein from the same mRNA transcript as BE4. GFP- positive cells in this experiment can arise only if they also produce full-length BE4 protein. Among GFP-positive cells, base editing efficiencies averaged 65+6.4%, 1.9-fold higher than the average editing efficiencies following transfection of the single plasmid expressing BE4 and GFP from separate promoters (FIG. 1E). These results strongly suggest that the fraction of cells expressing active base editors, and/or the amount of functional base editor protein produced by each cell, are major bottlenecks of base editing efficiency. [0312] To probe the possibility of sub-optimal nuclear localization, all six combinations of BE4 N- and C-terminal fusion were tested either to the SV40 NLS used in BE4, or to a bipartite NLS (bpNLS) previously shown to improve nuclear localization of Cas9 ²⁸ (FIG. 2A). All variants using one or two bpNLSs showed improvements in editing efficiency. The presence of a bpNLS at both the N- and C-terminus (referred to hereafter as “bis-bpNLS”) performed best, resulting in a 1.3-fold average improvement in BE4-mediated OG-to-T*A editing efficiency at five tested genomic loci (48+8.0% average editing compared to 37+5.6% for the C-terminal SV40 NLS used in BE4) (FIG. 2A; see FIG. 5A for p-values). These results together suggest that the use of a bis-bpNLS can significantly improve the editing efficiency of previously described BE3 and BE4 ^{6, 7} .

[0313] Next, it was speculated that improving codon usage might enhance base editor expression. To test this possibility, the improved bis-bpNLS form of BE4 (bpNLS-BE4- bpNLS) coded with eight distinct full-length and chimeric codon optimization strategies was compared. Previously reported BE4 and ABE used codon optimization from IDT (Integrated DNA Technologies) ^{3, 6} . First OG-to-T*A editing efficiencies of bis-bpNLS BE4 constructs that use codon optimization from IDT, GeneArt, Coller and co-workers ²⁹ , and GenScript were compared. At the five endogenous loci in HEK293T cells, all tested new codon optimizations resulted in improved editing efficiency compared to that of the original IDT codons. The best-performing variant used GenScript codons and resulted in an additional 1.8- fold higher editing over bis-bpNLS BE4 with IDT codons, enabling average editing in HEK293T cells of 62+7.8% (FIG. 2B; see FIG. 5B for p-values).

[0314] To more deeply dissect the effects of codon optimization, chimeric codon- optimized BE4 variants containing Cas9 nickase coding sequences previously reported by Kim and coworkers to improve expression in human cells, ³⁰ were also tested together with cytidine deaminase and UGI domains with codons from IDT or GenScript. The results reveal that both Cas9 nickase and APOBEC codon usage influence BE4 editing efficiency (FIG. 6A). None of the chimeric codon-optimized constructs tested (APOBEC/Cas9 codon usage = IDT/GenScript, GenScript/IDT, GenScript/Kim, or IDT/Kim) resulted in significantly increased BE4 editing efficiencies compared to the optimized bis-bpNLS BE4 editor using full-length GenScript codon optimization, referred to hereafter as BE4max (FIGs. 6A-6B). While the chimera of an IDT deaminase with a GenScript Cas9 nickase showed slightly improved average editing outcomes relative to BE4max at a single high dose of plasmid (FIG. 2B), when tested across an eight-dose plasmid titration at three genomic loci this chimera did not show improved editing compared to BE4max (FIG. 6B). Collectively, these findings establish that codon optimization can dramatically improve the activity of BE4- mediated base editing in human cells.

[0315] The above results implicate both APOBEC1 cytidine deaminase and Cas9 nickase expression as key determinants of base editing efficiency (FIG. 2B and FIG. 6A). To further explore strategies for enhancing APOBEC1 expression, ancestral sequence reconstruction (ASR) was performed by maximum likelihood using a set of 468 APOBEC homologs. ASR uses an alignment of known protein sequences, an evolutionary model, and a resulting phylogenetic tree to infer ancestral protein sequences at the nodes of the

phylogeny ³¹ . ASR has been previously shown to greatly improve the expression of a variety of proteins while retaining wild-type levels of biochemical activity ^{32 35} . In particular, maximum likelihood reconstructions have been reported to generate sequences with higher expression levels compared to Bayesian reconstructions ^{36, 37} . While the reasons for improved expression of ancestral proteins remain actively debated ^{32, 36 38} , surprisingly few cases have exploited this phenomenon for protein engineering ^{39 41} .

[0316] Using the set of 468 APOBEC homologs (“Sequences 5”, below) a maximum likelihood phylogeny was created and the most likely sequences at internal nodes were inferred (FIG. 2C). Five ancestral cytidine deaminases selected at increasing evolutionary distance from rat APOBEC 1 used in BE4 were characterized as base editors (FIG. 7). Two ancestors (Anc689 and Anc687) closest in evolutionary distance to the rat APOBEC 1 used in BE4 (36 and 45 amino acid differences vs. BE4, respectively) resulted in high editing efficiencies across the five genomic test loci in HEK293T cells, similar to those of BE4max (Anc689: 62+4.9%; Anc687: 57+6.7%; BE4max: 59+4.9%) (FIG. 2D).

[0317] To characterize in depth the base editing activities that result from these improvements, including under sub-optimal conditions, across eight different doses of base editor plasmid at three genomic loci, the editing efficiencies of the previously reported BE46 were compared with those of the three most promising BE4 variants generated in this work: BE4max, bis-npNLS BE4 using the chimeric IDT/GenScript codon optimization, and bis- npNLS BE4 with the Anc689 ancestral APOBEC domain and GenScript codons (referred to hereafter as AncBE4max) (FIG. 2E, p-values reported in FIGs. 8A-8C). The improvements offered by all three of these optimized editors over BE4 are dramatic, ranging from 1.7-fold at higher plasmid doses to >9-fold at lower plasmid doses (FIG. 2E). Ratios of desired point mutation to indels were also improved among all three of these optimized editors compared with the original BE4, as indel frequencies remained similar or lower while base editing improved substantially (FIG. 9). AncBE4max consistently offered the highest activity at target Cs across all three tested sites over a wide range of plasmid doses spanning four orders of magnitude (FIG. 2E; see FIG. 8C for p-values). BE4max resulted in editing efficiencies slightly below, or similar to, those of AncBE4max (FIG. 2E). These data suggest that AncBE4max and BE4max offer large improvements in editing efficiency over BE4, especially under sub-optimal conditions in which factors such as delivery limit overall editing efficiency. As many genome editing applications operate under sub-optimal conditions constrained by poor delivery, or by limited editing opportunities in time or space, it was anticipated that AncBE4max and BE4max will be useful in many settings.

[0318] Next, it was determined if the AncBE4max and BE4max result in increased mRNA and protein levels in human cells relative to the previously reported BE4. HEK293T cells were separately transfected with 250 ng of guide RNA and 750 ng of plasmid encoding BE4-P2A-GFP, BE4max-P2A-GFP, or AncBE4max-P2A-GFP. After 3 days, cells were subjected to reverse transcription using GFP-specific primers to evaluate the amount of base editor mRNA present in each sample. BE4max showed >3-fold higher mRNA expression compared to that of BE4, while AncBE4max showed >5-fold higher mRNA expression when compared to BE4 (FIG. 10A). To compare protein levels of BE4 versus BE4max and

AncBE4max, HEK293T cells were transfected as described above with plasmids encoding C- terminally epitope-tagged BE4, BE4max, and AncBE4max and performed western blots on the resulting cell lysates. The data reveal that both BE4max and, especially, AncBE4max result in substantial increases in full-length protein abundance relative to actin controls (FIG. 10B). Together, these results indicate that the optimizations that led to BE4max and

AncBE4max resulted in major increases in mRNA and protein levels of full-length base editors.

[0319] The relationship between improved base editor expression and improved editing efficiency was further illuminated by flow cytometry. HEK293T cells expressing BE4max-P2A-GFP and AncBE4max-P2A-GFP targeted to three test genomic loci were sorted. While the fraction of viable and transfectable (mCherry-positive) cells was very similar among BE4max-P2A-GFP (63+3.8%), AncBE4max-P2A-GFP (62+2.3%), and BE4-P2A-GFP (60+7.0%) across the three genomic sites tested, the frequency of GFP and mCherry double-positive cells, reflecting full-length base editor expression, was on average 1.7-fold higher for both BE4max-P2A-GFP and AncBE4max-P2A-GFP compared to BE4 (FIG. 10C). These improvements in editor expression were reflected in improvements in editing activity. Among mCherry and GFP double-positive cells, BE4max-P2A-GFP showed an average of 89+0.9% target OG-to-T*A editing across the editing window and

AncBE4max-P2A-GFP led to an average of 90+1.5% editing, while double-positive cells expressing BE4-P2A-GFP averaged 48+8.0% (FIG. 1E, FIG. 9). These large improvements demonstrate that isolating cell populations expressing BE4max and AncBE4max results in dramatically higher frequencies of edited cells, which may be especially useful for base editing applications that seek to create novel cell lines, agriculture strains, or animal models.

[0320] Adenine base editors (ABEs) use a laboratory-evolved deoxyadenosine deaminase and a Cas9 nickase to mediate the conversion of target A·T to G*C base pairs ³ . Because this conversion reverses the most common class of point mutations in living systems (OG to T·A) ⁴² , ABE have the potential to correct a far larger fraction (47%) of pathogenic SNPs than cytidine base editors (14%) (FIG. 1B). Encouraged by the above improvements in BE4 editing efficiency, next, these principles were applied to improve ABE. The applications began with ABE 7.10, the best-performing ABE variant for general use described to date ³ . While the use of the bis-bpNLS in ABE instead of the previously reported SV40 NLS resulted in little apparent improvement in average A*T-to-G*C editing efficiency in single dose experiments in HEK293T cells across five human genomic loci test sites (53+3.8% versus 50+3.8% respectively) (FIG. 3A), dose-titration experiments revealed that the bis- bpNLS offered substantially higher ABE editing efficiencies (typically -1.5- to 2-fold) at sub-optimal ABE doses (FIG. 3B). These observations indicate that replacement of the SV40 NLS with bis-bpNLS also can enhance ABE-mediated editing efficiency.

[0321] To test the effect of codon optimization on A*T-to-G*C editing efficiency, the original (IDT codons) form of bis-bpNLS-ABE was compared with the Genscript codon optimization form of the same protein at the five test sites in HEK293T cells. Consistent with the findings for BE4, Genscript codon optimization of bis-bpNLS ABE 7.10 (referred to hereafter as ABEmax) also resulted in substantial benefits to editing efficiency compared with IDT codon optimization. At high single-doses of base editor construct, ABEmax resulted in 1.3-fold higher editing levels than ABE (from 50+3.8% to 65+6.8%) across all five sites tested (FIG. 3A; see FIG. 11A for p-values). At sub-optimal doses, ABEmax improved editing efficiencies up to 7.9-fold over those of ABE 7.10 (from 3.2+1.0% to 25+4.4% average editing across three sites tested at low doses) (FIG. 3B; see FIG. 11B for p- values). Although indels from ABEmax remained rare occurrences (<l.6%), they were elevated from the virtually undetectable indel levels of ABE 7.10 ³ (FIG. 12). Together, these findings establish that improvements in nuclear localization and expression that benefit cytidine base editors are also applicable to ABEs.

[0322] With optimized BE4max, AncBE4max, and ABEmax in hand, it was sought to compare them to previously reported BE4 and ABE7.10 for their ability to edit disease relevant target loci in a variety of cell types. Patient-derived fibroblasts are commonly used for studying genetic diseases. Mutations in MDPU1, a gene central to N-glycan biosynthesis in the endoplasmic reticulum, gives rise to a condition known as congenital disorder of glycosylation type lf43. Patient-derived fibroblasts harboring the Leul l9Pro T>C mutation that drives this disease were nucleofected with plasmids expressing BE4, BE4max-P2A- GFP, or AncBE4max-P2A-GFP and the targeting sgRNA. A portion of BE4-treated cells sorted by FACS for an mCherry co-transfection marker, while a portion of cells treated with BE4max-P2A-GFP and AncBE4max-P2A-GFP were sorted for GFP-positive cells.

Unsorted cells for BE4 showed 13+1.2% correction of the disease-driving SNP in patient- derived fibroblast, while FACS-sorted BE4 samples showed 34+2.4% correction (FIG. 4A). Unsorted BE4max and AncBE4max resulted in 26+1.3% and 29+1.7% correction of the Leul l9Pro mutation, respectively, while sorted cells treated with BE4max and AncBE4max showed 69+2.5% and 75+2.2% SNP correction, respectively (FIG. 4A). Nucleofection efficiencies for all tested conditions were measured by flow cytometry and were consistent across samples, indicating that outcomes were primarily dependent on base editor expression and activity (FIG. 14A). Thus BE4max and AncBE4max resulted in 2.0- and 2.2-fold higher editing efficiencies, respectively, of a pathogenic human SNP in patient-derived fibroblasts.

[0323] To further test the improvements offered by BE4max and AncBE4max, a splice-modifying mutation was installed in mouse N2a neuroblastoma cells in the voltage gated sodium channel NaVl.7 (SCN9a gene), a target associated with familial

erythromyalgia, paroxysomal extreme pain disorder, and chronic insensitivity to pain ^{44, 45} . +1 G and -1 G was targeted in the splice acceptor of SCN9a intron 6 by nucleofection of plasmids encoding BE4, BE4max, or AncBE4max and the targeting sgRNA. BE4 treatment resulted in 9.3+4.4% editing of both +1 G and -1 G among unsorted cells, and 14+1.3% and 13+3.0% editing among sorted cells, respectively (FIG. 4B). BE4max resulted in 50+5.0% and 46+3.1% editing of the target +1 G and -1 G in unsorted cells, and 73+5.7% and

77+9.3% editing among sorted cells. Finally, AncBE4max resulted in 44%+l.7% and 43+0.47% editing of the target +1 G and -1 G in unsorted cells, and 94+7.7% for both target Gs among sorted cells (FIG. 4B). Together, these results demonstrate that BE4max and AncBE4max offer large improvements— here, 5- to 7-fold increases— in editing efficiency of a disease-relevant target in mammalian cells. Indeed, AncBE4max in one sorted sample resulted in 99.8% editing of both target G*C base pairs in SCN9a, demonstrating the very high likelihood of target base editing among cells containing AncBE4max protein (FIG. 13). Transfection efficiencies for all tested conditions were measured by flow cytometry and were consistent across samples, indicating that outcomes were primarily dependent primarily on base editor expression and activity (FIG. 14B).

[0324] Finally, the activity of ABE 7.10 versus ABEmax was compared at two blood disease-associated targets. A number of genetic blood disorders, including sickle cell anemia and b-thalassemia, are driven by mutations in the b-globin gene. Activating mutations in the promoters of HBG1 or HBG2 (g-globin) that are normally silenced after birth can rescue b- globin disorders ⁴⁶ . Two sgRNAs were designed to install different activating mutations in g- globin promoters. The first sgRNA should target ABE to introduce mutations at protospacer positions A5 and A8 (HBG promoter positions -116 and -113 relative to the transcription start site). The -116 A to G mutation may perturb the binding site for the g-globin repressor Bell la ⁴⁷ , while the -113 A to G mutation should perturb this binding site and is also a naturally-occurring SNP that confers the hereditary persistence of fetal hemoglobin ⁴⁸ . Once again, ABEmax substantially outperformed ABE 7.10, with both unsorted and sorted

HEK293T cells treated with ABEmax resulting in approximately double the conversion efficiencies at A5 and A8 than unsorted and sorted cells treated with ABE 7.10 (FIG. 4C).

For the second sgRNA, which installs the -175 T to C point mutation in the HBG promoter that is one of the strongest known HBG promoter SNPs upregulating fetal hemoglobin ⁴⁸ , 6.5+0.57% and 10+1.0% editing in unsorted and sorted HEK293T cells expressing ABE 7.10, was observed respectively, compared to 46+0.55% and 52+5.2% editing in unsorted and sorted cells expressing ABEmax, respectively (FIG. 4D). Thus ABEmax increased editing efficiency at the HBG -175T target by 5.2- to 7.l-fold over ABE 7.10. Notably, this mutation lies at position A3 of the protospacer, and thus is slightly outside the normal ABE editing window, demonstrating that ABEmax can support efficient editing of sites expected to be poor candidates for base editing by ABE 7.10. Transfection efficiencies for all tested conditions were measured by flow cytometry and were consistent across samples, indicating that outcomes were primarily dependent primarily on base editor expression and activity (FIG. 14C). Together, these results demonstrate that ABEmax substantially improves the efficiency of base editing at two loci relevant to human disease.

[0325] In summary, elucidation of factors that limit cytidine and adenine base editor efficiency resulted in optimization of nuclear localization and codon usage, as well as ancestral protein reconstruction, greatly improved the expression levels and editing efficiencies of both the BE4 C*G-to-T ^» A base editor and the ABE 7.10 A ^» T-to-G*C base editor. The editing performance improvements offered by BE4max, AncBE4max, and ABEmax extended to a variety of mammalian cell types across a number of previously unreported disease-relevant loci, as well as commonly tested loci. These improvements are especially pronounced when using base editors under sub-optimal conditions, such as those commonly found in some research and many therapeutic applications, or at sites that were previously edited with only modest efficiency. These developments greatly expand the capabilities of the current base editors for a wide range of applications, and BE4max, AncBE4max, and ABEmax are recommended for current base editing experiments.

Methods

[0326] General Methods. PCR was performed using either Phusion U Green

Multiplex PCR Master Mix (ThermoFisher Scientific) or Q5 Host Start High-Fidelity 2x Master Mix (New England Biolabs) unless otherwise noted. All plasmids were assembled by either the USER cloning method as previously described ⁴⁹ or by Gibson assembly ⁵⁰ . Plasmids for mammalian cell transfections were prepared using an endotoxin removal plasmid purification system, ZymoPURE Plasmid Midiprep (Zymo Research Corporation).

[0327] Cell culture conditions. HEK293T cells (ATCC CRL-3216) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Coming) supplemented with 10% fetal bovine serum (FBS) and 5% penicillin streptomycin (Pen Strep, ThermoFisher Scientific). Fibroblast cell lines were maintained in DMEM supplemented with 15% FBS. N2a cells were maintained in DMEM supplemented with 10% FBS. [0328] HEK293T transfection and genomic DNA preparation. HEK293T cells were seeded into 48-well Poly-D-Lysine coated plates (Coming) in the absence of Pen Strep antibiotic. 12-15 hours after plating, cells were transfected with 1 pL of Lipofectamine 2000 (ThermoFisher Scientific) using 750 ng of base editor plasmid, 250 ng of guide RNA plasmid, and 20 ng of fluorescent protein expression plasmid as a transfection control. Unless otherwise stated, cells were cultured for 3 days before they were washed with PBS

(ThermoFisher Scientific). Genomic DNA was extracted by addition of 150 pF of freshly prepared lysis buffer (10 mM Tris-HCl, pH 7.5, 0.05% SDS, 25 pg/mF proteinase K

(ThermoFisher Scientific)) directly into each transfected well. The resulting mixture was incubated for 1 hour at 37 °C before a 30-min enzyme inactivation step at 80 °C. Guide RNA sequences for HEK2, HEK3, HEK4, RNF2, EMX1, Site 2, Site 5, Site 13, Site 16 were previously reported ^{2, 3} ’ ⁶ .

[0329] HEK293T base editing dose titrations. HEK293T cells were seeded as described above and transfected with a mixture of base editor plasmid, guide RNA plasmid, pUC, and GFP. 250 ng of guide RNA plasmid and 20 ng of GFP transfection control plasmid were used for all samples. Base editor and pUC plasmids were combined in different amounts to maintain a constant amount of total DNA per transfection.

[0330] Fluorescence-activated cell sorting. Flow cytometry analysis was carried out using an Aria Fortessa III. HEK293T cells were transfected with guide RNA expression plasmids, fluorophore expression plasmids, and editor expression plasmids. In trans samples were sorted for mCherry-positive cells. Both the in cis and P2A samples were sorted for both GFP and mCherry double-positive cells. A stringent mCherry-positive gate was used to avoid mCherry false positives. Over 15,000 cells were collected for each experimental sample. Genomic DNA for sorted and unsorted FACS samples was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer’s instructions.

[0331] Nucleofection of fibroblasts and genomic DNA extraction. Cells were nucleofected using the Primary P2 Cell Fine 4D-Nucleofector X Kit (Fonza) according to manufacturer’s protocol. l.25xl0 ⁵ cells were nucleofected in 20 pF of P2 buffer

supplemented with 750 ng of editor, 250 ng of guide RNA plasmid, and 20 ng of mCherry nucleofection marker. Cells were nucleofected in a l6-well nucleocuvette strip using the DT- 130 program. Following a 3-day incubation, cells were flow sorted and genomic DNA was extracted as described for HEK293T cells above.

[0332] High-throughput DNA sequencing (HTS) of genomic DNA. HTS of genomic DNA from HEK293T cells was perform as described previously ^{2, 3} ’ ⁶ . For fibroblasts, 34 cycles of amplification were used for PCR1. Primers for PCR 1 of HEK2, HEK3, HEK4, RNF2, EMX1, ABE Site 2, ABE Site 5, ABE Site 13, ABE Site 16, and HBG loci were used as previously described ^{3, 6 19} . PCR 1 primers for type 1F congenital glycosylation disorder, SCN9a, and all previously used loci are listed in Sequences 2 below.

[0333] General HTS analysis. Sequencing reads were demultiplexed using the MiSeq Reporter (Illumina) and Fastq files were analyzed using using open source analysis tools. FASTQ files were aligned to the reference genome using the burrows-wheeler aligner (bwa-mem) ⁵¹ . Statistics for each base were calculated using the pysamstats utility available at

[0334] github.com/alimanfoo/pysamstats. All reads for a given base were aligned to the reference sequence. Total reads were the sum of all base calls, insertions, and deletions at any given nucleotide position. Percent representation of each base was calculated as reads of a given base divided by total reads. Indel frequencies were quantified with a custom Matlab script as previously described ^{3, 20} .

[0335] Quantitative RT-PCR and quantitative PCR. HEK293T cells were transfected with base editor-P2A-GFP plasmids and incubated 3 days before harvesting DNA and RNA from each sample. DNA samples were harvested using the genomic DNA prepartion protocol described above. RNA was isolated and amplified using the Cells-to-Ct (Thermofisher) kit according to the manufacturer’s protocol except the DNase treatment step used 2X DNase for twice as long to ensure complete degradation of plasmid DNA. Levels of mRNA were calculated by normalizing base editor mRNA levels to b-actin levels by AACt. Plasmid DNA levels were calculated to ensure that mRNA levels were not skewed by transfection efficiency. Plasmid DNA levels were calculated by normalizing amplification of the BGH poly-adenylation present on the base editor plasmid to b-actin levels.

[0336] Western blotting. HEK293T cells were transfected with 750 ng of base editor-3X HA tag plasmid and 250 ng of guide RNA plasmid . After 3 days, cells were lysed using RIPA buffer with PMSF and cOmplete Protease Inhibitor Cocktail (Sigma- Aldrich). Samples were boiled and quantified using a BCA assay. 10 pg of protein was loaded per well into a l2-well 4-12% Tris gel (Novex). Blots were transferred to nitrocellulose paper for 7 min at 20 V before blocking and incubation with anti-HA (Cell Signaling Technology) and anti-Actin antibodies (Cell Signaling Technology). Blots were visualized using an Odyssey imager.

[0337] APOBEC sequence collection. APOBEC protein sequences used in phylogenetic analyses were identified through searches of the Uniprot database ⁵² with the BLASTP algorithm ⁵³ using selected query sequences. All sequences from these searches that returned BLASTP E-values < 10 ⁷ were downloaded from Uniprot. To reduce phylogenetic complexity, sequences were curated based on character length and pairwise sequence identity within each dataset. The dataset used for the construction of the non-redundant phylogeny was generated using four query sequences: UniProt IDs P41238, H2P4E7, E1BTD6, and H2P4E9. Multiple sequences were necessary to generate full coverage due to the low sequence identity across the family, which is <25% between some members. Limits were chosen to remove truncated and partial sequences and those featuring large insertions or terminal extensions. Sequences greater than 97% identical, determined by pairwise alignment within the dataset, were also removed. This level of identity provides a high level of detail within the tree while accelerating computational time by removing redundant taxa. The final dataset contains 468 taxa (Sequences 5 below).

[0338] Phylogeny construction. A multiple sequence alignment of the dataset was generated with the program MAFFT using the FFT-NS-I xlOOO algorithm ⁵⁴ . Model selection used the Bayesian information criteria (BIC) to determine the evolutionary model that best fit the input alignment ⁵⁵ . 228 models where tested. The Jones Taylor Thornton (JTT) substitution matrix with empirical frequencies (F) and free rates with five categories (R5) was the model that best fit the data. A maximum likelihood (ML) phylogenetic tree was inferred with IQ-TREE ⁵⁶ using the best fit model (JTT+F+R5). The starting trees were generated by randomized maximum parsimony and searched by fast hill-climbing Nearest Neighbor Interchange (NNI). Tree topology, branch lengths, and rate parameters were optimized.

Branch supports were estimated with Ultrafast boot strapping, implemented in IQ-TREE ⁵⁷ (FIG. 15).

[0339] Ancestral sequence reconstruction. Sequences at internal nodes in the phylogeny were inferred using the codeml program from the PAML software package ⁵⁸ . Posterior amino acid probabilities at each site were calculated using the JTT substitution matrix, given the ML tree and estimated background frequencies generated by IQ-TREE. bl and C-termini of ancestral sequences were modified manually to match those of Rat APOBEC1.

[0340] Note 1. Python script to analyze pathogenic SNPs within the ClinVar database.

[0341] import numpy as np

[0342] import pandas as pd

[0343] #download latest ClinVar from

//ftp. ncbi.nlm.nih.gov/pub/clinvar/tab_delimited/

[0344] #convert to csv

[0345] ClinVar = pd.read_csv('20l8-04-23-variant_summary.csv')

[0346] #restrict to SNPs

[0347] ClinVar = ClinVar[ClinVar.Type == 'single nucleotide variant']

[0348] #restrict to pathogenic

[0349] ClinVar = ClinVar[ClinVar.ClinicalSignificance == 'Pathogenic']

[0350] #remove nans

[0351] ClinVar = ClinVar[ClinVar.ReferenceAllele != 'na']

[0352] ClinVar = ClinVar[ClinVar.AltemateAllele != 'na']

[0353] #drop duplicates of AllelelD

[0354] ClinVar = ClinVar.drop_duplicates('#AlleleID')

[0355] #total SNPs in ClinVar

[0356] total_SNPs = len(ClinVar)

[0357] def SNP_count(cv, ref, alt):

[0358] ClinVar_ref = cv[cv. Reference Allele == ref]

[0359] ClinVar_ref_alt = ClinVar_ref[ClinVar_ref. Alternate Allele == alt]

[0360] return len(ClinVar_ref_alt)

[0361] counts = np.array([[", 'A', T, 'G', 'C'],

[0362] ['A', 0, SNP_count(ClinV ar, 'A', 'T'), SNP_count(ClinVar, 'A', 'G'),

SNP_count(ClinVar, 'A', 'C')],

[0363] ['T', SNP_count(ClinV ar, T, 'A'), 0, SNP_count(ClinVar, T, 'G'),

SNP_count(ClinVar, T, 'C')], [0364] ['G', SNP_count(ClinV ar, 'G', Ά'), SNP_count(ClinVar, 'G', T), 0, SNP_count(ClinVar, 'G', 'C')],

[0365] ['C, SNP_count(ClinV ar, 'C, Ά'), SNP_count(ClinVar, 'C, T'),

SNP_count(ClinVar, 'C, 'G'), 0] ])

[0366] np.savetxt('ClinVar_SNPs.csv', counts, fmt='%5s', delimiter=',')

[0367] Sequences 1. Target protospacer sequences used in this study.

Target Cs and As are bold, with a subscripted number denoting spacer position. PAM sequences are italicized.

[0368] Sequences 2. Primers used in this study.

All oligonucleotides were purchased from Integrated DNA Technologies (IDT). Primers used for generating sgRNA plasmids

[0369] The MPDU 1 guide plasmid was cloned by digesting a modified version of PFYF1320 ¹ in which BsmBI restriction cut sites were installed via KLD cloning. The primers below were phosphorylated and annealed to enable ligation into BsmBI cut backbone. All guides were designed to include a 5’-G to enable transcription from the hU6 transcription. CCACC was included at the 5’ end of the forward primer, and AAAC was included at the 5’ end of the reverse primer to complement the overhands generated by restriction digest. The HEK2, HEK3, HEK4, RNF2, EMX1, ABE site 2, ABE site 5, ABE site 13, ABE site 16, SCN9a, HBG site 1, HBG site 2 sgRNAs were prepared by KLD cloning as previously described ² using the primers listed below.

Primers for MPDU1 sgRNA

[0370] JLD 85

[0371] JLD 86

Primers for SCN9a, HBG site 1, HBG site 2, and previously used sgRNAs

[0372]

165)

[0373] fwd_HEK293_site 2

[0374] fwd_HEK293_site 3

[0375] fwd_HEK293_site 4

[0376] fwd_RNF2

[0377] fwd_EMXl

[0378] fwd_ABE_site 2

[0379] fwd_ABE_site 5 G ATG AG AT A AT GAT G AGT C AGTTTT AG AGCT AG A A AT AGC A AGTT A A A AT A AGG C (SEQ ID NO: 172)

[0380] fwd_ABE_site 13

GAAGATAGAGAATAGACTGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

C (SEQ ID NO: 173)

[0381] fwd_ABE_site 16

GGGAATAAATCATAGAATCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

C (SEQ ID NO: 174)

[0382] fwd_SCN9a

GTTAGTCCTTAAAATGTAGGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG GC (SEQ ID NO: 175)

[0383] fwd_HBG_ _site 1

GCTTGACCAATAGCCTTGACAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG GC (SEQ ID NO: 176)

[0384] fwd_HBG_ _site 2

GAT ATTT GC ATT GAG AT AGTGGTTTT AG AGCT AG A A AT AGC A AGTT A A A AT A AG GC (SEQ ID NO: 177)

Primers to amplify genomic loci for HTS of mammalian cell culture experiments [0385] SCN9a HTS Fwd

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGAACACACTGAGACAG AAC (SEQ ID NO: 178)

[0386] SCN9a HTS Rev

TGGAGTTCAGACGTGTGCTCTTCCGATCT GCACTCCTAGTTAGGCTTGTG (SEQ ID NO: 179)

[0387] Type lf HTS Fwd

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCCCTGGATGGATGGGC TATGG (SEQ ID NO: 180)

[0388] Type lf HTS Rev

TGGAGTTCAGACGTGTGCTCTTCCGATCTGGCTTTCCCAGACCTGAGTTCCC (SEQ ID NO: 181)

[0389] HBG HTS Fwd ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTGGCCTCACTGGATAC TC (SEQ ID NO: 182)

[0390] HBG HTS Rev

TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGACAAAAGAAGTCCTGGTATC

(SEQ ID NO: 183)

[0391] fwd_HEK293_site 2_HTS

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGCCCCATCTGTCAAA CT (SEQ ID NO: 184)

[0392] rev_HEK293_site 2_HTS

TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAATGGATTCCTTGGAAACAATGA

(SEQ ID NO: 185)

[0393] fwd_HEK293_site 3_HTS

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTGGGCTGCCTAGAAA GG (SEQ ID NO: 186)

[0394] rev_HEK293_site 3_HTS

TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTTGTCAACC (SEQ ID NO: 187)

[0395] fwd_HEK293_site 4_HTS

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAACCCAGGTAGCCAGAG AC (SEQ ID NO: 188)

[0396] rev_HEK293_site 4_HTS

TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCGAACGGAG (SEQ ID NO: 189)

[0397] fwd_RNF2_HT S

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACGTCTCATATGCCCCTTG

G (SEQ ID NO: 190)

[0398] rev_RNF2_HTS

TGGAGTTCAGACGTGTGCTCTTCCGATCTACGTAGGAATTTTGGTGGGACA (SEQ ID NO: 191)

[0399] fwd_EMX 1 _HT S

ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCTCAGCCTGAGTGTT GA (SEQ ID NO: 192) [0400] rev_EMXl_HTS )

[0401] fwd_ABE_site 2_HTS

[0402] rev_ABE_site 2_HTS

[0403] fwd_ABE_site 5_HTS

[0404] rev_ABE_site 5_HTS

[0405] fwd_ABE_site l3_HTS

[0406] rev_ABE_site l3_HTS

[0407] fwd_ABE_site l6_HTS

[0408] rev_ABE_site l6_HTS

[0409] Sequences 3. Amino acid sequences of BE4, rAPOBECl, ancestral APOBECs, ABE, and P2A-GFP.

[0410] Within base editor sequences, NLS sequences are bold, APOBEC and TadA sequences are italicized, linkers are double underlined, Cas9 nickase sequence is underlined, and UGI sequences are bold and italicized. [0411] BE4max and AncBE4max

[0412] The portion indicated by [APOBEC] in the above sequence of SEQ ID NO 202 may include any APOBEC sequence, or variant thereof, provided herein. For example, in some embodiments, the [APOBEC] may comprise any one of SEQ ID NOs: 4-9, 203-209, or 220-687.

[0413] Exemplary BE4max(with nickase):

MKRTADGSEFESPKKKRKVSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLY

EINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFFSWSPCGECS R

AITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS S GVTIQIMTEQES GY CWRNFV

NYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCH Y

QRLPPHILW ATGLKS GGSSGGSSGS ETPGTSES ATPES S GGS S GGS DKKY S IGLAIGTN

S V GW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET AE ATRLKRT ARRR

YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY H

EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI Q

LV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLIAQLPGEKKN GLF GNLIALS L

GLTPNFKSNFDLAED AKLQLS KDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAILLS

DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA G

YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LH

AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN FE

EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR

KPAFLS GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEIS G VEDRFN AS LGT

YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ L

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI Q

KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE

N QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQN GRDM

YVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK

MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA

V V GT ALIKKYPKLES EFV Y GD YKV YD VRKMIAKS EQEIGKAT AKYFF Y S NIMNFFKT

EITLAN GEIRKRPLIETN GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE V QTGGFS

KES ILPKRN S DKLIARKKD WDPKKY GGFDS PT V AY S VLV V AKVEKGKS KKLKS VKE

LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE LQ KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR VILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT STKE VLD ATLIHQS ITGLYETRIDLS QLGGDS GGS GGS GGSTNLSDIIEKETGKQLVIQE S ILMLPEE VEE VIGNKPES DILVHT A YDES TDEN VMLLT S D APE YKP W ALVIQDS N GE NKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHT A YDES TDEN VMLLT S D APE YKPW ALVIQDS N GENKIKMLS GGS KRT ADGS EFEPKK KRKV (SEQ ID NO: 693)

[0414] Exemplary BE4 sequence:

MSSETG PVAVDPTLRR RIEPHEFEVF FDPRELRKET CLLYEINWGGRHSIWRHTSQ NTNKHVEVNF IEKFTTERYF CPNTRCSITW FLSWSPCGEC S RAITEFLS R YPH VTLFIYI ARLYHHADPR NRQGLRDLIS SGVTIQIMTE QESGYCWRNF VNYSPSNEAH WPRYPHLWVR LYVLELYCII LGLPPCLNIL RRKQPQLTFF TIALQSCHYQ RLPPHILWAT GLKSGGSSGG SSGSETPGTS ESATPESSGG SSGGSDKKYS IGLAIGTNSV GWAVITDEYK VPSKKFKVLG NTDRHSIKKN LIGALLFDSG ETAEATRLKR T ARRRYTRRK NRICYLQEIF SNEMAKVDDS FFHRLEESFL VEEDKKHERH PIFGNIVDEV AYHEKYPTIY HLRKKLVDST DKADLRLIYL ALAHMIKFRG HFLIEGDLNP DNSDVDKLFI QLVQTYNQLF EENPINASGV DAKAILSARL SKSRRLENLI AQLPGEKKNG LFGNLIALSL GLTPNFKSNF DLAEDAKLQL S KDT YDDDLD NLLAQIGDQY ADLFLAAKNL SDAILLSDIL RVNTEITKAP LSASMIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLRKQRTFD NGSIPHQIHL GELHAILRRQ EDFYPFLKDN REKIEKILTF RIPYYVGPLA RGNSRFAWMT RKSEETITPW NFEEVVDKGA SAQSFIERMT NFDKNLPNEK VLPKHSLLYE YFTVYNELTK VKYVTEGMRK PAFLSGEQKK AIVDLLFKTN RKVTVKQLKE DYFKKIECFD SVEISGVEDR FNASLGTYHD LLKIIKDKDF LDNEENEDIL EDIVLTLTLF EDREMIEERL KTY AHLFDDK VMKQLKRRRY TGWGRLSRKL INGIRDKQSG KTILDFLKSD GFANRNFMQL IHDDSLTFKE DIQKAQVSGQ GDSLHEHIAN LAGSPAIKKG ILQTVKVVDE LVKVMGRHKP ENIVIEMARE NQTTQKGQKN SRERMKRIEE GIKELGSQIL KEHPVENTQL QNEKLYLYYL QNGRDMYVDQ ELDINRLSDY DVDHIVPQSF LKDDSIDNKV LTRSDKNRGK SDNVPSEEVV KKMKNYWRQL LNAKLITQRK FDNLTKAERG GLSELDKAGF IKRQLVETRQ ITKHVAQILD S RMNTKYDEN DKLIREVKVI TLKSKLVSDF RKDFQFYKVR EINNYHHAHD A YEN A V V GT A FIKKYPKFES EFVYGDYKVY DVRKMIAKSE QEIGKATAKY FFYSNIMNFF KTEITFANGE IRKRPFIETN GETGEIVWDK GRDFATVRKV FSMPQVNIVK KTEVQTGGFS KESIFPKRNS DKFIARKKDW DPKKYGGFDS PTVAYSVFVV AKVEKGKS KK FKSVKEFFGI TIMERS SFEK NPIDFLEAKG YKEVKKDLII KLPKYSLFEL EN GRKRMLAS AGELQKGNEL ALPSKYVNFL YLASHYEKLK GSPEDNEQKQ LFVEQHKHYL DEIIEQISEF SKRVILADAN LDKVLSAYNK HRDKPIREQA ENIIHLFTLT NLGAPAAFKY FDTTIDRKRY TSTKEVLDAT LIHQSITGLY ETRIDLSQLG GDSGGSGGSG GSTNLSDIIE KETGKQLVIQ ESILMLPEEV EEVIGNKPES DILVHTAYDE STDENVMLLT SDAPEYKPWA LVIQDSNGEN KIKMLSGGSG GSGGSTNLSD IIEKETGKQL VIQESILMLP EEVEEVIGNK PESDILVHTA YDESTDENVM LLTSDAPEYK PWALVIQDSN GENKIKMLSG GSPKKKRK (SEQ ID NO:688)

[0415] Exemplary BE4 sequence with His tag:

MHHHHHHHHGS GGS GS S ETGP V A VDPTLRRRIEPHEFE VFFDPRELRKET CLLYEINWGGRHS IWRHTS Q NTNKHVEVNF IEKFTTERYF CPNTRCSITW FLSWSPCGEC S RAITEFLS R YPH VTLFIYI ARLYHHADPR NRQGLRDLIS SGVTIQIMTE QESGYCWRNF VNYSPSNEAH WPRYPHLWVR LYVLELYCII LGLPPCLNIL RRKQPQLTFF TIALQSCHYQ RLPPHILWAT GLKSGGSSGG SSGSETPGTS ESATPESSGG SSGGSDKKYS IGLAIGTNSV GWAVITDEYK VPSKKFKVLG NTDRHSIKKN LIGALLFDSG ETAEATRLKR TARRRYTRRK NRICYLQEIF SNEMAKVDDS FFHRLEESFL VEEDKKHERH PIFGNIVDEV AYHEKYPTIY HLRKKLVDST DKADLRLIYL ALAHMIKFRG HFLIEGDLNP DNSDVDKLFI QLVQTYNQLF EENPINASGV DAKAILSARL SKSRRLENLI AQLPGEKKNG LFGNLIALSL GLTPNFKSNF DLAEDAKLQL S KDT YDDDLD NLLAQIGDQY ADLFLAAKNL SDAILLSDIL RVNTEITKAP LSASMIKRYD EHHQDLTLLK ALVRQQLPEK YKEIFFDQSK NGYAGYIDGG ASQEEFYKFI KPILEKMDGT EELLVKLNRE DLLRKQRTFD NGSIPHQIHL GELHAILRRQ EDFYPFLKDN REKIEKILTF RIPYYVGPLA RGNSRFAWMT RKSEETITPW NFEEVVDKGA SAQSFIERMT NFDKNLPNEK VLPKHSLLYE YFTVYNELTK VKYVTEGMRK PAFLSGEQKK AIVDLLFKTN RKVTVKQLKE DYFKKIECFD SVEISGVEDR FNASLGTYHD LLKIIKDKDF LDNEENEDIL EDIVLTLTLF EDREMIEERL KTY AHLFDDK VMKQLKRRRY TGWGRLSRKL INGIRDKQSG KTILDFLKSD GFANRNFMQL IHDDSLTFKE DIQKAQVSGQ GDSLHEHIAN LAGSPAIKKG ILQTVKVVDE LVKVMGRHKP ENIVIEMARE NQTTQKGQKN SRERMKRIEE GIKELGSQIL KEHPVENTQL QNEKLYLYYL QNGRDMYVDQ ELDINRLSDY DVDHIVPQSF LKDDSIDNKV LTRSDKNRGK SDNVPSEEVV KKMKNYWRQL LNAKLITQRK FDNLTKAERG GLSELDKAGF IKRQLVETRQ ITKHVAQILD SRMNTKYDEN DKLIREVKVI TLKSKLVSDF RKDFQFYKVR EINNYHHAHD AYLNAVVGTA LIKKYPKLES EFVYGDYKVY DVRKMIAKSE QEIGKATAKY FFYSNIMNFF KTEITLANGE IRKRPLIETN GET GEIV WD K GRDFATVRKV LSMPQVNIVK KTEVQTGGFS KESILPKRNS DKLIARKKDW DPKKYGGFDS PTVAYSVLVV AKVEKGKS KK LKSVKELLGI TIMERSSFEK NPIDFLEAKG YKEVKKDLII KLPKYSLFEL EN GRKRMLAS AGELQKGNEL ALPSKYVNFL YLAS HYEKLK GSPEDNEQKQ LFVEQHKHYL DEIIEQISEF SKRVILADAN LDKVLSAYNK HRDKPIREQA ENIIHLFTLT NLGAPAAFKY FDTTIDRKRY TSTKEVLDAT LIHQSITGLY ETRIDLSQLG GDSGGSGGSG GSTNLSDIIE KETGKQLVIQ ESILMLPEEV EEVIGNKPES DILVHTAYDE STDENVMLLT SDAPEYKPWA LVIQDSNGEN KIKMLSGGSG GSGGSTNLSD IIEKETGKQL VIQESILMLP EEVEEVIGNK PESDILVHTA YDESTDENVM LLTSDAPEYK PWALVIQDSN GENKIKMLSG GSPKKKRK (SEQ ID NO: 689)

[0416] Rat APOBEC 1

S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKETCLLYEINW GGRHS IWRHT S QNTN KH VE VNFIEKFTTERYFCPNTRCS GG WFLS WS PC GEC S RAITEFLS R YPH VTLFIYIARL YHH ADPRNRQGLRDLIS S G VTIQIMTEQES G Y C WRNFVN Y S PS NE AHWPR YPHLW V RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPH ILWATGLK (SEQ ID NO: 203)

[0417] Anc689 APOBEC

S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKETCLL YE IKW GT S HKIWRHS S KNTT KH VE VNFIEKFT S ERHFCPS T S CS ITWFLS WS PC GEC S KAITEFLS QHPN VTLVIY V AR LYHHMDQQNRQGLRDLVN S GVTIQIMT APE YD Y CWRNFVNYPPGKEAHWPRYPPL WMKLYALELHAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLP PHILWATGLK

(SEQ ID NO: 204) [0418] Anc687 APOBEC

S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKE ACLLYEIKW GT S HKIWRN S GKNTT KH VE VNFIEKFT S ERHFCPS IS C S ITWFLS WS PC WECS KAIREFLS QHPN VTLVIY V AR LF QHMD QQNRQGLRDLVN S G VTIQIMT AS E YDHC WRNFVN YPPGKE AHWPR YPPL WMKLYALELHAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLP PHILWATGLK

(SEQ ID NO: 205)

[0419] Anc686 APOBEC

[0420] S S ET GP V A VDPTLRRRIEPEFFNRN YDPRELRKET YLLYEIKW GKES KI WRHTSNNRTQHAEVNFLENFFNELYFNPSTHCSITWFLSWSPCGECSKAIVEFLKEHP N VNLEIY V ARLYLCEDERNRQGLRDLVN S G VTIRIMNLPD YN Y C WRTF V S HQGGDE DYWPRHFAPWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLP PHILWATGLK (SEQ ID NO: 206)

[0421] Anc655 APOBEC

S S ET GP V A VDPTLRRRIEPF YF QFNNDPR ACRRKT YLC YELKQDGS T W VWKRTLHN KGRHAEICFLEKISSLEKLDPAQHYRITWYMSWSPCSNCAQKIVDFLKEHPHVNLRIY VARLYYHEEERYQEGLRNLRRSGVSIRVMDLPDFEHCWETFVDNGGGPFQPWPGLE ELNSKQLSRRLQAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQR LPPHILWATGLK

(SEQ ID NO: 207)

[0422] Anc733 APOBEC

S S ET GP V A VDPTLRRRIEPFHF QFNNDPR A YRRKT YLC YELKQDGS TW VLDRTLRNK GRHAEICFLDKINSWERLDPAQHYRVTWYMSWSPCSNCAQQVVDFLKEHPHVNLRI FAARLYYHEQRRYQEGLRSLRGSGVPVAVMTLPDFEHCWETFVDHGGRPFQPWDG LEELN S RS LS RRLQ AGILGLPPCLNILRRKQPQLTFFTIALQS CH Y QRLPPHILWATGLK (SEQ ID NO: 208)

[0423] P2A-GFP

GS G ATNF S LLKQ AGD VEENPGP_M V S KGEELFT G V VPILVELDGD VN GHKFS V S GEG EGDATY GKLTLKFICTTGKLPVPWPTLVTTLTY GVQCFSRYPDHMKQHDFFKS AMP EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYN SHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLST QS ALS KDPNEKRDHM VLLEF VT A AGITLGMDELYKS GGS PKKKRK V (SEQ ID NO: 209) [0424] Exemplary ABE7.10 with His tag:

MHHHHHHHHG SGGSGSEVEF SHEYWMRHAL TLAKRAWDER EVPVGAVLVH NNRVIGEGWN RPIGRHDPTA HAEIMALRQG GLVMQNYRLI DATLYVTLEP CVMCAGAMIH SRIGRVVFGA RDAKTGAAGS LMDVLHHPGM NHRVEITEGI LADECAALLS DFFRMRRQEI KAQKKAQSST DSGGSSGGSS GSETPGTSES ATPESSGGSS GGSSEVEFSH EYWMRHALTL AKRARDEREV P V G A VLVLNN RVIGEGWNRA IGLHDPTAHA EIMALRQGGL VMQNYRLIDA TLYVTFEPCV MCAGAMIHSR IGRVVFGVRN AKT G A AGS LM DVLHYPGMNH RVEITEGILA DECAALLCYF FRMPRQVFNA QKKAQSSTDS GGSSGGSSGS ETPGTSESAT PESSGGSSGG SDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRT ARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGW G RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK N YWRQLLN AK LITQRKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLAS AGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGDSG GSPKKKRKV (SEQ ID NO: 690)

[0425] Exemplary ABE:

MSEVEF SHEYWMRHAL TLAKRAWDER EVPVGAVLVH NNRVIGEGWN RPIGRHDPTA HAEIMALRQG GLVMQNYRLI DATLYVTLEP CVMCAGAMIH SRIGRVVFGA RD AKT G A AGS LMDVLHHPGM NHRVEITEGI LADECAALLS DFFRMRRQEI KAQKKAQSST DSGGSSGGSS GSETPGTSES ATPESSGGSS GGSSEVEFSH EYWMRHALTL AKRARDEREV P V G A VLVLNN RVIGEGWNRA IGLHDPTAHA EIMALRQGGL VMQNYRLIDA TLYVTFEPCV MCAGAMIHSR IGRVVFGVRN AKTGAAGSLM DVLHYPGMNH RVEITEGILA DECAALLCYF FRMPRQVFNA QKKAQSSTDS GGSSGGSSGS ETPGTSESAT PESSGGSSGG SDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRT ARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK N YWRQLLN AK LITQRKFDNL

[0426] ABEmax TOLONEKLYLYYLONGRDMYVDOELDINRLSD YD VDHIVPOS FLKDDS IDNKVLTR S DKNRGKS DN VPS EE V VKKMKN YWROLLN AKLIT ORKFDNLTKAERGGLS ELD KA GFIKROLVETROITKHVAOILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFOF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEO EIGKAT AKYFFY S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWD KGRDF AT VRK VLS MPO VNIVKKTE V OT GGFS KES ILPKRN S DKLIARKKD WDPKKY GGFDS PT V AY S VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELOKGNELALPSKYVNFLYLASHYEKLKGSPEDNEOKO LFVEOHKHYLDEIIEOISEFSKRVILADANLDKVLSAYNKHRDKPIREOAENIIHLFTLT NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGD KRTAD GSEFEPKKKRK V [optional P2A-GFP] (SEQ ID NO: 210)

[0427] Sequences 4. Codon optimized nucleotide sequences of BE4, rAPOBEC, Ancestral APOBECs, ABE, and P2A GFP.

[0428] Exemplary ABEmax:

MKRT ADGS EFES PKKKRKV S E VEFS HE YWMRH ALTLAKR A WDERE VP V G A VLVHN NRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGA MIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV LH YPGMNHRVEITEGILADEC A ALLC YFFRMPRQ VFN AQKK AQS S TDS GGS S GGS S G S ETPGT S ES ATPES S GGS S GGS DKKY S IGLAIGTN S V GW A VITDE YK VPS KKFKVLGN TDRHS IKKNLIG ALLFDS GET AE ATRLKRT ARRR YTRRKNRIC YLQEIF S NEM AKVDD SFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDN S DVD KLFIQLV QT YN QLFEENPIN AS G VD AKAI LS ARLS KS RRLENLIAQLPGEKKN GLF GNLI ALS LGLTPNFKS NFDLAED AKLQLS KD TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE HHQDLTLLKALVRQQLPEKYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI LTFRIP Y Y V GPLARGN S RF A WMTRKS EETITPWNFEE V VDKG AS AQS FIERMTNFD K NLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPAFLS GEQKKAIVDLLFKTNR KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP QS FLKDDS IDNKVLTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT LKS KLV S DFRKDF QF YKVREINN YHH AHD A YLN A V V GT ALIKKYPKLES EF V Y GD Y KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKY GGFDS PT V AY S VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AK G YKE VKKDLIIKLPKY S LFELEN GRKRMLAS AGELQKGNELALPS KY VNFLYLAS H Y EKLKGS PEDNEQKQLFVEQHKH YLDEIIEQIS EF S KRVILAD ANLD KVLS A YNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI DLS QLGGDS GGS KRT ADGSEFEPKKKRKV (SEQ ID NO: 694)

[0429] Within base editor sequences, NLS sequences are bold, APOBEC and TadA sequences are italicized, linkers are double underlined, Cas9 nickase sequence is underlined, and UGI sequences are bold and italicized.

[0430] BE4max and AncBE4max

ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAGCGG AAAGTC APOBEC TCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAGACACC AGGAACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGGCGGCA

GC GAGA AGAAGTAC AGCATCGGCCTGGACATCGGC ACC AACTCTGTGGGCTGG

GCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGC

AACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGAC

AGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATA

CACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGAT

GGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGA

AGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT

GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGA

CAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATC

AAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGAC GTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAA

AACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTG

AGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAA

GAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTC

AAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACC

TACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGAC

CTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGA

GAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGAT

ACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGC

TGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCG

GCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCA

TCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGG

ACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCC

ACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCT

GAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTA

CGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAG

CGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTC

CGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGA

GAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGA

GCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAG

CGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGT

GACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTC

CGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCA

CGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGA

GGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGAT

GATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAA

GCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGAT

CAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTC

CGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGAC

CTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCA

CGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCA

GACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGA GAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGA

AGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGC AGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAA GCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACT GGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTT CTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCG GGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACT ACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATC TGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCA AGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGG ACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGA AAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTT TTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAA CGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTT CGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGA GCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAA _{CTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTG} ATC GAG AC A A AC GGC G A A ACC GGGG AG ATC GTGTGGG AT A AGGGCCGGG ATTTT GCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACC GAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGC GATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTC GACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGC AAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAA AGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAA GAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTG GAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAA CGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTAT GAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAA CAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAG AGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAG CACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACC

CTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACC

[0431] The portion indicated by APOBEC in the above sequence of SEQ ID NO 211 may include any nucleic acid sequence encoding an APOBEC, or variant thereof, provided herein. For example, in some embodiments, the APOBEC may comprise any one of SEQ ID NOs: 37-42 or 212-217.

[0432] Rat APOBEC 1

T

C

G

G

G

G

C

G

G

C

G

G

C [0433] Anc689 APOBEC

AGCAGTGAAACCGGACCAGTGGCAGTGGACCCAACCCTGAGGAGACGGATTGAG

CCCCATGAATTTGAAGTGTTCTTTGACCCAAGGGAGCTGAGGAAGGAGACATGC

CTGCTGTACGAGATCAAGTGGGGCACAAGCCACAAGATCTGGCGCCACAGCTCC

AAGAACACCACAAAGCACGTGGAAGTGAATTTCATCGAGAAGTTTACCTCCGAG

CGGCACTTCTGCCCCTCTACCAGCTGTTCCATCACATGGTTTCTGTCTTGGAGCCC

TTGCGGCGAGTGTTCCAAGGCCATCACCGAGTTCCTGTCTCAGCACCCTAACGTG

ACCCTGGTCATCTACGTGGCCCGGCTGTATCACCACATGGACCAGCAGAACAGG

CAGGGCCTGCGCGATCTGGTGAATTCTGGCGTGACCATCCAGATCATGACAGCCC

CAGAGTACGACTATTGCTGGCGGAACTTCGTGAATTATCCACCTGGCAAGGAGG

CACACTGGCCAAGATACCCACCCCTGTGGATGAAGCTGTATGCACTGGAGCTGC

ACGCAGGAATCCTGGGCCTGCCTCCATGTCTGAATATCCTGCGGAGAAAGCAGC

CCCAGCTGACATTTTTCACCATTGCTCTGCAGTCTTGTCACTATCAGCGGCTGCCT

CCTCATATTCTGTGGGCTACAGGCCTTTGA AA (SEQ ID NO: 213)

[0434] Anc687 APOBEC

TCATCAGAAACAGGACCAGTCGCCGTGGACCCAACACTGAGGAGAAGGATTGAG

CCCCATGAATTTGAAGTCTTTTTCGACCCCAGGGAGCTGAGGAAGGAGGCATGCC

TGCTGTACGAGATCAAGTGGGGCACAAGCCACAAGATCTGGCGCAACAGCGGCA

AGAACACCACAAAGCACGTGGAAGTGAATTTCATCGAGAAGTTTACCTCCGAGC

GGCACTTCTGCCCCTCTATCAGCTGTTCCATCACATGGTTTCTGTCTTGGAGCCCT

TGCTGGGAGTGTTCCAAGGCCATCCGCGAGTTCCTGTCTCAGCACCCTAACGTGA

CCCTGGTCATCTACGTGGCCCGGCTGTTTCAACACATGGACCAGCAGAACAGGC

AGGGCCTGCGCGATCTGGTGAATTCTGGCGTGACCATCCAGATCATGACAGCCTC

AGAGTACGACCATTGCTGGCGGAACTTCGTGAATTATCCACCTGGCAAGGAGGC

ACACTGGCCAAGATACCCACCCCTGTGGATGAAGCTGTATGCACTGGAGCTGCA

CGCAGGAATCCTGGGCCTGCCTCCATGTCTGAATATCCTGCGGAGAAAGCAGCC

CCAGCTGACATTTTTCACTATCGCACTGCAGAGCTGTCATTACCAGAGACTGCCT

CCTCATATCCTGTGGGCTACAGGCCTTTGAAA (SEQ ID NO: 214)

[0435] Anc686 APOBEC

AGCAGCGAGACAGGACCCGTGGCAGTGGACCCTACACTGAGGAGGAGGATTGA

GCCCGAATTTTTCAACAGGAACTACGACCCCAGAGAGCTGCGGAAGGAGACATA

CCTGCTGTATGAGATCAAGTGGGGCAAGGAGTCCAAGATCTGGCGGCACACCTC TAACAATAGAACACAGCACGCCGAGGTGAACTTCCTGGAGAACTTCTTTAATGA

GCTGTACTTTAATCCTTCTACCCACTGCAGCATCACATGGTTCCTGAGCTGGTCCC

CATGCGGCGAGTGTTCTAAGGCCATCGTGGAGTTTCTGAAGGAGCACCCCAACGT

GAATCTGGAGATCTACGTGGCCAGGCTGTATCTGTGCGAGGACGAGAGGAACAG

GCAGGGCCTGCGGGATCTGGTGAATAGCGGCGTGACCATCAGAATCATGAACCT

GCCTGACTACAATTATTGTTGGCGCACATTCGTGTCCCACCAGGGAGGCGACGAG

GATTATTGGCCAAGGCACTTTGCACCATGGGTGCGCCTGTACGTGCTGGAGCTGT

ATTGCATCATCCTGGGCCTGCCCCCTTGTCTGAACATCCTGCGGAGAAAGCAGCC

CCAGCTGACATTCTTCACCATCGCACTGCAGAGTTGTCATTACCAGCGACTGCCT

CCTCATATCCTGTGGGCTACAGGCCTTTGAAA (SEQ ID NO: 215)

[0436] Anc655 APOBEC

TCATCAGAGACCGGACCTGTGGCAGTGGACCCAACCCTGCGACGGAGAATCGAG

CCCTTTTACTTTCAGTTCAACAACGACCCAAGAGCCTGCCGGAGAAAGACCTACC

TGTGCTATGAGCTGAAGCAGGACGGCTCTACCTGGGTGTGGAAGCGGACACTGC

ACAACAAGGGCAGACACGCCGAGATCTGCTTCCTGGAGAAGATCAGCTCCCTGG

AGAAGCTGGACCCTGCCCAGCACTACAGGATCACATGGTATATGTCTTGGAGCCC

ATGCTCCAACTGTGCCCAGAAGATCGTGGATTTTCTGAAGGAGCACCCACACGTG

AATCTGCGGATCTACGTGGCCAGACTGTACTATCACGAGGAGGAGAGGTATCAG

GAGGGCCTGAGGAACCTGAGGCGCTCCGGCGTGTCTATCAGAGTGATGGACCTG

CCCGATTTCGAGCACTGCTGGGAGACATTCGTGGATAACGGAGGAGGACCTTTCC

AGCCATGGCCCGGCCTGGAGGAGCTGAATAGCAAGCAGCTGTCCCGGAGACTGC

AGGCAGGAATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGAGGCGCAAGCAGC

CCCAGCTGACATTTTTCACCATCGCACTGCAGAGTTGTCATTATCAGCGACTGCC

TCCTCATATCCTGTGGGCTACAGGCCTTTGAAA (SEQ ID NO: 216)

[0437] Anc733 APOBEC

AGCAGCGAGACCGGACCTGTGGCAGTGGACCCAACCCTGAGAAGACGCATTGAG

CCATTTCATTTTCAGTTTAACAACGACCCCAGAGCCTACCGGAGAAAGACCTACC

TGTGCTATGAGCTGAAGCAGGACGGCTCCACCTGGGTGCTGGATCGGACACTGA

GAAACAAGGGCCGGCACGCCGAGATCTGTTTCCTGGACAAGATCAATTCCTGGG

AGAGGCTGGATCCCGCCCAGCACTACCGCGTGACATGGTATATGAGCTGGTCCC

CTTGCTCTAACTGTGCCCAGCAGGTGGTGGATTTCCTGAAGGAGCACCCACACGT

GAATCTGCGGATCTTTGCCGCCAGACTGTACTATCACGAGCAGAGGCGCTATCAG

[0438] P2A-GFP

[0439]

[0440] ABEmax

[0441] GAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTC

TATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCT

CGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAA

GAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAA

GTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAA

GCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCAT

CCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGA

TTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTC

CGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGG

ATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTG

GACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAG

AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTC

ACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAG

CCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAG

ACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATC

GAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCC

TGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACA

ATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTG

AGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACG

ACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTG

AGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTG

GATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACG

ACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGG

GCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGA

AGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCC

GGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACC

CAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCAT

CAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCT

GCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGT

GGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGT

GCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAG

CGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGA

[0442] Sequences 5. APOBEC homologs used for ancestral reconstruction.

[0443]

WNKKWQITMYLTMQPCHSTDTGGTKEDQSCCEVMIKAKEKLGNVEIVIKPTHLCQV GKGKPEKGVRKLKTTGIELECMKEGDWKY Y AQPEENPYDTS KTEELHN QQLEQ

(SEQ ID NO: 220)

[0444] >D8M4Y9_BLAHO/46-382

CMATGTKCVGAKAQSPEGFVVNDCHAEVLCRRSFIHYLIKEIQKVFEPCEGDFHLQV KRD YHF YM YIS QS PC GY GS E YETN GK AMEQS C S DKLCLLIS PIYLT GIVIC WDE ARM KN ALADRIE V S AIVW S FFKE V V Q YKEIKRERYEQ AKES LMK (SEQ ID NO: 221)

[0445] >11 F5T9_ AMPQE/52-472

S LGTGS KCIGEHKMS LEGWLVNDS H AEIIARRGF VKYLIGQLKS IFERS PIKGQ Y S IKN GGD AS IFLS TKHS S C SDKMMRLID KP V YLHS IT VELYN QE AMERALV GRLCLS KELL GS IVW S DFKELV QNDLP V Y YD WKQS S Y QKARTLFN (SEQ ID NO: 222)

[0446] >E9GMF4_DAPPU/61-428

S MGT GS KC V GQNKLS KDGDILNDS H AE V V ARRGFLR YM YHQMEILFT VD AIS KKFL QREGVSFIFFSSHTPCGDASIIVKENSMCNSCSDKLAKFLSKPIYVSHVIICPYSQSVME RALINRFEHS AN QLS S IS WQKFV S LLSDNLPIYNKAKRLC YFEMWEQTRA (SEQ ID NO: 223)

[0447] >K7Jl68_NASVE49-389

SLCTGTKCLSGVELRSTGNKLSDSHAEILARRAFLRYLYHQIELIFYLEDNGIKLRDVS FHFFSSQTPCGDCSIIKLTGEDLSCSDKLAKLMIPTIKFESIVICPYSLESMQRGIFQRF D PS S IVWKTFLTIYDPQHPKY YHC KQ Y S Y QNLWREKS (SEQ ID NO: 224)

[0448] >F4X2X4_ACREC/49-389

ALATGTKCLGDSELSKGGSRLSDSHAEVLTRRAFIRYLYDQIDLIFSRNDKNKIELNS NIS FHFFS S QTPCGDC S IFKLETN S C S DKIAKLLIPPIKLES ITIS PF S LD AMERGLYKRFN NIS IIW QS FLQIFDIRHPKY YD WKQW S Y QNEWKQLKH (SEQ ID NO: 225)

[0449] >E9IK04_SOLIN/50-395

ALAT GTKCLGDS ELS ES GS RLS DS H AE VLTRRAFIR YLYD QIDLIF S RNDDNEIELNNN ISFHFFSSQTPCGDCSIFKEETTSDSCSDKMAKFMIPPIKFESITICPFSFDAMERGFYK R FNNISIVWRSFLQIFDIRHPKYYDWKQWS YQNEWKRLRR (SEQ ID NO: 226)

[0450] >E2AUE8_CAMFO/49-39l

ALATGTKCLGESELINDGSRLSDSHAEVLARRAFIRYLYDQIDLVFSWSHKNKIELNS N V S FHFF S S QTPC GDCS IFKHEIY S C S DKM AKLLIPPIKLKS ITICPF S LD AMERGLYKR FNNTSIIW QTFLQIFDIRHPKYYDWKQWS YQS KWKQLKL (SEQ ID NO: 227)

[0451] >E2B4R0_HARS A/49-389

ALATGTKCLGESELTNSGSRLSDSHAEVLTRRAFIRYLYDQIDLVFKRNDKNKIELNS AISFHFFSSQTPCGDCSIFKEKTSNSCSDKIAKLMIPSIRLESITICPFSFD AMERGLYKR FNN S S IIW QTFLQ VLDIKHPKYYDWKQWS YQNKWRQLQF (SEQ ID NO: 228)

[0452] >B0WPXl_CULQU/53-394

SLGTGTKCLGADQLSEKGDILNDSHAEVMARRGFLRFVMQQMTSIFEFNSESRKFSC KS GIS FHFFTTHS PC GD AS IYET S RS S C S DKM ARLLERPIYLES V VITDFC KE AIERALW KRWDPDVVEGGGIVWATFAAVWKGADMDYVDVKNRSYREQWDALRV (SEQ ID NO: 229)

[0453] >B4NPW3_DROWE50-393

SLGCGTKCIGQDKQCPKGYILNDSHAEIIARRAFLRYLYHELNKIFEWDHELVCYKLK KH YE YHFLCTHTPC GD ACIGQLDTE V S C S DKLARLIHEPIYFT S FNF S E AN V ADIERAI FKRW QHKEFRN GLS WLCFLELLRIRKS LY GTC KQLA Y QE A W QQLKS (SEQ ID NO: 230)

[0454] >B4JCX0_DROGR/56-405

SLATGTRCIGS S KLC ARGFILNDCH AEVLARRGFLRYLNNEIMKIFS WQT AERCFTLN ENLVFHFLSTQTPCGDACILESNIDTLSCSDKLSRLLNKPIYFTSFNFADAHLKSLQRA FY QRWNGRSCKNSFIWFNFFNFFNFRQKFY AQCKAFAY QFVW QQFKC (SEQ ID NO: 231) [0455] >B4M8L0_DRO VI/67-417

ALATGTRCIGASKLCAKGFVINDSHAEVLARRAFLRYLHNQLLKIFSWKSAAGCFTL NEQLEYHFLSTQTPCGDACIVDSEILSCSDKLSRLIDKPIYFSSLNFAEARLESLQRAV Y QRWRGRNCSTS LIWLNFLDLLHLREKFY AECKALA Y QLAWRQLKC (SEQ ID NO: 232)

[0456] >B4KIX6_DROMO/54-403

ALATGTRCIGASKLCNRGYILNDCHAEVLARRAFLRYLQNELLNVFIWKPSTGCFAL NEQFEYHFFSTQTPCGDACIVDDACGVSCSDKFARVIDKPIYFISYNFTEANPESFERA IYRRW QGRECNS S LIWINFMDLLS VREKLY S ELKALS Y QLA W QQLKS (SEQ ID NO: 233)

[0457] >Q29K7 l_DROPS/55-392

ALGCGTKCIGYTRHCPKGFILNDSHAEVLARRAFLRYLYHELEHIFQWAAKRGSFDL SAHVEYHFFSTQTPCGDACIVESVVVESCSDKLARLLSKPIYFSTLNFTEARQESVER AIYKRW QQGRDS KN GLVWLKFLNILHLREKFY ASCKDLC Y QQAWCQLKR (SEQ ID NO: 234)

[0458] >B3MN3 l_DRO AN/55-389

ALGLGTKCIPHTKLCENGFILNDSHAEVLARRAFQRYLYHELGQIFHWNCDSQCYDL DDHVEYHFLSTQTPCGDACIVEEDRSCSDKLSRLISKPIYFSTLNFADAHFESLNRAIY KRWEGRNFS N GLVWLTFLDCIS LS EKF Y AT C KS WS Y QE A WLQLKD (SEQ ID NO: 235)

[0459] >B4HX96_DROSE/54-382

SLGCGTKCIGESKLCPKGLILNDSHAEVLARRGFLRFLYQELKQIFHWNSELSTFDMD EHVEFHFLSTQTPCGDACILEEQVSCSDKIARLISKPIYFSSLNFDDAQLESLERAIFKR FECRSFKNGLIWLTFLELVKLSEKFY ASCKNLAY QFAWREIKE (SEQ ID NO: 236)

[0460] >H2YAA2_CIOS A/58-486

AIGSGTTCLGESQMRPDGLILHDSHAEVIARRSFIRYLYHEIKSIFNKLIDGKCCLKPGI S FHF YT S NTPCGD ATIF VT VN QP VNT S C S DKILRLV S TP V YMRS IV V ADIQ ALRR AF Y GRMDLN S LDFS ALS WRMFKE VMECELKS YRN C KLLA YFNTW S D VKD (SEQ ID NO: 237)

[0461] >C3Y8L3_BRAFL/55-488

AMGTGSKCIGRSKMRLEGDVLHDSHAEIIARRAFLRYLYHQLQMVFTTPGADCRCG LKPG VKFHLFT S HTPC GD AS IFKGS KS TIDQS CS DKLARFMTEP V YF S TIV V CP Y S S Y A MYRGIVDRCAGIQDLAAIMWETFKQLLNERRPAY GDYKRAAHHTAKTCFLR (SEQ ID NO: 238)

[0462] >F 1 R076_D ANRE/54-459

SLGTGTKCIGRSAMSMKGDVLNDSHAEVIARRGSVRYLTEQLLKVFCAGSEKGKWR LKAG V S FLFFT S QTPC GD AS IFMS GSEPCETSC S DKLARYLQE ALYF S A VLV S P Y S HP ALRR ALHTRC S H VKDLA AIS WHS FLK V V A AELPE YWD YKQ A A Y QLA WTQLRL

(SEQ ID NO: 239)

[0463] >H2S4J7_TAKRU/61-472

SLATGTKCIGRTALSPNGDVLNDSHAEVIARRGCVRYLIQELHRVFCRAEQQGKWKL KPGVSFHFFTSHTPCGDASIIMSDSQPCPGSCSDKMARYLQEALYFTSVVVCPYSQEV MHR ALV ARCLH V S DLA AIS WHS FLS LV S S S LPS YWD YKRAS Y QQ A W QQLHS (SEQ ID NO: 240)

[0464] >AD AT l_XENTR/59-465

AMGTGTKCIGQAKLRKTGDVLQDSHAEIIAKRSFQRYLLHQLSLLFIPGTEKGKWML RPEIS FVFFTS HTPC GD AS IIVIS HELGHGS CS DKM ARFLQQPIYLS A V V V CPFS QD AM ER ALYNRCHKVLS LA A VS WNTFRELV QKQRS E YWD YKA A A Y QE A WNCLRQ (SEQ ID NO: 241)

[0465] >U3JIM9_FICAL/63-494

AMGTGTKCIGRNKMRKTGDILNDSHAEVVAKRSFQRYLLHQLWLIFSPGTETGKWK LKPNIIFIFF S S HTPCGD AS IFIS EPELS KN S C S DKLARFLQ YP V YLS AVIV CP Y S QE AMR RAVIERCQHVSSLAAISWHKFQRLMTEDLPDYWDYKQAAYQEAWRVLRS (SEQ ID NO: 242) [0466] >ADATl_CHICK/63-496

ALGTGTKCIGLNKMRKTGDVLNDSHAEVVAKRSFQRYLLHQMRLIFIPGTETGKWK FKPNIIFIFFC S HTPC GD AS IIIRETEFS KS S CS DKFARFFQ YP V YES AVIV CP YS QE AMQ RAVIERCRHISLLAAISWHEFQKLVTENLPDYWDYKEAAYQEAWKALRS (SEQ ID NO: 243)

[0467] >U3IY34_ANAPL/63-495

AMGTGTKCIGQNKMRKTGDILNDSHAETVAKRSFQRYLLHQMCLIFIPGTETGKWK LKPNIIF VFF S S HTPC GD AS IIIS EPELS KN S CS DKLARFLQ YP V YLS AIIVCP Y S QE AMR RAIIERC QH V S FLA AIS WH AF QKLVTENLPD YWD YKE A A Y QE A WKVLRS (S EQ ID NO: 244)

[0468] >F7EENO_ORN AN/78-517

AMGTGTKCIGQSKMRKDGDILNDSHAEVIAKRSFQRYLLHQIEAIFIPGTEVGKWKL KPDLSFVFFSSHTPCGDASIIMLDVEPCPRSCSDKLARLLQDPVYLAAVVICPYSPEAV RRA VIDRCRHVSPLA AIS WGAFQKLV GDKQPA YWD YKE AA Y QETWKALQR (SEQ ID NO: 245)

[0469] >F7G3V9_MONDO/64-497

SMGTGTKCIGLSKMRKNGDILNDSHAEVIAKRSFQRYLLHQLRSIFMPGTKTGMWK LKPDLHFVFFSSHTPCGDASIIMLELEPCSHSCSDKLARFLEEPVYLAALVICPYSPEA MKRA VIDRCQHV S S LAAIS WNLFQNLV S S KRPE YWE YKEAA Y QE AWKTLQK (SEQ ID NO: 246)

[0470] >G 1 T9N 5_RAB IT/63 -492

SMGTGTKCIGQSKMRKSGDILNDSHAEIIAKRSFQRYLLYQLHMIFVPGTQRGLWKL RPDLSFVFFSSHTPCGDASIIMLEFEPCCSSCSDKVARFLEEPIYLSAVVICPYSQEAMQ RALLGRC QN V S ALA AIS WRS F QKLLS DKWPD Y QEHKE A A Y QE A WRTLRK (SEQ ID NO: 247) [0471] >GlQ6Y l_MYOLU/63-485

SMGTGTKCIGQSKMRKSGEVLNDSHAEVIARRSFQRYLLHQPHLIFIPGTQRGLWKL RPD VFLF V S S HTPC GD AS IIMLEFEPC Y S S C S DKLARFLEEP V YLS A V VICPY S QE AMQ RAPVRRCRMSPLYAAISWRSFQELLSDKWPDYQEYEEASYQEAWSALRK (SEQ ID NO: 248)

[0472] >F6Z5 W 8_HORSE/63 -490

SMGTGTKCIGQSKMRKSGDILNDSHAEIIARRSFQRYLLHQLHLIFVPGTRRGLWKLR PDLLF VFF S S HTPC GD AS IIMLEFEPCCS S C S DKLARFLEEPIYLS A V VICPY S QE AMQR ALT GRCHNIS ALA AIS WRS FQKLLS DKWPD Y QE YKE A A Y QE A W S ALRK (SEQ ID NO: 249)

[0473] >M3YEQl_MUSPF/71-497

SMGTGTKCIGQSKMRKSGDILNDSHAEVIARRSFQRYLLHQLHLIFVPGTQRGLWKL RPDLSFVFFSSHTPCGDASIIMLEFEPCCSSCSDKLARLLEEPVYLSAVVICPYSQEAM HRALT GRCQN V S ALA AIS WRS F QKLLS DKWPD YHE YKE A A Y QE A W S ALRK (SEQ ID NO: 250)

[0474] >ADATl_MOUSE/63-492

SMGTGTKCIGQSKMRESGDILNDSHAEIIARRSFQRYLLHQLHLIFVPGTQRGLWRLR PDLSFVFFSSHTPCGDASIIMLEFEPCCSSCSDKMARFLEKPIYLSAVVICPYSQEAMR RALTGRCEETLVLAAISWRSFQKLLSDEQPDYQEYKDAAYQEAWGALRR (SEQ ID NO: 251)

[0475] >HOVKI8_CAVPO/62-489

SMGTGTKCIGQSKMRKSGDVLNDSHAEVIARRSFQRYLCHQLQLIFVLGTQKGQWK LRPGISFVFFSSHTPCGDASIIMLGFEPCYSSCSDKVARFLEEPIYLSAVVICPYSQEAM HRALIGRCQNISALAAISWRSFQKLLSDMWPNYQEYKQAAYQEAWSALRK (SEQ ID NO: 252)

[0476] >G7QlN8_MACFA/63-495

SMGTGTKCIGQSKMRKSGDILNDSHAEVIARRSFQRYLLHQLQLIFVPGTQKGLWKL RRDLFFVFF S S HTPC GD AS IIMLEFEPCC S S CS DKM ARFLEEPIYLS A V VICP Y S QE AM QRALTGRCQN V S ALA AIS WRS F QKLLS DKWPD Y QD YKE A A Y QE A W S TLRK (SEQ ID NO: 253)

[0477] >I3MW85_ICTTR/63-492

SMGTGTKCIGQSKMRKSGDILNDSHAEVIARRSFQRYLLYQLQLIFVPGTQRGLWKL RPNLLFVFFSSHTPCGDASIIMLEFEPCCGSCSDKMARFLEEPIYLSAVVICPYSQEVM QRALIGRCQNVLALSAISWRSFQKLLSDKWPDYQEHKEAAYQEAWSALRK (SEQ ID NO: 254)

[0478] >D8SETQ7_SELML/51-383

ALGTGTKCLGGSQRSLAGDTINDCHAEVIARRTLLKLLYTDIGSNGRLRMRPEIRLHL YISQSPCGDACGVDSCSDKIARFLNEPVYISTITVSDLACIEALQRATFGRLCSVARNY SISWRSFRHLLTHFFPGYLECKKLAYLEAKEILLH (SEQ ID NO: 255)

[0479] >M0SLB0_MUS AM/69-408

ALGT GTKCIGGS LLS PTGD V VNDS H AEIIARRS LLRYFY AEIER AFDT S CC GQTKYRM NPGWGLHLYITQLPCGVFSYPTLRELSCFNKITRILQPVYLSTLTVSVKTNYLEKAIYD CMERLHIKY S IC WE VFV S LQS RLS LQ YHGLKAM A Y QS S LKMLR (SEQ ID NO: 256)

[0480] >M 1B Y G7_SOLTU/64-418

SLGTGTKCIGRSRRSSKGDVVNDSHAEIIARRALLRYLYSEIQDMFESDGLGTKKLK MKHGW QLHLYIS QLPC G V AS PGS ELAHDS MS C S DKIARFLEPIYIF S VTIS IIEDE VMR AIHERVLPLS NKY S IC WES FLS LS HNC VGE YRELKDKS YNLAS ETFK (SEQ ID NO: 257)

[0481] >K7KC56_SOYBN/58-388

AMGTGTKCLGRSLLRTCGDVVHDSHAEVIARRALIRFFYTQIQPFDPGCLNKGKYSL KKD WKFHM YIS QFPC GD AS FS VS PFS CS DKIARFFQP V YFS S IT VFRFEDS FKRA YE RMFPFS NE Y S IC WE VFFS FGPENFIG YREFKDG A YHFAS KIFK (SEQ ID NO: 258) [0482] >F4HU58_ARATH/50-411

ALGTGTKCVSGSLLSPRGDIVNDSHAEVVARRALIRFFYSEIQRSIDSSCPGEVKYKLK S GCLLHLYIS QLPC GY AS TS S PLYKKIS CS DKI ARVLQP V YIS TIT VF S LADHLRRS LYE RILPLSDEYSLCWELFLKETHGHKREYRELKNKAYYLMSKIFK (SEQ ID NO: 259)

[0483] >W4XXD3_STRPU/717- 1055

S LGTGNRC VT GDKLS MEGRT VNDS H AEIITRR AFLR YLYN QLQ AILT QGTN GKLRLL PDVSLHLYISTAPCGDGAQFSRTDENESSCSDKVASFIEPMYLSSISLSLYHHGHLARA

V CCRV S S ELDNLLS IN W S E Y QQTCRLFDRT YHD AKLS A Y YT AKQ YLKC (SEQ ID NO: 260)

[0484] >IlGJ0l_AMPQE/629-965

SFGTGTRTASGDLLDLKGEVVFDSHAEIIARRGLKMFLYQELQVIFEANEDGKLRVK KS IKFHLYIS T APCGD G AQFS RLDNRLS S C S DKIGS F VEP V YLF S LS LS LHHHGHLS R A

V CCRFHELGP YLS MN WNEFLS LS S VS GHT Y AE AKS LAY QE AKGLLHK (SEQ ID NO: 261)

[0485] >A7REZ9_NEMVE/ 161-491

S LG AGNRC VT GQRLS MEGKV VNDS H AEIIARRS LLRFFY AQLH AIFEKNN S RRLA VR QG V S FHLYIS T APC GDG ALFTPRENTDLS C S DKICRFIEP V YLES LTLYLYDHGHLAR A V CCRLQDLG AELS VNWES FKELCQRTDRE Y S Q AKQM AF QN AKRELFE (SEQ ID NO: 262)

[0486] >KlPWV0_CRAGE737- 1070

SIGTGNRCITGPQLSLEGNTVNDSHAEIITRRGFIRFMYKQLQALFEPSPSGKLRLKDNI TFHLYISTAPCGDGALFSPRDSNNASCTDKLCRLMDPVYLDSLTLLLYDHGHLARAM CCRLARDINSLLSVNWDNFKQVCADLGKQYGKAKTAAFQTAKKALIK (SEQ ID NO: 263)

[0487] >TU5P4_STRMM/754-l087

SMGTGNRCISGERLSQEGLVVNDSHAEIVTRRGFLRFLYKQLIEIFEPSENRKFRVKPD VTFHLYIS T APCGDG ALF S KTD V S ELS C S DKIARFVDPIYLT S ITLYL YDHGHLS R A V C CRLSELDTLHSLSWASFKEACIKHQRDYYLVKTAAFQKAKKVLLE (SEQ ID NO: 264)

[0488] >M4A7Z3_XIPMA/994- 1320

S LGTGNRC VKGEELS LKGET VNDCH AEIIS RRGFIRFLY S ELLKIFEP AEENKLKIKPDI TFHLYISTAPCGDGALFDCSEKSCSDKILRFLHPIYLKSITLYLYSHGHLTRAVCCRLA RAF S QN S S VNWCLFRS LC QRC GRT Y AQ ARTS AFQLAKQQFFE (SEQ ID NO: 265)

[0489] >M3XGSO_LATCH/812- 1140

SIGTGNRCVKGEELSLKGETVNDCHAEIISRRGFIRFLYSELMKFLEVVSDGRMKIKT GVTFHLYISTAPCGDGALFDCSEKSCSDKILRFLHPVYLSSVTLYLYSQGHLARAICC RMS RAF QEGS S VNW QLFQQICTKTDRKY QE AKE A AF QKAKVHF V Q (SEQ ID NO: 266)

[0490] >U3IQ8 l_ANAPL/729- 1061

SIGTGNRCVKGEELSLKGETVNDCHAEIISRRGFVRFLYSELMKIFEPAGKKRLKIKSN VTFHLYVSTAPCGDGALFDCSDKSCSDKILRFIEPVYLSSVTLYLYSQGHLTRAICCR M VR VLQKRS S VNW ALF QQLC AKNKRKY S E AREA AY QE AKQRFF S (SEQ ID NO: 267)

[0491] >F6ZMB0_XENTR/760- 1089

SIGTGNRC VKGEELS LRGET VNDCH AEIVSRRGFIS NTIXS QLMKIFEE AEGDLLRVRP GVTFHLYISTAPCGDGALFDCSDKSCSDKILRFMEPVYLSSLTLYLFSQGHLTRAICCR MSRAFQNQSSVNWTLFQQLSVLRGRHYSDVKATAYQTAKGQLFR (SEQ ID NO:

268)

[0492] >F7 GM Y 9_M ACMU/591 -920

SLGTGNRCVKGDSLSLKGETVNDCHAEIISRRGFIRFLYSELMKIFEPAKGGEKLQIKK TVSFHLYISTAPCGDGALFDCSDKSCSDKILRFLQPIYLKSVTLYLFSQGHLTRAICCR VTR AFEDGT S VNWLLFKKLC S FR YRR Y GE AKKA A YET AKN YFKK (SEQ ID NO: 269) [0493] >C3YCU6_BRAFL/320-659

AMGT GN S C VMGKNIGTDGRTLNDCH AE V V VRRS LLR YLYRELKNIFEKHS D S LLVL KEG V S FHLYLTT APC GD A ATHITRDTRV QS C S DKLAKFIEP V YLS S VTFTN YDHGQLS RALCCRIDDHISADISLNWMRFKAMCTLARRQYNQAKMMCYQEAKRQLYD (SEQ ID NO: 270)

[0494] >H3ABU2_LATCH/l79-499

ALGTGNSCYAGWLAFDGRLLHDCHALVVVRRTLQRFLYKQLLLIFCSSPGGSLSLRP GIFFHLYLGKVPEGAARTTFMYAPRNPSESDKMTRFIQPIYITSVVLHFYDRNTVSQVI NKRLEG ALIRKS S LNW S S FRKV AEKTGRS YHS VKLRA Y QKVKKQ VN C (SEQ ID NO: 271)

[0495] >H9G4E7_AN OCA/55-372

ALGTGDICYEGWMEFNGRRVHDMHGMVVARRVLLRYFYKQLLMIFCPAGDGILAL KPKYFLHLYLS RTPS GAS ENFHLAP V V QS GS DKLTRIILP V YIT S V VLP Y QDHT VLHE V VNDRV QLGPGN GLS LNW S MFRKV V QEMKRE YHKGKV Q A Y QS AKLQM YT (SEQ ID NO: 272)

[0496] >GlNZA7_MYOLU/269-59l

ALGTGS S S C AGWLEF S GRQLHDCHGLV V ARR ALLRFLYRQLLLVLTPQPGPGF VLKP RIFIHF YIS NTPKG A AHDIYPP A AS AS DKLS RFLPPLY ATS LVLPCHDPPTLS S AIHTRL DRV S GS CLS LNWR AFRQ A AQ ALGKS YEE AKA Y QE ARQQLS L (SEQ ID NO: 273)

[0497] >FlM8B2_RAT/237-552

ALGT GS S S C AGWLEF S GRRLHDCHGLVIARRALLRFFFRQLLLVLTPQPGS GF ALKPG VFLHLY V S NTPKG A AHDIYLAS PS AS DKLARFLPPLY AT S LVLPCHDPPTLNRAIHS R LDS VLGS CLS LNW A AFRQ V ARALEKP YE A AKA YRE ARQQLS L (SEQ ID NO: 274)

[0498] >HOXOC9_OTOGA/275-589

ALGTGNSCCAGWLEFSGRQLHDCHALVVARRALLRFLYRQLLLVLAPQPGPGFALK PRIFLHLY V S NTPKG A AHDIY S AS TS AS DKLARFLPPLY STS LVLPCHDPS TLS R AIHS R LDGALGPVLSLNWRAFRQAAGALGKPYEAAKAYQEARRRLSL (SEQ ID NO: 275) [0499] >F6QYL4_MACMU/258-573

ALGTGSSCCAGWLEFSGQQLHDCHGLVIARRALLRFLFRQLLLVLAPQPGPGFTLKP RVFLHLYIS NTPKG A ARDIYPT S PS AS DKLARLV S PLY STS LILS CHDPPTLS RAIHTRL DS VLGPCLS LNWR AFHQ V AR A V GKP YE A AKA Y QE ARRQLS L (SEQ ID NO: 276)

[0500] >H3 A635_LAT CH/258-579

ALGTGDFNYSQCICRDGRVVHDSHGVVMARRSLLRFLYRQLLLIFCIEPTSKLTIKPN ANIHLYLNQLPKGAAQIKSQLRPQSQSASDKLTRFIQPVYISSVLIANCTDTRGLEIAV KQRVDD ALT S RLS LNW S RFNLLAKES NREYHD AKIMS Y QE AQCLLKS (SEQ ID NO: 277)

[0501] >H2RE88_RONAB/193-512

AIGT GE YN Y S QDIKPN GRVLHDTH A V VT ARRS LLRYFYRQLLLIFCTEPT S NLTLKQN INICLYMNQLPKGSAQIKSQLRPHSESSSDKLTRFIQPVYISSILVGNCSDTRGLEIAIK Q RVDD ALT S KLS LNW S RFNLLAKE AKKYH A AKCMS Y QE AKC KLKS (SEQ ID NO:

278)

[0502] >H0VR27_CAVPO/289-609

AVGTGEYNCSQCIKPNGRVLHDTHGVVTARRSLLRYFYRQLLLIFCTEPASDLTLKQ DINIYLYMN QLPKGS AQIKS QLRPN S ES S S DKLTRFIQP V YIS NILV V GN C SDTRGLEIA IKQR VDD ALT S RLS MNW S RFNLLAKE AKRDYH A AKCMS Y QE AKTLLKS (SEQ ID NO: 279)

[0503] >GlNDQ0_MELGA/l65-484

ALGTGDCNYSNDYSPEGRVVHDSHAIVTARRSLLRYFYRHLLLLFCTAPDSKLTLKR NISIYLYMSQLPKGSAQIKTQFCPHSESASDKLTKFIEPVYINTILVGNCRSLKGLEIAI R QRIDD ALT S KLS LNWSRFKCLAGRAKRT YHE AKVKS Y QEAKKLLHS (SEQ ID NO: 280)

[0504] >U 3 IGD7_ AN APL/256- 575

ALGT GQCN Y S QD Y QPN GRVLHDS H AIVT ARRS LLRYFYRHLMLIFCT APGS KLVLK QNTNIFLYMN QLPKGS AQLKS QLHPQS ES ATDKLTKFIEP V YIN S IL V GN C KDTRGLEI A VKQRIDD ALT VELS LNW S RFKS LAKE AERN YHKAKIQS Y QE AKKLLHS (SEQ ID NO: 281)

[0505] >H0YUT0_TAEGU/l65-484

ALGTGECNYSRRFESCGRLLHDSHAVVTARRSLLRYFYRHLLLIFCTAPGSKLTLKR NITLS LYMN QLPKGT AQLES E VHPQS ES AGDKLTKFIEP V YIS NILV GS C KDTKGLDIT IKQRLDDELT S KLS LNW S RFRMLARE AGRD YHE AKVKS Y QE AKS LLQS (SEQ ID NO: 282)

[0506] >W 5M4T 6_LEPOC/238-558

AIGT GDTNTN QH AT AN GRLLHDS H A V VT ARRS LLR YLYRHLLLIF QLDLN S QLTLKR NITIHLYMN QLPKGS AQLPPRLHPCS N S AS DKITQFIEPIY V GS ILIAS C S D VRGLEIA V KQRVEGIT S KLS MNW S RFNLV AKE AERE Y YE ARMS S Y QE AKIVLKT (SEQ ID NO: 283)

[0507] >FlRC56_DANRE/229-549

ALGT GS S NTKAS P APT GRILHDS H A V VT ARRS LMRFLYRNLLLVF QQDETTKLS FKN HITLHLYLS QLPKG AS QIPS QLRPLS N S ATDK VMQFIEPIYMS S IFIGS C S DIRGMEM A V N QRVDGIT SALS LNW S RFNLV AKES QRE YRE AKMM A Y QE AKS MLKS (SEQ ID NO: 284)

[0508] >W5LAE0_ASTMX/254-574

ALGTGNLNTKESLTPSGRILHDSHAVVTARRSLMRYLYRHLLLIFQQDQNTKLSLKS HITLHLYLN QLPKG A AQIPS HLRPLS N S ATDKITQFIEPIY V S S ILIES C S DTRGME V AIN QRVDGITS KLS LNW S RFNLV S KE AQRE YRE AKMM A Y QE AKS VLKS (SEQ ID NO: 285)

[0509] >H2N2Y7_ORYLA/l66-486

ALGT GGFNTRES IS S D GRIVHDS H A V VT ARRS LMR YLYRNLLM V Y QHKS N S NLS LKT GIS LHLY VN S LPKG A A VIPS KLYPLS S S T ADKITQFIEPIY V QS ILIS CCTD VRGME V S V CQRVEGVTSQLGINWHRFKLVAKEAQRHYREAKRMAYQEAKSVLRA (SEQ ID NO: 286)

[0510] >G3PWS7_GASAC/l62-482

ALGT GNFN AKES AST GGRIVHDS H ALV S ARRS LMRFLYRHLLM VFEQS GS GLS LKS G ITLHLY VN QLPKG A AQIPS QLRPLS N S ATDKLTQFIEP V Y VHS ILV V GC S D VRGLKMS V S QRVEGIT S QLGINWHRFKLV AKE ARRL YRE AKRM A Y QE AKN VLR A (SEQ ID NO: 287)

[0511] >E4WSB5_OIKDE481-800

ALGTGTKTMTGDYISSIGTAVIDCHGEIISRRNLKRYFYAELQKIFERKDGQFALRDGI KFHLYINTTTC GD AR VFNPN S DFES S C S DKVMRLIEPIYLS S IS LGLYHRQHFPR AMFE RIDDIGLDF S INW QLYLLG Y ATKIYLD AKNLS YQS VKN GLYE (SEQ ID NO: 288)

[0512] >XlWRM4_ACYPE235-549

CLATGTKCLSGNYLSLSGESLHDCHAEILTRRCLLKFFYKELMEIFVFSGDKKFKLAE DIAFHLYINT APC GE ARVF S FCDEN S C S DKLTLFIEPT YLES IS IS VFNINHMKRTIY GR VENSIDKYCVNWKEYIKLCSLSTLIYDLLKNKNYIAAKNQILS (SEQ ID NO: 289)

[0513] >H3EEX4_PRIP A/256-594

SLATGNKCIKATSLSFDGCAVNDCHAEILTRRGLVRWLYTQVQLLVEQGEEGGKLR LRKRFS LHLFIS T APC GDGR V Y QF GS NKDN S C S DKVLKFLHPLLICLS IYLS S LALHFT RES CIAR AC Y GR V ARFKP V S ANWTRFA ALTP YKD VKRS AY S KT QFELIR (SEQ ID NO: 290)

[0514] >H2W2W9_CAEJA/l69-490

SLSTGNKGLRGDKVVSDGTALIDCHAEILARRGLLRFLYSEILKIFMKKGTLVLRPGIS FHLFINT APCG AGRIDNKAKS VKAS C S DKLLRFVEPIY Y S S IA VEKNN VERLQRA VF G RAASSSVNWELMVHVCAITQTYDELKAGCYETEKKAFIA (SEQ ID NO: 291)

[0515] >A8WK36_CAEBR/l68-488

SLATGNKGLRGDQITSDGSALIDCHAEILARRGLLRFLYSEVLKIFETGKLALRQGISF HLFINT APC GT AR VDRKVRT AE AS C S DKLLRFIEP V Y Y AS IA VEQNNFVRMNKA V YT RAS S GS MNWELM VKT CFLTKT YEEMKAGC Y S A AKKS FIM (SEQ ID NO: 292)

[0516] >G0MG92_CAEBE/l70-489

SLATGNKGLSGDKIRSDGSALIDCHAEILAKRGLMRFLYSEVLKIFLESGLILKLGITF HLFINTAPCGTARVDKKMRNPESSCSDKLLRFMNPIYYSSIAVEKSNFERMNRAVND RAAGGSMNWELMKTVCTLAKIYEELKAGS YAATKKEFY A (SEQ ID NO: 293)

[0517] >E3 NFH7_C AERE/ 177-503

SLSTGNKGLRGDKIVNDGSALIDCHAEILARRGLLRFLYSEVIKIFERGTLVLKKGILF HLFINTAPCGTARVDRRMKTAEASCSDKLLRFIEPIYYTSIAVEQNNFDRLNKAVFAR A AN GS MNWELMM ATC KLTQT YDELK AGS YLN AKQS FIK (SEQ ID NO: 294)

[0518] >ADR2_CAEEL/l67-486

ALSTGNKGLRGDKIVNDGTALIDCHAEILARRGLLRFLYSEVLKIFTKGKLVLKPGISF HLFINT APC G V ARIDKKLKT S DNS C S DKLLRFIDPIY Y S S IA VELNN ADRLRKA V Y S R A AT S S MNWELMITICTLTKT YEELK AGS Y A A AKKS FIT (SEQ ID NO: 295)

[0519] > A7 S FG 1 _NEM VE/255-570

SIGTGTKFIS GEYISDKGY AVNDCHGEIIARRGLRKFLYN QLELIFELKPS GYGLKDQV EFHLYIS TS PC GD ARVFS PHEPEEKS CS DKIC KFIEP V YFT S IILS LYR YNHM ARAM YE RV GP VEDHS LNWTKFLALW ARQ YDE AKLS AY QKAKAS LMM (SEQ ID NO: 296)

[0520] >T 111 QO_RHOPR/253-578

SVGTGTKCVGGEHISVKGAVLNDSHAEIISRRGLVRYFYSQLLLIFIRNEDGQFCLKPE IRFHLYISTSPCGDARIFSPHDNTSSCSDKVAKFIEPIYLHSIVVSLFHQTHLRRALYGR I ENTLIGHS INWIKFHELLRKT GIT Y S D AKEL V YKD AKT YMIA (SEQ ID NO: 297)

[0521] >D6X3 T2_TRIC A/272-590

S VTT GTKCIS GEHIS MN GC S LNDMH AEILS RRCLIT YF YDQLELIFT QRED GKYKLKP GLDFHLYINT APC GD ARIFS PHEE AS S C S DKICRFIEPIYLKS IVLS LMRE AHM YRALC GRIENTIQGFA V VWKRFARLV GS VTEIY CD VKD A V YKA AKTNLYE (SEQ ID NO: 298)

[0522] >E2B L35_HARS A/335-651

C VTTGTKC V S GEHLS VS GGA VNDCHAE VVARRCLCEYLYKQLELILEPAKKGFKLK QGIQFHLYINT APC GD ARIF S PHEES AS CS DKIARFIEPIYFHS IVLS LLNPS HM YR A V C GRIENTIQG Y S VNWRRFFNLLGTIED V YLE AKLS V Y S LAKRQLKD (SEQ ID NO: 299)

[0523] >B4MGV3_DROVE350-668

S V S TGTKC V S GEHMS VN G A VLNDS H AEIV S RRCLLK YLY AQLDLIFVRNTD GQ YKL KS G VHFHLYINT APC GD ARIF S PHET G AS CS DKIARIIEP V YLHS IVLS LLHPEHM YRA VCGRIEKSIQGFGINWDKYGFLMKGMQYGETKADVYQTAKQELFS (SEQ ID NO: 300)

[0524] >Ql7I09_AEDAE/283-6 l2

SLATGTKCVSGEHMSVTGSVINDSHAEIIARRGLLDFFYTQLDLIFVAPTDGTYKLKD GIHFHLYINT APC GD AR VFS PHENMD AS C S DKIS RVIEPIYLHS IVLS LLHP AHM YRAI CGRIEN S IQGFSINWRRFAA VIEHARY GETKMC VY QQAKKELFA (SEQ ID NO: 301)

[0525] >K 1 QN Q5_CR AGE353-676

SVSTGTKCIN GEYMSDQGLA VNDCHAE VIGRRSLMRYLYFQLGKIFQEKEGGFMLK PNIHFHLYISTSPCGDSRIFSPHEQEASCSDKIARFIEPVYFDSLILSLYHGDHLSRAVY S RISNIENFAVNWKRFLQLCGLTGQSYADAKVTVYQTAKQQMYL (SEQ ID NO: 302)

[0526] >C3XSL3_BRAFL/39-363

SLATGTKCINGEYMSDQGMALNDCHAEIVSRRSLLRYLYSQLDLVFEPKEDGKYRL KDNIQFHLYIS T S PCGD ARIFS PHETD AS C S DKIARFVEPIYLS S IILS LYHGDHLS RA V Y QRLGELEQF S VNW QC WNKLF GTTGRH Y S E AKLLN Y Q A AKQEM VK (SEQ ID NO: 303)

[0527] >H2QL56_PANTR/370-731

SVSTGTKCINGEYMSDRGLALNDCHAEIISRRSLLRFLYTQLELIFQKSERGFRLKENV QFHLYISTSPCGDARIFSPHELEAASCSDKIARFVEPIYFSSIILSLYHGDHLSRAMYQR I SNIEDFSVNWCRWMRVHGHLLRSYHESKLAAYQAAKARLFT (SEQ ID NO: 304)

[0528] >F7DBJ2_HORSE/369-690

SISTGTKCINGEYMSDRGLALNDCHAEIIARRSLLRFLYTQLELIFQKSERGFRLKENV QFHLYISTSPCGDARIFSPHELEASCSDKMARFVEPIYFSSIILSLYHGDHLSRAMYQRI SNIEDFSVNWCRWMRVHGNLLRSYHESKLAAYQAAKACLFK (SEQ ID NO: 305)

[0529] >H3CUl3_TETNG/420-74l

CVSTGTKCINGEYMSDRGLALNDCHAEIIARRSLIRYLYSQLEFIFVHYEKGYRLKDN V QFHLYIS TS PC GD ARIF S PHE VE AS C S DKIARFMEPIYF S S IILS LYH ADHLS R AM Y QR IADIEDFS VNW S RW VRLHS S ILS N YHE AKQ A A YHS AKQ ALIK (SEQ ID NO: 306)

[0530] >H2N2D8_ORYLA/315-637

CIS T GTQCID GEHLS EDGLT VNDCH AE V V ARRCLVRFLY S QLELIFRRGEC GRFRLKD KVQFHLYVSASPCGDACIFSPHDVETSCSDKMARFTEPLYFSSIIVSLYHAAHMSRAM YGRIGPLEGFSENWCRWMQLHSSFLRVYHQAKRAAYCSAKHTLYR (SEQ ID NO: 307)

[0531] >I3JK85_ORENI/388-7 lO

C VS T GTKCIGRE YMS NRGLALNDCH AEIV ARRS LIRFLY S QLE YIFMRCDN S QFRVKE N V QFHLYIS TS PC GD ARIFS PHEVD AS CS DKIARFTEPIYFS S IILS LYH ADHLS R AM Y Q RITEIEEF S VNW S RWMNLHC S MLWIYHDTKQ A A YHS AKETLFR (SEQ ID NO: 308)

[0532] >H2N9L5_PONAB/404-725

ALSSGTKCISGEHLSDQGLVVNDCHAEVVARRAFLHFLYTQLELIFVRLKEGYRLRE NILFHLY V STS PCGD ARLHS P YETDFS CTDKI ARF VEP V YLQS IV VS LHHT GHLAR VM S HRMEG V GQFS VNW ARW AQLY GTRTPS YCE AKLG A Y QS VKQQLFK (SEQ ID NO: 309)

[0533] >F6VCE0_XENTR/367-729

ALS S GTKCIN GEYLNDQGLVVNDCHAEIIVRRAFLHFLYTQLELIFIRLKEGYRLRENI MFHLY V STS PCGD ARLHS P YES DFS CTDKITRFIDPIYLQS IIV S LHHTGHLS R VMS HRI EDLGNFS LNWTRW ARLY GTRIGNYCD AKLAHY QS VKQQMFK (SEQ ID NO: 310)

[0534] >H3C3F5_TETNG/393-7 l5

ALSTGTKCINGEYLSDQGLVVNDCHAEVTARRALLRFLYSQLEFIFVRHKERYRLRD NIHFHM YIS T S PCGDGRLN S P YES DHS CTDKITRF VEP V YLHS VTIS LRHT GHLGR VLN QRLERLGPMS VNWTRW S RLYRTH AS S Y AE AKM A A Y QS VKQQWFR (SEQ ID NO: 311)

[0535] >W5LIl8_ASTMX/382-706

ALSTGTKCINGEYMSDQGLVVNDCHAEVTTRRALLRFLYSHLELKLVQQGRGFRLR V C VIYHLM AF V ADCQT ARFAS PFFLS F S CTDKIARFVEP V YLS S LTV S LRHTGHLS RT LS QRLERLGPYS VNWTRWTRLYRAC APGYCEAKQAA Y QT AKEHW VR (SEQ ID NO: 312)

[0536] >X2BQ53_DANRE/4l 1-730

ALSSGTKCINGEYISDQGLVVNDCHAEITTRRALLRFLYSQLELIFVRHKDSLRLREN VLFHM YIS TS PC GD AR VN S P YES DFS CTDKIS RFVEP V YLY S LT VS LRHT GHFS RMMN HRMEKAGP Y S VNW ARW VRLYRVRG AS YCE AKQ A A Y QT VKLQWLK (SEQ ID NO: 313)

[0537] >W5N363_LEPOC/428-750

AISTGTKCIN GE YIS DRGLVVNDCHAEIIARRAFVRFLY S QLELIFIRHKE AYRLRENIL FHMYISTSPCGDARLNSPYEADFSCTDKITRLVEPVYLHSLIVSLHHTGHLSRIVTHRL DRMGHFS VNW ARW AKLYRTQVPNYCEAKLAA Y QT VKQQLFK (SEQ ID NO: 314)

[0538] >H2LP25_ORYLA/349-671

SLATGTKCLDLEDESDSGRILRDCHAEVISRRALVRFLYAQLELIFVRNKDNFRLQEGI LFHMYVSSSPCGDARLNCPYEAAFSCTDKLAKLVEPIYLHSLTVTLCHTGHLARGMA RRLTP VKHIS LNWTRWLQLQRGP V S GFS AC KMS AY QR ALRRF GS (SEQ ID NO: 315) [0539] >13 KM J4_ORENE382-706

SLATGTKCLDLGGVSDS GCTLTDCHAE VV S RRALVRFLY S QLELIFIPNKS CGDFRLR EGVHFHMYVSSSPCGDARLNCPYEAAFSCTDKLAKLVEPVYLQSLTVTLSHTGHLG RAM ARRLAPIKHIS VNW ARWLRLQQGP VIG Y CAS KMS A Y QRA V QQFS S (SEQ ID NO: 316)

[0540] >M3ZX42_XIPMA/284-606

SLATGTKCLDLDRLNEDGWTLRDCHAEVLSRRALVRFFYSQLELIFVPDKDSTHFRL QEGIRFHM Y V S LS PCGD ARLNCP YEP AF S CTDKIAKLVEPIYLHS LT VRLS HTGHLGR A VTRRLAR VKH VC VNW S C WLRLENTP V S GY Q AS KMG A Y QKAMQQF S N (SEQ ID NO: 317)

[0541] >G3 PZQ 1 _G AS AC/403-718

SLATGTKCRDSDGVGDREGTLSDCHAEVLSRRALVRFLYAQLELIFVSNTAGGFRLR DG VFFHM Y AS S S PCGD ARLN CPHE A AF S CTDKIAKLVEP V YLHS LT VTLS HT GHLGR A V ARRLAP VRRIKIIA ARS RD VLTRHLN S YS GS KM A AY QRAMQQFT G (SEQ ID NO: 318)

[0542] >H3 CRD2_TETN G/277-601

SLATGTKCRDRGSAADGAGSLSDCHAEVISRRALLRFLYSQLELVLTPKPGGYRLRD GLLFHM Y V S C S PCGD ARLNCP YE A AFS CTD KLAKLIEP V YLQS LT VTLS HT GHLS RA LTRRLAPVRRTSSNWARWRRLQQAHLDGYAGWKAAAYQRVMQTFSS (SEQ ID NO: 319)

[0543] >H2S0X8_TAKRU/296-626

SLATGTRCPGRAGARDRAGTLSDCHAE VIS RRALLRFLYW QLELIFTPN S GGCRLRD G VLFHMFV S S S PC GD ARLNCP YE A AFS CTD KLAKLIEP V YLHS LT VTLS HT GHLS RA M ARRLAP VRQIS TNW VRW QQLQQPQLN GY S GWKG A A YRRMMQNFIS (SEQ ID NO: 320)

[0544] >K 1 RR67_CR AGE 135-316

KYPSRTYYKQTCIVCNITTGENTLFKLAKSDGKHAEELLLEELEKLVPKDSLTITIFMN

[0545] >K 1 QUE8_CRAGE4- 176

[0546] >A0A0W0VFJ2_9GAMM/61-217

[0547] >A0A0W0XR23_9GAMM/33-206

[0548] > A0 A0W OS 272_9G AMM/47 -207

[0549] >A0A098GDCl_TATME41-209

[0550] >A0A0M0LPN3_9EUKA/47 -262 [0551] >ClEl86_MICCC/53-299

FFHCS PTPE ARKTKG V V V A ALRRGDLRS NS S H AEE Y V VRDEELVR A V APED AGTLT LY QRLQPCHGS S DNRGS C S D A V AGLHRELLG V S LRV A VS YT YR AH VRGFREGIRVF A ADG VTLE ALN AED W ALCDED Y A A AF GG VFT AAV AHRRAMDE (SEQ ID NO: 328)

[0552] >A0A0M0K2Z4_9EUKA/249-414

PPPNIFLAQTREREAETGLRLLLYMTYQPCHHSGGRPKEALGTCSESLRDFYIKELGV ALELVLADVYKATEELHREGMRMLLAEGITMRAMQPADWDLCDPEWAKRGAGPF SKH (SEQ ID NO: 329)

[0553] >RlE5J9_EMIHU/273-492

CGKV V V A V ALEHWR AGS S EN VH AES FVIADETILS A V V AELTLYLS Y QPCHHS GGR GGGEARQSCTEELIAYHERELNVPLSVVVADLYKVMEELMREGIRRLMSPGMSMRA TGEED W ALCD QS YERRGGS AF GPH V ALR AKLD (SEQ ID NO: 330)

[0554] >A0A0M0LPN3_9EUKA/390-614

RGRRDGPVIVARVLGQSADLYVAYARGRSENVHAEEFMLADPQLLALLDAGGARIL RLYMSYQPCHHSGGRKTPEDARKSCSERLRAFYEAEMAISLELVVADLYKAMEDLM RQGILLM V APG VTMR ATDEND W ALCDP A Y ATRGGS AF S RH (SEQ ID NO: 331)

[0555] >A0A0M0JB66_9EUKA/269-499

VDTRDKTKAQ VIV AR VLGQN ADLY V AY ARGRS EN VH AEEFMLADPQLLALLD AS G ARILRLYMSYQPCHHSGGRKTPEDARKSCSERLRAFYEAEMAISLELVVADLYKAM EDLMRQGILLLV APG VTMR ATDEND W ALCDP A YETRGGS AFS RHE (SEQ ID NO: 332)

[0556] >A0Al83IJ2l_9BILA/l 99-397

S CKS FDERG AS TM V ACLRTTN GT Y QE YR ATDNDDRHPEEIFHKDMLQ ART Y VLPLT HIIVYLPTSPCFHQDCEPQCDVLDACAEQLAIVYRQAKKTDLKMSVKFLASYIGDLY KQGITMMMNAGIDVEPLGMRDWIELVHNDSTYYFSWKGLLTSYIRQSEIYI (SEQ ID NO: 333) [0557] > AO A077Z854_TRITR/ 190-389

RKLQYRGSSALVAQLTETATYRAFVVSEKTIHVEQKFYHQLMDAANFTMPLREVIL YLPTSPCFHQDCDPLCDVLDACAEALSICYKQIRREELQMTVKFLASYVGDLYKQGI MSMME AGLT VEPLNMKD WIS LV S T AGT Y YRD WEN S LAD Y VMQT QLYI (SEQ ID NO: 334)

[0558] >A0A085NPE0_9BILA/206-389

AQLTETATYRAFVVSEKTVHVEQKFYHHLMDAANFTMPLRELILYLPTSPCFHQDCD PLCD VLD AC AE ALS IC YKQIQREELQMT VKFLAS YV GDLYKQGIMS MMEAGITVEPL NMKD WIS LV S T AGT Y YRD WEN S LAD Y VMQT QLYI (SEQ ID NO: 335)

[0559] >A0A0V0XXU8_TRIPS/513-708

LNEQVQKWEHRGASVMVAKLSDQQVYADYFVGDGKPQHVEQIFYTELMNADQCQ MATLRQICLFMPTSPCFHQDCEPQCDVLDACAETLAIVYKQLQNSDLQMTVKFLAS Y V GDLYKQGILCMLQ AGIS VEPLNRKD W Q ALVEPDHTR A WDS LLTN Y AMQS QF YI

(SEQ ID NO: 336)

[0560] >A0A0VlMSE5_9BILA/488-683

LNEQ V QKWEHRG AS VM V AKLS DQQ V Y AD YFV GDS KPQH VEQIF YTELMN AD QC Q MTTLRQICLFMPTSPCFHQDCEPQCDVLDACAETLAIAYKQLQNSDLQMTVKFLASY VGDLYKQGILCMLQAGISVEPLNRKDWQALVESDHSRSWDSLLTNYAMQSQFYI

(SEQ ID NO: 337)

[0561] >E5S9I2_TRISP/l08-302

PNEQ V QKWEHRG AS VM V AKLS DQH V Y AD YFV AETKPQH VEQIF Y AELIN AD QCRM TTLRQICLFMPTSPCFHQDCEPQCDVLDACAETLAIAYKQLQNPDLQMTVKFLASYV GDLYKQGILCMLQ AGIS VEPLS RKD W Y ALVES DHTRGWDS LLTN Y AMQS QF YI

(SEQ ID NO: 338)

[0562] >AOAl83CD28_GLOPA/474-665

VPKHVQNCEHKHESGLIVTLGDGYIYGDFFHDSNPHVEEQLVAAIYDLSKYKVDLYE IVIFV S KSPCFHQDCDPKCE VVD AC AKLLGLLLS KVRKVD VRMTVKFLYPHLGDLY KQGILS MLQS GIK VEPLLMKD W S A VMD W AGE YLQW GS HLDR A V AQS Q AFI (SEQ ID NO: 339)

[0563] >H3FBK6_PRIPA/51-311

FHVPKHVQSCEHKHESGLIVTLGEDYIYGDFYHESSPHVEEQLVAAIYDLSKYKVDL YEIVIFV S KSPCFHQDCDPKCEVVD AC AKLLGLLLS KVRKVD VKMTVKFLYPHLGDL YKQGILCMLQAGIKVEPLLMKDWSAIMDWTGEYLQWNNHLDKAVAQSQS (SEQ ID NO: 340)

[0564] > AO AON 5 AZH9_9B ILA/ 14-264

FHIPKHVQSCEQKHESGLIVTLGDDYVYGDFYHESNPHVEEQLVAAIYDLSKYKVDL YEIMIYV S KSPCFHQDCDPKCE VVD AC AKLLGLLLS KVRKVDIRMT VKFLYPHLGDL YKQGILCMLQAGIKVEPLLMKDWSAVMDWSAEYLQWNNHLDKAVAQSQAFI (SEQ ID NO: 341)

[0565] >A0A0B2VFT 1 _TOXC A/ 106-363

FHVPKHVQSCEQKHESGLIVTLGEDYIYGDFYHESSPHVEEQLVAAIYDLSKYKVDL YEMVIYVSKSPCFHQDCDPKCEVVDACAKLLGLLLSKVRKVDVKMTVKFLYPHLG DLYKQGILCMLQTGIKVEPLLMKDWS A VMDW S GE YLQWNNHLDKA VAQS QS F

(SEQ ID NO: 342)

[0566] >A0Al58Q5D3_DRAME/l 17-306

IPKHV QSCEHKNES GLIVTLGDD YIY GDFYHES SPHVEEQLV AAIYDLS KYKVDLNE V VIYV S KSPCFHQDCDPKCEVVD AC AKLLGLLLS KIRKVD VRMT VKFLYPHLGDLYK QGILCMLQ AGIKVEPLLMKD W S T VMD W S GE YLQWNNHLDKA V AQS QS F (SEQ ID NO: 343)

[0567] >A0A0N5CVT8_THECL/l56-346

VPKHV QNCEHKNES GLIVTLGHD YVY GDFYHES S LHVEEQLAAAIYDLS KYKVDLF EAVIYVSKSPCFHQDCDPKCEVVDACAKLLGLLLS KIRKVD VKMTVKFLYPHLGDL YKQGILCMLQ AGIKVEPLLLKD W S A VMD WTGE YLQWNNHLD KA V AQS QS F (SEQ ID NO: 344)

[0568] >JOXJI9_LO ALO/ 138-380

FHLPKHVQSCEHKNETGLILTLGDDYIYGDFYHESSLHVEEQLVAAIYDLSKYKVDL FE A VIY V S KS PCFHQDCDPKCE V VD AC AKLLGLLLS KIRKVD VKMT VKFLYPHLGDL YKQGIFCMFQ AGIKVEPFFFKD W S A VMD WTGE YFQWN S HFDKA V AQS QS F (SEQ ID NO: 345)

[0569] > AO A0K0EN 59_S TRER/201-391

[0570] VPKHVQNCEHKHESGLIVTLGEDYIYGDFYHESGPHVEEQLVAAIYDL S KYKIELYEIVIFV S KSPCFHQDCDPKCEVVD AC AKLLGLLLS KVRKVD VKMTVKFL YPHLGDLYKQGILCMLQAGIKVEPLLMKDWSAIMDWAGEYLQWNNHLDKAVAQS QSF (SEQ ID NO: 346)

[0571] >H2W477_CAEJ A/255-446

VPKHV QNCEHKHES GLIVTLGED YVY GDFYHES GPHVEEQLVAAIYDLS KYTVDLH EIQIFV S KSPCFHQDCEPKCEVVD AC AKLLGLLLS KVRKVD VKMT VKFLYPHLGDLY KQGILCMLQ AGIKVEPLLMKD W C AIMD W S GD YLQWNNHLD KA V AQS QLFI (SEQ ID NO: 347)

[0572] >A0A0K0DC Y 3_AN GCA/ 157-328

IPKHVQNCDHKHESGLIVSLGDDYIYGDFYHESGPHVEEQLVANIYDLSRYKVDLHEI VIFV S KSPCFHQDCDPKCEVVD AC AKLLGLLLS KIRKVD VKMT VKFLYTHLGDLYK QGILCMLQ AGIKV GFFS VLQKA V AQS QLFIN (SEQ ID NO: 348)

[0573] > A0 A 158PLA6_AN GC S/ 132-323

IPKHVQNCDHKHESGLIVSLGDDYVYGDFYHESGPHVEEQLVANIYDLSRYKVDLHE VVIFV S KSPCFHQDCDPKCEVVD AC AKLLGLLLS KIRKVD VKMT VKFLYTHLGDLY KQGILCMLQ AGIKVEPLLMKD W C AIMD W S GD YLQWNNHLD KA V AQS QLFI (SEQ ID NO: 349) [0574] >U 6NTW 9_H AEC 0/157-347

VPKH V QN CDHKHES GLIV S LGEN YIY GDFYHES GPH VEEQLV ANIYDLTKYN VELHE IVIFV S KSPCFHQDCDPKCE VVD AC AKLLGLLLS KVRKVD VKMT VKFLYPHLGDLY KQGILCMLQ AGIKVEPLLMKDW C AIMD W S GD YLQWNNHLD KA V AQS QLFI (SEQ ID NO: 350)

[0575] > AO A 158R0Z7_NIPB R/52- 302

FSVPKHVQNCDHKHESEDYIYGDFYHESGPHVEEQLVANIYDLTKYNVELHEIVIFVS KS PCFHQDCEPKCE VVD AC AKLLGLLLS KVRKVD VKMT VKFLYPHLGDLYKQGILC MLQAGIKVEPLLMKDW C AIMDW S GD YLQWNNHLD KAVAQS QLFI (SEQ ID NO: 351)

[0576] >A0A0C2D4B7_9BILA/ 130-292

VPKH V QN CDHKHES GLIV S LGED YIY GDFYHES GPH VEEQLV ANIYDLS K YN VELHE IAIFV S KSPCFHQDCDPKCE VVD AC AKLLGLLLS KVRKVD VKMT VKFLYPHLGDLY KQGILCMLQ AGIKV C GHFF V CF A YLF V (SEQ ID NO: 352)

[0577] > AO A 183 IMD0_9B ILA/ 141-363

FAVTKNTKQCGQKNETAAIVTLGGGNIEFHHESSLHPEEQLFTALMELSEYQVNLEE VVLYCSKSPCYHQDCNPLCEVIDACAKLLVLLLYKVRTVDVNLTVRFLYPHLGDLY KQAIMYMLQHGINVEPLLMSDWSAIVEWGCNYLDWNEHLDQAVARGQS (SEQ ID NO: 353)

[0578] >A0A0N5DEG0_TRIMR/83 -275

FLVSRSTQQCEHKHESAIVVTLGEDYVYIEYAHESGLHCEEQLMNALEELAMYTVNL YEVVVYTSRSPCFHQNCEPRCAVIDACSKLLSLFLLKLRRVDLRMTVRFLFPHLGDL YKQGILCMLQHGIKVEPLLMKD W S AIMD W S GD YLA WN Q YLDH A V AKS QS F (SEQ ID NO: 354)

[0579] >A0A085LQT 5_9B ILA/ 165-355

ISRTTQHCEHKHESAIVVTLGEDYVYVEYLHESSLHCEEQLLYALEELAKYAINLYEV

LVYTSRSPCFHQNCEPRCAVIDACSMLLSLFLFKLRRVDLRMTVRFLFPHLGDLYKQ GILCMLQHGIKVEPLLMKDW S GIMDWAGD YLTWN QHLDQA VAKS QSF (SEQ ID NO: 355)

[0580] >A0A077ZDL7_TRITR/l75-365

VSRTTQHCEHKHESATVVTLGEDYVYVEYLHESSPHCEEQLLFALEELAKYAINLYEI LVYTSRSPCFHQNCEPRCAVIDACSMLLSLFLFKLRRVDLRLTVRFLFPHLGDLYKQG ILCMLQHGIKVEPLLMKD W S GIMD W AGD YLA WN QHLDQ A V AKS QS F (SEQ ID NO: 356)

[0581] >E5 S VP6_TRIS P/ 124- 300

SRQGQQCEQKHESVILVTLGEDYVYVEFCHESSRHCEEQLAIALHELVNRSPCFHQD CEPRCDVIDACSKLLALLLTKVRKADIRMTVRFLFPHLGDLYKQGILCMLQHGIKVE PLLMRD W S AIMD W AGD YLGWNEHLDQ A V AKS QS F (SEQ ID NO: 357)

[0582] >A0A0VlMU5l_9BILA/l71-360

SRQGQQCEQKHESVILVTLGEDYVYVEFCHESNRHCEEQLAIALHELVKYKINLYEIL IYSSRSPCFHQDCEPRCDVIDACSKLLALLLTKVRKADIRMTVRFLFPHLGDLYKQGI LCMLQHGIKVEPLLMRD W S AIMD W AGD YLGWNEHLDQ A V AKS QS F (SEQ ID NO: 358)

[0583] >A0A067 QK96_ZOONE/50-213

WTAFYINGRPKLKKCITLCHVVFNETATAEQWEISYSHGPHAEIKVLRNIKARELCLG YTRIVTLFLSYSPCANCANFIIEFSRTRPQCTVYIRFTCLFRHPEEIHRDGLRRLNAPGI S LGVFTV YEWRRLAEAGMPFRPWDS KWK (SEQ ID NO: 359)

[0584] >H3B7Z9_LATCH/l8l-343

FY GKFNNTRKN GRNMLCFS LEGENKPWKW GY AHN S KHAENIVLREVAS YKPFLNH FYISYGPCSNCCDKILDFLQKFEKIKIMIKISRLYKDESSVFQNSIKKLHQMGVSVQVM NRGDFEQCFKGFVQGDFQPWPALEPTSEKCAANLEA (SEQ ID NO: 360)

[0585] >H3B7Z9_LATCH/4-l68

FEAEFNNTVNSFRTLLCFSLQQENKTWNWGYAHNNDSHAEILVLREIEKYEKADHEI

[0586] >W 5M7 AO_LEPOC/25 - 192

[0587] >GlKV46_ANOCA/8-l75

[0588] >G3VS78_SARHA/l4-l79

[0589] >K7E403_MONDO/ 13-175

[0590] >A0A091 G380_9 A VES/2- 125

[0591] > AO A091 WIF8_OPIHO/3 - 124 [0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598] _

[0599]

[0600] >A0A093DVS l_9AVES/2-85

[0601] >A0A091 Q6M3_LEPDC/5 - 125

[0602] >A0A093HKT8_STRCA/2- 124

[0603] >A0A099Z 1 L5_TIN GET/2- 122

[0604] >H9GLZ8_ANOCA/51-241

[0605] >M7B 925_CHEM Y 6-257

[0606] >H3 B 3 J 0_LAT CH/47 -227 ISYITLYSNYSPCNESNHYCIGKMYDFLISYPSTRLDIYFSQLYHTDFPESREALRSLAG PRVTIS PIS GGT WES F VN GQ AFYNPTRAFKHN AY (SEQ ID NO: 382)

[0607] >F6ZR34_XENTR/47-234

FYEAFGFPYTPENKQLIFYEVKDFSGTNIQKGQVTNSNIHAESILFEDSGYLDALHHGS V GYITLY ANYTPCNE Y GHY CIS KMYNFLLKYEDTRLDIYFS QLYHVESPAARQALRS LAS PRVT VNPLS EGIW QTF AKGLS FYEP ARAS S S N AS TIHLIT G (SEQ ID NO: 383)

[0608] >F6VET78_ORN AN/48-231

FYQTFGFPHMPQPEHLTFYELKTFSGAPVQKGQATSQSIHPESMLFEEGGYLDSVYD DSIGHIILFSNYTPCNEAGHCCISQMFDFLMTYPDITLSIYFSQLYHTEFPASRKALRSL AS PRV S VNPIS GGIWH AF V S GG ALRHPS RALGHN A YE IN A (SEQ ID NO: 384)

[0609] >G3WLB3_SARHA/47-227

FY QIF GFP Y S PQTQHLTFYELKTS S GS LV QKGH AS GEDTHPES MLFEMDS YLE A VNN DNIEH VFLY S NY CPCNE ANHCCIS KM YNFLMR YP AIS LNIYF S QLYHTEFP V S RE ALR S LAS PQVTVNPMS GGIWH YFV S DE ALHQP ARTLRHN AYE (SEQ ID NO: 385)

[0610] >H0VBH8_C A VPO/47-214

FHQIFRFP YTPS TKHLTF YELKTFS GS LV QKGH AS N GHTHPES MLFEMN G YLDS ANN S S IKHIILY S NN S PCNE ANHCCIS KM YNFLTM YPD VTLS V YF S HLYHT GFP AS RE ALRS LAS PQ VTLS PIS GGIWHCFVTGG A AF QP (SEQ ID NO: 386)

[0611] >G5B444_HETGA/47-231

FHQ VFGFPHTPPTKHLTFYELKT S S GTLV QKGH AS N GDTHPES MLFEMN G YLDS AS N STIKHIILYLNNFPCNEANHCCISKMYNFLMMYPDITLSIYFSQLYHTKFPTSREALRS LAS PQ VTLS PIS GMIWHS FVMGGP AF QPGRALRHN AYE (SEQ ID NO: 387)

[0612] >ABEC4_MOUSE/47-263

FHQTF GFPWTPQTKHLTF YELRS S S KNLIQKGLAS N GHNHPE AMLFEKN G YLD A VH N S NIRHIILY S NN S PCNE AKHCCIS KM YNFLMN YPE VTLS VFFS QLYHTEFPT S RKALQ S LAS PQ VTLS PICGGLWH AF V S NGS VPQPGRILRYNT YEIN S IIA (SEQ ID NO: 388) [0613] > AB EC4_RAT /47 -256

FHQTF GFPWTPQTKHLTF YELRS S S GNLIQKGLAS N GHTHPES MLFERDG YLDS LHD S NIRHIILY S NN S PCDE ANHCCIS KM YNFLMN YPE VTLS VFFS QLYHTEFPT S RE ALRG LAS PQ VTLS AIS GGIWQS FVS GGLA VRPGRTLRYN A YEINCITE (SEQ ID NO: 389)

[0614] >F7D9l l_HORSE/47-232

FY QIF GFP YMPQTKHLTF YELKTT S GS LV QKGH AS S GNTHPES MLFELN G YFDS ANN DSIRHIILYSNNSPCNEANHCCISKMYNFLIMYPHVTLSIYFSQLYHTEFPASREALRSL AS PQ VTLS PIS GGIWHS F V S GGP VFQPGR ALRHN A YEIN AI (SEQ ID NO: 390)

[0615] >GlNXW l_MYOLU/47-231

FY QIF GFP YTPQTKHLTF YELKT S T GGLV QKGH AS STS THPES MLFETN G YFD W ANN GGIRHIILY S NHS PCNE ADHCCIS KM YNFLTT YPD VTLS IYFS QLYHTDFP AS RE ALRS LAS PKVTLS PIS GGIWHFF V S GGS VFQPGR ALRHN A YEIN A (SEQ ID NO: 391)

[0616] >F 1 S 657_PIG/ 47-239

FY QLF GFP YTPQTKHLAFYELRPS S GS LV QKGH AS S GDTHPES MLFERN G YFDS ANN N GIRHIILY S NNS PCNE ANHCCIS KM YNFLRM YPD VTLS IYFS QLYHTEFP AS RE ALRS LAS PQ VTLS PIS GGIWHS FVS GGWLFQPGR ALRHN A YEIN AITG (SEQ ID NO: 392)

[0617] >S 9WFU 5_C AMFR/54- 199

FY QLF GFP Y VPQTKHLTFYELKT S PGS LV QKGHEN S GDTH AES MLS EMN G YFDS AN HQGIRHIILY S NN S PCDE ANHCCTS KM YNFLTM YPD VTLS IYFS QLYHTEFP AS REAL RSLASPQVTVSPP (SEQ ID NO: 393)

[0618] >HOWM53_OTOGA/47-232

FY QIF GFP YIS QTKHLTF YELKT S S GS LV QKGH AS RGDTHPES MLFEMN G YLDS ANN DGIRHIILYSSNSPCNEANHCCISKMYNFLKVYPD VTLS VYFSQLYHTEFPASRE ALRS LAS PQ VTLS PIS GGIWHS FVSGGS VFQPGR ALRQN A YEIN AI (SEQ ID NO: 394) [0619] >G3MWJ4_BOVIN/47-233

FY QIF GFP YTPPTKHLTF YELKT S S GS LV QKGH AS S GDTHPES MLFE VN G YFDS ANND CIRHIILYSSNSPCNEANHCCISKMYTFLAKYPDITLSVYFSQLYHTEFPASREALRSLA S PQ VTLS PIS GGIW Y S FVS GG A VFQPGR ALRHN AHEIN AIT (SEQ ID NO: 395)

[0620] >W 5P5 G7_S HEEP/ 47-235

FY QIF GFP YTPPTKHLTF YELKT S S GS LV QKGH AS S GDTHPES MLFEMN G YFDS AS ND GMRHIILYSSNSPCNEANHCCISKMYTFLAKYPDITLSIYFSQLYHTEFPASREALRSL AS PR VTLS PIS GGMW Y S FV S GG A VFQPGR ALRHN AHEIN AIT G (SEQ ID NO: 396)

[0621] >G 1 L6U 8_AILME/47 -234

FY QIF GFP YTPQTKHLTF YELKT S S GS LV QKGH AS S GNTHPES MLFEMN G YFDS ANN N AIRHIILY S NNS PCNE ANHCCIS KM YNFLIL YPDITLS IYFS QLYHTEFP AS RE ALRS L AS PQ VTLS PIS GGMWHS FVS GGS VF QPGRALRHN A YEIN AITG (SEQ ID NO: 397)

[0622] >M3 YK05_MUSPF/47-239

FHQIF GFP Y VPQTKHLTFYELKT S S GS LV QKGH AS S ENTHPES MLFEMN G YFD A AKN N AIRHIILY S NNS PCNE ANHCCIS KM YNFLIM YPD VTLS IYF S QLYHTEFP AS RE ALRS LAS PQ VTLS PIS GGIWHS F VS GGP VFQPRRALRHN A YEINRITG (SEQ ID NO: 398)

[0623] >M3WE63_FELC A/47-233

FY QIF GFP Y APQTKHLTFYELKT S S GS LV QKGH AS S GNTHPES MLFEMN G YLDS ANN N AIRHIILY S S NS PCNE ADHCCIS KM YNFLIM YPD VTLS IYFS QLYHTEFP AS RE ALRS L AS PQ VILS PIS GS IWHS FVS GGS VFPPGRAQRHN A YEIN AIT (SEQ ID NO: 399)

[0624] >E2RD46_C ANLF/47-236

FY QIF GFP Y VPQTKHLTFYELKTS S GS LV QKGH AS S GNTHPES MLFEMN G YFDS ANN NTIRHIILYSNNSPCNEANHCCIGKMYNFLITYPDVTLSIYFSQLYHTEFPASREALRSL AS PR VTLS PIS GGIW Y S F V S GGS VFQPGR ALRHN A YEIN AITG (SEQ ID NO: 400)

[0625] >L9L463_TUPCH/47-264

FY QIF GFP YTPQTKHLTF YELKTS S GS LV QKGH AS S GQIHPES MLFEMN G YLDLANN

[0626]

[0627]

[0628]

[0629]

[0630]

[0631] [0632] >A0A093I8Y9_STRCA/l-74

ITLYLSWSPCRNCCYEMQYFLKKHPNVNICIYLARLYYTEDEEICKALKDLSEKVIISV MKIED YIY CWKTFV (SEQ ID NO: 408)

[0633] >A0A09lMHU6_9PASS/l-63

ITWYLSWSPCVNCCYKIRDFLNRHSYVTIRIYVARLCYRGFHRNRKGLRNLVSLRVT VNVME (SEQ ID NO: 409)

[0634] >A0A093S6Z3_9PASS/l-65

ITWYLSWSPCVNCCNEILDFLERHENVNIDIHVARLYFKDSKRTHRALKELARSTVSI NVMNME (SEQ ID NO: 410)

[0635] >R4GDA3_ANOCA/l8-l73

QRNFDPREFPECTLLLYEIHWDNNTSRNWCTNKGLHAEENFLQIFNEKIDIRQDTPCSI TWFLS WSPC YPCS QAIIKFLEAHPNV S LEIKA ARLYMHQIDCNKEGLRNLGRNRVSIM NLPD YRHC WTTF V VPRNED YWPQDFLATN Y S REL (SEQ ID NO: 411)

[0636] > A0 A091 ETZ4_C ORB R/ 1 - 64

ITWYLSWSPCMTCCYIIRNFLVRHPNVNIEIHVARLYNTRWAGTRRGLRELARLRVTI DVME (SEQ ID NO: 412)

[0637] >U 3 JXR8_FIC AL/ 12-171

FDPRT YPS ET YLLCELQW GGS GRFWIHW ARNDEDS H VEH YFLEQIFEPRS YS VCDIT WYLSWSPCANCCDIIQEFLEEQHNVNLDIRVARVYNEHIRENRAALRQLANFQAAIR AMDVEDYMYCWDTFLQQGGYFDFTAGSFRSAVERTRLRLE (SEQ ID NO: 413)

[0638] >H0ZSB5_TAEGU/l-76

DMTWYLSWSPCGECCDIIQDFLEEQPNVNINIRVARLYYTDRASNRKGLRELASSPV TLEIMD AED YNY C WETFI (SEQ ID NO: 414) [0639] >A0A09lR868_9GRUEl-65

[0640] >A0A09lQH63_MERNU/l-65

[0641] > AO A091 IKM 1 _C ALAN/ 1 -70

[0642] >A0A094K7N8_ANTCR/l-65

[0643] >A0A09lGQ55_BUCRH/l-65

[0644] > A0 A 151 P7C9_ALLME 18-177

[0645] >A0A093CT04_TAUER/l-65 [0646] >A0A091 V 1 W3_PHORB/ 1 -65

[0647] >A0A091 V 9F4_NIPNE 1-65

[0648] >A0A 151 P6H5_ALLME36- 174

[0649] >A0A099ZZR5_CHA V 0/1-65

[0650] >A0A093J6I5_FULGA/l-65

[0651] >A0A091 PTE0_LEPDC/ 1-75

[0652] >A0A093JP51 EURHL/1-65

[0653] > A0 A091 RW G2_NES NO/ 1 - 65 [0654] >A0A0Q3WRD0_AMAAE/72-248

LKYHFDPREVXRDTYLLCILRWGETGTPWSHWVKNRYHAEVYFLEKIFQTRKSSKNI NCSITWYLSWSPCAKCCRKILNFLKKHSYVSIKIHVARLFRIDDKETXQNLKNLGSLV VTVSVMEXEDYTNCWKTFIRGHADGDSWIDDLKSEIRKNRLKFQ (SEQ ID NO: 430)

[0655] >A0A091 PR75_HALAL/ 1-65

ITWYLSWSPCADCCHKILKFLKKHSNVNIDIHVARVYYAEDEKVRQGLKNLVSLAV TIAVMETK (SEQ ID NO: 431)

[0656] >A0A087QNJ4_APTFO/l-65

ITWYLSWSPCADCCRKILNFLKKNSNVNIDIYVARLYYTEDEKIRQGLQNLVSLAVTI AVMETE (SEQ ID NO: 432)

[0657] >A0A087VRL2_BALRE/l-66

ITWYLSWSPCADCCHKILNFLKRHSNVNIDIYVARLYYIEDEEIRQCLKNLVSLAVTIA VMKIE (SEQ ID NO: 433)

[0658] >A0A093F6R6_GAVST/l-69

ITWYLSWSPCANCCRKILRFLRKHSNVNIDIHVARLYYIEDENIRQGLKSLVNLAVTI AVMEIEGKVF (SEQ ID NO: 434)

[0659] >A0A091 TH63_PHALP/ 1-65

ITWYLSWSPCADCCHKILNFLKKHSNVNIIIYVARLYYKEDEKIRQGLKNLVNLAITIA VMEIE (SEQ ID NO: 435)

[0660] >A0A093 QYH8_PHAC A/ 1-65

ITWYLSWSPCEECCCKILNFLKKHSNVSICIYVARLYHIEDEKIRQGLKNLVNFTVTV AVMGIE (SEQ ID NO: 436)

[0661] >Clipboard_ _. Contents

FEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNT RCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGV TIQIMTEQES GY C WRNFVN Y S PS NE AHWPRYPHLW VRL (SEQ ID NO: 437)

[0662] >L5KGJ 8_PTEAL/30- 187

FEVFFDPRELRKEACLLYEIQWGTSHKIWRNSGKNTTKHVELNFIEKFTSERHFCSSV SCSIIWFLSWSPCWECSKAIREFLSQRPTVTLVIFVSRLFQHMDQQNRQGLRDLINSG VTIQIMRASEYDHCWRNFVNYPPGKEAHWPRYPPLWMKLYA (SEQ ID NO: 438)

[0663] >L5LUG3_MYODS/37-228

IFDPRELRKE ACLLYEIKW GT GHKIWRHS GKNTTRH VE VNFIEKIT S ERQFCS S TS C S II WFLS WSPCWECS KAITEFLRQRPGVTLVIYV ARLYHHMDEQNRQGLRDLVKS GVTV QIMTTPEYD Y C WRNFVNYPPGKDTHCPIYPPLLMKLY ALELH (SEQ ID NO: 439)

[0664] >F6WR88_HORSE/9-l68

FEAFFDPRELRKEACLLYEIKWGMSHNIWRYSGKNTTKHVEINFIEKFTSERHLRPSIS CS IVWFLS WS PC WECS KAIREFLS QHPN VTLVIY V ARLF QHMDRLNRQGLRDLIN S G VTIQIMRT S EYDHC WRNFVN YPPGKEAHWPRY S LLWMKLY ALEL (SEQ ID NO: 440)

[0665] >F 1 S LW 4_PIG/25- 176

VFFDPRELRKETCLLYELQW GRS RDTWRHT GKNTTNH VERNFLAKIT S ERHFHPS VH CSIVWFLSWSPCWECSEAIREFLDQHPSVTLVIYVARLFQHMDPQNRQGLRDLVNHG VTIQIMGAPEYDYCWRNFVNYPPGKEAHWPRFPPVWMT (SEQ ID NO: 441)

[0666] >W 5N VH9_S HEEP/27 - 179

FDPRNFC KE A YLLYEIQW GN S RD VWRHS GKNTTKH VERNFIEKIAS ERHFRPS IS CS IS WYLSWSPCWECSKAIREFLNQHPNVTLVIYIARLFQHMDPQNRQGLKDLFHSGVTIQ VMRDPEYD Y C WRNFVNYPQGKE AHWPRYPPLWMNLY A (SEQ ID NO: 442)

[0667] >M3WB96_FELC A/23 - 178

FEVFFDPRELRKEACLLYEIKWGTSHRIWRNSGRNTANHVELNFIEKFTSERHFCPSV S CS ITWFLS WS PC WEC S KAIRGFLS QHPS VTLVIY V S RLFWHLDQQNRQGLRDLVN S GVTVQIMRVPEYDHCWRNFVNYPPGEEDHWPRYPVVWMKL (SEQ ID NO: 443) [0668] >F 1 PUJ5_C ANLF/23 - 183

FEGFFDPRELRKETCLLYEIQWGTSHKTWRNSGKNTTNHVEINFMEKFAAERQYCPS IRC S IT WFLS WS PC WEC S N AIRGFLS QHPS VTLVIY V ARLFWHTDPQNRQGLRDLIN S GVTIQIMTVPEYDHCWRNFVNYPPGKEDHWPRYPVLWMKLYALELH (SEQ ID NO: 444)

[0669] >13 N301 _ICTTR/ 12-175

FE VFFNPG VLRKETCLLYEIQW GT S RKIWRN S S KNTTNH VE VNFIEKFT AERHFCPS IS CS ITWFLS WSPC WECS KAIREFLS QHPNMTLVIYTARLFQHMDQQNRQGLRDLIN S G VTIQMMTVSDSLTCWLHFLSHRVYVILMKKCGNLV (SEQ ID NO: 445)

[0670] >G 1 T VM9_RAB IT/50-205

FE VFFDPQELRKE ACLLYEIKW GAS S KTWRS S GKNTTNHVEVNFLEKLTSEGRLGPS TCCSITWFLSWSPCWECSTAIREFLSQHPGVTLVIFVARLFQHMDRRNRQGLKDLVT S G VT V Q VMS V S E YC YC WENF VN YPPGKA AQWPRYPPRW (SEQ ID NO: 446)

[0671] >H0 V V 31 _C A VPO/20- 179

FE AS FDPRQLQKE ACLLS E VRW GAS PRT WRES GLNTT S H VEINFIEKFT S GRS LRP AIR CSVTWFLSWSPCWECARAIREFLHQHPNVSLVIYVARLYWHVDEQNRQGLRDLVTS G VRV QIMSDSEYS HC WRNF VNFPPGQE AGWPRFPPM WTTLY A (SEQ ID NO: 447)

[0672] >H0W 6W 5_C A VPO/ 16- 179

FE A YFDPRQLRKE ACMLS E VRW GAS PRT WRES S LNTT S H VEINFIEKFT S GRS LRP A V RCSMTWFLSWSPCWECARAIREFLHQHPNVSLVIYVARLYWHVDEQNRQGLRDLV TSGVRVQIMSDSEYRHCWRNFVNFPPGQEAGWPRFPPMWTTLYA (SEQ ID NO: 448)

[0673] >F7F6M6_CALJ A/24- 178

YIS YDPKELC KETCLL YEIKW GMS WKIWRS S GKNTTNH VEINFIEKFT S ERHFHLS V S CS ITWFLS WSPC WECS QAIREFLS QHPGVTLVIYVARLFQHMDQQNRQGLRDLVN S G VTIQMMT V S E Y YHC WRNFVN YPPGEE AH WPRHPPLWLMLY (SEQ ID NO: 449) [0674] > AO A096MWB4_PAP AN/24- 177

DIF YDPRELRKE ACLLYEIKW GMS PKIWRS S GKNTTNH VE VNFIEKLT S ERRFHS S IS C S ITWFLS WS PC WEC S Q AIREFLS QHPG VTLVIY V ARLFWHTDQQNRQGLRDLVN S G V TIQIMT AS E Y YHC WRNF VN YPPGEE AHWPRYPPLWMML (SEQ ID NO: 450)

[0675] >G 1 QZV 0_N OMLE/24- 177

DVFYDPRELRKEACLLYEIKWGMSQKIWRSSGKNTTNHVEVNFIKKFTSEGRFQSSIS CS ITWFLS WSPCWECS QAIREFLS QHPGVTLVIYVARLFWHMDQQNRQGLRDLVN S GVTIQIMRASEYYHCWRNFVNYPPGDEAHWPRYPPLWMML (SEQ ID NO: 451)

[0676] >H2Q5C6_PANTR/23- 177

FDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERHFHPSI SCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQQNRQGLRDLVNS GVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMML (SEQ ID NO: 452)

[0677] >K7 G211 _PELS E 189- 300

ERSLNPLTHCSVTWFLSWSPCWKCSQSVVEFRKAYPKVNLEIYVARLFRHEEECNRQ GLRDLVMN G VTIRVMNLS A YN Y C WRTFV S HQGDD YWPWHLT (SEQ ID NO: 453)

[0678] > A0 A 151 P6M4_ALLMI/21-173

FQEN YMPS T WPKVTHLLYEIRW GKGS KVWRNW C S NTLTQH AE VNCLEN AFGKLQF NPPVPCHHWFLSWSPCCQCCRRILQFLRAHSHITLVIKAAQLFKHMDERNRQGLRDL VQS G VH V Q VMDLPD YR Y C WRTFV S HPEGEGDFWPWFF (SEQ ID NO: 454)

[0679] >A0A09lEQ78_CORBR/9-l42

FQINYSPSQHRRGVYLLYEIRWRRGSIWRNWCSNTHRQHAEVNFLENCFKDRPQVP CSHWFLSASPCGKCSKRILEFLKSRPYVTLKIYAAKLFRHHDIRNREGLCNLGMHGV TIHIMNLED Y S Y C WRNF V V Y (SEQ ID NO: 455)

[0680] >U3JXR8_FICAL/251-385

FQRNYSPSQNGRVVYLLYEIRWKGGSIWRNWCSNNPEQHAEINFLENRFNDRPQTSC S ITWFLS TS PC GKCS KRILEFLRS HPN VTLKI YT AKLFRH YEIRNRQGLRNLIMN G V A V HIMNLED Y S Y C WTNF V AHQ (SEQ ID NO: 456)

[0681] >H0ZSB3_TAEGU/8-l42

FQRNYSPRQHGRVVYLLYEIRWRRGSIWRNWCLNNHEQHAEVNFLENHFNDRPQTP CSITWFFSTSPCGKCSRRIFDFFRSHPNVTFVIYAAKFFKHHDIRNRQGFRNFNMNGV TIRIMNVEDYRY CWRNFVA Y (SEQ ID NO: 457)

[0682] >A0A093PWR2_9PASS/7- 142

FKRNYFPGQHPQVVYFFYEIRWRNGSIWRNWFSNNRNQHAEVNFFENCFSDVPPAP CSITWFFSTSPCGKCSRRIFEFFRTHRNVTFEIYAAKFFRHQDIRNRQGFCNFVMNGV TIHIMNFAD Y S Y C WKRF V AY (SEQ ID NO: 458)

[0683] >A0A09lMEP8_9PASS/7-l42

FQRNYLPDQHPQAVYLLYEFRWRRGSIWRKWCSNNRAQHAEVNFLENCFNGIPPVP CS ITWFLS TTPC GNCS RRILEFLRLHPN VTLEIY A AKLFRHTDIRNRKGLYNLAMN G VI IRIMNLAD Y S Y C WRNFV AY (SEQ ID NO: 459)

[0684] >A0A09lRF60_9GRUEl 1-129

RN YLPGC YPKV V YLL YEIKWRRGTTWRNW C S NS RNLH AE VNFLENCFKA VPS VS C S MTWFLSAIPCGKCSRRILEFLKVYPNVTLEIYAAKLFKHLDIRNRQGLRNLAMNGVII RIMNL (SEQ ID NO: 460)

[0685] >A0A091 IIG0_C ALAN/8- 142

FKRN Y QPGRRPN V V YLLYEIRWRRGTIWRN W C S NEFPQH AEDNFF QNRFN A VPS VS C S ITWFLS TTPC GRC S KRILEFLRLHPN VTLKIY A ARLFRHLDNRNRQGLRKLAS N G V IIQIMGLPDYSYSWKKFVAY (SEQ ID NO: 461)

[0686] > A0 A091 QEK6_MERNET/8 - 142

FKTN Y S PDHRPRVV YLLYEIRWRRGTIWRN W C S NNIDQHAE VNFLENCFKA KPS V S C S ITWFLS T APC AKC S RRILKFLT AHPKVTLEIY A AKLFRHLEIRNRQGLMDLA VN G V ILRIMNLAD Y S Y C WKQFV AY (SEQ ID NO: 462) [0687] >A0A093GVH6_PICPB/l0- 142

KLN Y VP V GRPRV V YLLYEIRW S RGS IWRNW C S NS S TQH AE VNFLEN CFKAMPS VS C S ITWFLS TTPCGN C S RRILEFLR AHPKVTLAIH A AKLFKHLD VRNRHGLKALATDG V VLHIMS IAD YRY C WTKFV AY (SEQ ID NO: 463)

[0688] > AO A091 PS V 3_H ALAL/7 - 142

FKRNYLPGQHPKVV YLLYEIRW S RGTIWRNWCS NN S T QH AE VNFLENCFKATPS V S CSITWVLSTTPCGKCSRRILEFLRVHPNVTLEIYAAKLFKHLDIRNRKGLRDLAMNGV IIRIMNLS D YS Y C WKTF V AY (SEQ ID NO: 464)

[0689] > AO A091 M4D7_C ARIC/8 - 142

FKRN YLPGQHPE V V YLLYEIKWN S GTIWRNWCS NNPT QH AE VNFLENHFN VMS S VS CS ITW GISTTPCGKCS RRILEFLTTHPNVTLEIY AAKLFKHLDIRNRQGLRNLAMN GV VICIMNLADYSYFWKTFVAY (SEQ ID NO: 465)

[0690] >A0A099ZZX4_CHA V 0/10- 142

IRNYLPDKHPNVV YLLYEIRW S RGTIWRNWCS NN S T QH AE VNFLENCFKAMPS VS C SITWFLSTTPCGRCSRRILKFLRVHPNVTLKIHAAKLFKHLDMRNRQGLKNLAMHGV IIRIMNLAD Y S Y C WKTFV AH (SEQ ID NO: 466)

[0691] > AO A094MFH 1 _ANTCR/ 11- 142

RNYLPVQYPNMVYLLYEIRWSTGTIWRNWCSNNSTQHAEVNFLENRFNSRPSVSCSI TWVLSTTPCGKCSTKILEFLRLHPNVTLKIYAAKLFKHLDIRNRQGLRNLAMNGVIIRI MNLAD Y S Y C WKTF V AY (SEQ ID NO: 467)

[0692] >A0A093FY7 l_TYTAL/8-l42

FKRNFLPGQHPKV V YLM YEIRWIRGT A WRS WC S NN S KQD AE VNLLENCFKAMPS V FC S VTW VLFTTPCGKCFRRILEFLR VHS N V ALERY A AQLFRHLDICNW QGIRS LAMN G VIIHIMNLAD Y S Y C WKRFV AY (SEQ ID NO: 468) [0693] >A0A091 TCM6_PHALP/7 - 142

FKRN Y S PGQHPKV V YLLYEIRW S RGTTWRNWCS NN S T QH AE VNFLENCFKAMPS VF CSITWVLSTTPCGKCSRRIQEFLRVHPNVTLEIYAAKLFKHLDRRNRQGLRNLAMNG VIIRVMNLAD YRY CWKRFVAY (SEQ ID NO: 469)

[0694] >A0A093CIQ8_9AVES/5-l30

FKINNLPGQHPRVVCLLYAIRWSRSTLWKSWCSNNSTQHAEVNFLENCFKGNPSVFC FMTWFFHTTPHGKCCRRTPEFLGVHPNVTLKIRAAKLFKHLDRYNQQGLRNVAMN GVVIRIINL (SEQ ID NO: 470)

[0695] >A0A093F3R4_GA VST/8- 142

FKRN YLPAQHPKVV YLLYEIRW S RGTIWRNWCS NN S T QHAE VNFLENCFKAMPS V S CSITWFLSTTPCGKCSRRILTFLREHPNVTLEIYAAKLFKHLDVRNQQGLRNLDRNGV IIRIMNF AD Y S Y C WKRFV AY (SEQ ID NO: 471)

[0696] >A0A093LP85_FULGA/9- 142

FKRNFLPS KYPKVV YLLYEIRW S S GTIWRS WC S NN S TQH AE VNFLEN CFKAMPS VS C SITWVLPITPCGKCSKKILEFLSVHPNVTLEIYAAKLFRHLDIRNQQGLRNLAMNGVII RIMNLAD Y S Y S WKRFV AY (SEQ ID NO: 472)

[0697] >A0A091 S SF0_9 A VES/7 - 142

FKRN YLPGQHPKVV YLLYEIRW S RGTIWRS WCS NN S KQH AE VNFLENCFKARPS V S CSITWVLSTTPCGKCSRRILEFLRVHPNVTLEIYAAKLFKHLDIRNQQGLRNLAMNGV IIRIMNLAD Y S Y C WKRFV AH (SEQ ID NO: 473)

[0698] >A0A093JI54_EURHL/9-l42

[0699] FKRN YMPS QYPKVV YLLYEIRW S RGT VWRNWCS N S FTQH AE VNFLE N YFKPMPS V S CS GG W VLS TTPC GKCS RRILEFLR VHPN VTLEIY A AKLFKHLDIRNRQ GLRDLAMNGVTIRIMNLADYSFCWKRFVAY (SEQ ID NO: 474)

[0700] >A0A087QNJ5_APTFO/8- 142

FKRN YLPGQHPKVV YLLYEIRW S RGTIWRNWCS NN S T QHAE VNFLENCFKAMPS V S

[0701] >A0A093RC0l_PHACA/9- 142

[0702] >A0A091 V7F8_NIPNE 10-142

[0703] >A0A091 XJLO_OPIHO/7 - 142

[0704] > A0 A091 RET 17_NES N 0/8-142

[0705] >A0A0Q3 WQET 9_AM AAE/56- 187

[0706] >A0A094LEL8_9AVES/8-l4l [0707] >A0A087VMP5_B ALRE/8- 142

FKRN YLPGKHPR V V YLLYEIRW S RGTIWRS WCS NN AT QH AEINFLETCFLART S VS C S ITW VLS TTPC GKCS RRILEFLN A YPN VTLEIY A AKLFRHLDNRNRQGLRNLAMKG V RIHIMNLAD Y S YFWKIF V AY (SEQ ID NO: 482)

[0708] > A0 A091 GLR0_B UCRH/7 - 142

FTRN YLPN QHPRV V YLLYEIRWRRGTIWRNWCS NN S T QH AEIKFLEN CFN ATT S VS C S IIWFLS TTPCGKC S TRILEFLRAHPN VTLEIY A AKLFKHHDNRNRRGLWNLAMN G V KLHIMNP AD Y S Y C WKMFV AY (SEQ ID NO: 483)

[0709] >I3M955_ICTTR/250-4l7

FHLQFNNLHRPCRRKTYLCYQLRLGSLCDQDYFQNKDLHAEIRFIKKIRSLDLDQSH N YE VT C YLTW S PCPDC AQELV ALTRS HPH VRLTLFT S RLYFHWLWRFQEGLRLLWR S GV QIRVMS LREFTHC WVKFVNHGGCPFEPWDGLEQRS QS IQNRLNR (SEQ ID NO: 484)

[0710] >G3I2J2_CRIGR/230-389

NRHRVRYQRKTYLCYLLEQNGQQPLKGCLQNKGKHAEILFIDEMRSLELGQVQITC YLTWSPCPNC AQELAAFKSDHPDLVLRIYTSRLYFHWRRKY QEGLCCLWRS GIQVD VMDLPQFADCWTNFVNQSPFWPWNNLEKNSRCIQRRLQR (SEQ ID NO: 485)

[0711] >F7EWS7_RAT/229-389

NSHRVRYRRKSYLCYQLERNGQEPLKGYLLYKGQHVEILFLEKMRSMELSQVRITC YLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFYWRKKFQKGLCTLWRSGIHVDV MDLPQFADCWTNFVNQRPFRPWNELEKNSWRIQRRLRR (SEQ ID NO: 486)

[0712] >ABEC3_RAT/241-405

FY S QFYN QRVHGVKPYLC Y QLEQNGQAPLKGCLLSEGQHAEILFLD KIRS MELS QVII TCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILV DVMDLPQFTDCWTNFVNKRPFWPWKGLEIISRRTQRRLHR (SEQ ID NO: 487) [0713] > AO A091 EM42_FUKD A/548-715

FRIQFNNAYKPHRRVTYLCYQVQKNGNLLTKGCLRTKGYHAESRFIKRICSLGLDQA QS YQVTCFLTWSPCPRC AQELVLFKSSHPHLYLRIFTARLYFHWRKS YQEGLQRLCR AQVPVAVMGYPEFAHCWYNFVDHPGPFEPWYKMEYYSKCIKKRFQR (SEQ ID NO: 488)

[0714] >G5 A YU 5_HETG A/236-403

FRVQFNNAYKPRRRVTYLCYQLQENGDPLTKGCLRTKGYHAESRFIKRICSMDLGQ DQSYQVTCFLTWSPCPHCAQELVSFKRAHPHLRLQIFTARLFFHWKRSYQEGLQRLC RAQVPVAVMGHPEFAYCWDNFVDHPGPFEPWAKLEYYSSCLKRRLQQ (SEQ ID NO: 489)

[0715] >T0NHJ 8_C AMFR/587-753

FNKQFGN QPRPYRRKTYLC Y QLKGN GS ILAQGC VRNKQRHAEIRFIDKINFMNLNPN QSYEIICYVTWSPCPTCAEKLVDLINDQVHLKLQIFASRLYFHWVRKYQIGLQYLWA SQVTVAVMNRQEFKDCWEKFVDNGKDFQGWYKLEEYNRSISRRLNR (SEQ ID NO: 490)

[0716] >B7Tl55_BOVIN/34-200

FKQQFGN QPRPYRRKTYLC Y QLKQNDLTLDRGCFRNKQRHAEIRFIDKIN S LDLNPS QS YKIIC YITW S PCPN C ANELVNFITRNNHLKLEIF AS RLYFHWIKS FKMGLQDLQN A GIS V A VMTHTEFEDC WEQFVDN S RPFQPWDKLEQ Y S AS IRRRLQR (SEQ ID NO: 491)

[0717] >F7IF99_CALJA/lO- 175

NIQLTNPYPKRTYLCYQLMPNGSTPTRGYFKNKNRHAEICFIDEIESMGLDKTQCYEV TC YLTW S PCPS C AQKLV AFAKAQ VHLNLRIFAS RLY YHWLLS C KKGLQLLWKS QIP VEVMGLPEFTDCWENFVVHGPPPFNPSEKLQELGSRSIKRRLDK (SEQ ID NO: 492)

[0718] >A0A096NK5 l_PAP AN/9- 179

FSLQFNNKRRPYPRKALLCYQLTPNGSTPTRGYLKNKKNHAEIRFINKIKSMGLDETQ

CYQVTCYLTWSPCPSCAGKLVDFLKAHRHLNLSIFASRLYYHWRPNYQEGLLLLCG SQVPVEVMGLPEFTDCWENFVDHEPPSFNPSEKLEELDSRAIKRRLER (SEQ ID NO: 493)

[0719] >A0A0D9R222_CHLSB/9-l79

FS LQFNNKHHPYRRKALLC Y QLTPN GSTPTRGQLQNKKDHAEIRFINKIKS MGLDET QC Y Q VTC YLTW S PCPS C ARELVDFIKAHNHLNLS IF AS RLY YHWRPH Y QEGLLLLC G SRVPVEVMGLPEFTDCWENFVDHKPPSFNPSEKLDELDSQAIKRRLER (SEQ ID NO: 494)

[0720] >F6SJ45_MACMU/9-l79

FSLQFNNKRRPYPRKALLCYQLTPNGSTPTRGHLKNKKDHAEIRFINKIKSMGLDETQ CYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLLLLCGS QVPVEVMGLPEFTDCWENFVDHKPPSFNPSEKLKELDSQAIKRRLER (SEQ ID NO: 495)

[0721] >GlRYZ5_NOMLE/9- l79

FRLQFNNKRRPYPRKALLCYQLTPNGSTPTRGYFKNKKRHAEIRFINKIKSMGLDETQ CYQVTCYLTWSPCPSCAQELADFIKAHDHLNLRIFASRLYCHWCRRQQEGLRLLCGS QVPVEVMGFSEFADCWENFVDYEPLSFNPSEMLEELDSRAIKRRLEK (SEQ ID NO: 496)

[0722] >H2P4F3_PONAB/9- 179

FS LQFNNKRRPYPRKALLC Y QLTPN GSTPTRGYFKNKKCHAEIRFINEIKS MGLDETQ CYQVTCYLTWSPCPSCVRELVAFIKAHDHLNLRIFASRLYCHWCRRQQEGLRLLCGS Q VP VE VMGS REFADC WENF VDHKPLS FNPS EMLEELDS RAIKRRLER (SEQ ID NO: 497)

[0723] >H2QLQ0_PANTR/9- 179

FRLQFNNRRRPYPRKALLCYQLTPNGSTPTRGYFENKKCHAEICFINEIKSMGLDETQ CYQVTCYLTWSPCSSCAWKLVDFIQAHDHLNLRIFASRLYYHWCKPQQEGLRLLCG SQVPVEVMGLPEFNDCWENFVDHEPLSFDPCKMLEELDSRAIKRRLER (SEQ ID NO: 498) [0724] >M4W 6S4_HUMAN/9- 179

FRLQFNNKRRPYPRKALLCYQLTPNGSTPTRGYFENKKCHAEICFINEIKSMGLDETQ CYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCG SQVPVEVMGFPEFADCWENFVDHEPLSFNPYKMLEELDSRAIKRRLER (SEQ ID NO: 499)

[0725] >F7AL68_MONDO/6-l73

FLYHFKNVRWAGRHETYLCYVVKRDSATSFLDFGYLRNKGCHVELIFLRYISAWDL DPSRCYRVTWFTSWSPCYDCARHVANFLRCYPNLTLRIFTARLYFCEDKKPEGLRRL HRAG V QIAIMTFKD YF Y C WNTF VENERTFKA WEGLHEN S VRLS RQLRR (SEQ ID NO: 500)

[0726] >G3W3P5_SARHA/l 1-178

FLYHFKNVRWAGRHETYLCFVVKRDSATSFLDFGYLRNKGCHVELLFLQYISAWDL DPSRCYRVTWFTSWSPCYDCARHVANFLRCYPNLSLRIFTARLYFCEDKKPEGLRRL HQAGVPIAIMTFKDYFYCWNTFVENERTFKAWEGLHENSVRLSRQLRR (SEQ ID NO: 501)

[0727] >F7AOKl_HORSE/l 1-178

FLYHFKNVRWAGRHETYLCYVVKRDSATSFLDFGHLRNKGCHVELLFLRYISDWDL DPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKPEGLRRL HRAG V QIAIMTFKD YFY C WNTF VENERTFKA WEGLHEN S VRLS RQLRR (SEQ ID NO: 502)

[0728] >G3SM9l_LOXAF/9-l76

FLYQFKNVRWAGRHETYLCYVVKRDSATSFLDFGHLRNKGCHVELLFLRYISDWDL DPGRCYRVTWFTSWSPCYDCARHVTDFLRGYPNLTLRIFTARLYFCEGRKPEGLRRL HRAGV QIA VMTFKD YFY C WNTFV ANERTFE AWEGLHENS VRLTRQLRR (SEQ ID NO: 503) [0729] >L9KY 18_TUPCH/51-185

MYPLWGRHETYLCYVVKRDSATSFLDFGHLRNKGCHVELLFLRYISDWDLDPDRCY RVTWFTSWSPCYDCARHVADFFRGYPNFTFRIFTARFYFCEDQKPEGFRRFHRAGV QLAIMTFK (SEQ ID NO: 504)

[0730] >W5NV85_SHEEP/l 1-181

FIYQFKNVRWAGRHETYLCYVVKRDSPTSFLDFGHIRNKGCHVELLFLRYISDWDLD PGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKPEGLRQL HRAG V QIAIMTFKD YF Y C WNTF VENERTFKA WEGLHEN S VRLS RQLRR (SEQ ID NO: 505)

[0731] >HOW743_CAVPO/l 1-178

FLYQFKNVRWAGRHETYLCYVVKRDSATSCLDFGHLRNKGCHVELLFLRYISDWDL DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKPEGLRRL HRAGVQIAVMTFKDYFYCWNTFVENEKTFKAWEGLHENSVRLSRQLRR (SEQ ID NO: 506)

[0732] >G3QLD2_GORGO/l 1-168

FLYQFKNVRWAGRRETYLCYVVKRDSATSFLDFGYLRNKGCHVELLFLRYISDWDL DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKPEGLRRL HRAG V QIAIMTFKENERTFKA WEGLHEN S VRLS RQLRR (SEQ ID NO: 507)

[0733] >AICDA_HUMAN/l 1-178

FLYQFKNVRWAGRRETYLCYVVKRDSATSFLDFGYLRNKGCHVELLFLRYISDWDL DPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKPEGLRRL HRAG V QIAIMTFKD YFY C WNTF VENERTFKA WEGLHEN S VRLS RQLRR (SEQ ID NO: 508)

[0734] >G5BPM7_HETGA/64-2l9

FLYHFKNVRWAGRHETYLCYVVKRDSATSFLDFGYLRNKGCHVELLFLRYISDWDL DPGRC YRVT WFTS WS PC YDC ARH V ADFLRGNPNLS LRIFT ARLT GW GP AGLMS PAR PS D YFY CWNTFVENERTFKA WEGLHEN S VRLS RRLRR (SEQ ID NO: 509) [0735] >L5KIU3_PTEAL/6- 173

[0736] >FlSFW5_PIG/l 1-179

[0737] >S7N9P5_MYOBR/lO-l77

[0738] >GlTZP8_RABIT/6-l73

[0739] >G3V7Y8_RAT/l 1-178 [0740] >G 1 KTX0_AN OCA/ 12-179

FLYHFKNLRWAGRHETYLCYVVKQNSATSCLDFGYLRNKGCHVEVLFLRYISTWDL DPRHCYRITWFTSWSPCYDCARHVADFLSAYPNLSLRIFAARLYFCEERNPEGLRRL HRAG AQIAIMTFKD YF Y C WNTF VENKTTFKA WEGLHEN S VRLARRLRR (SEQ ID NO: 515)

[0741] >F7EGY 6_XENTR/ 13-181

FLYHYKNLRWAGRHETYLCYIVKRYSSVSCLDFGYLRNRGCHAEMLFLRYLSVWV GHDPHRAYRVTWFSSWSPCYDCAKRTLEFLKGHPNFSLRIFSARLYFCEERNPEGLR KLQKAGVRLAVMSYKDYFYCWNTFVESERRFEAWDGLHENSVRLARKLRR (SEQ ID NO: 516)

[0742] >H3 ALQ6_LATCH/20- 149

FLYHYKNVRWAGRHETYLCYIVKRYNPASYLDFGFLRNKGCHVEMLFLRFLTGWNI DPTLP Y S VT WFTS W S PC YDC S QH VTHFLR V YPNLRLRIFT ARLYFCEENNPEGLRNL HMAGVQLGVMT (SEQ ID NO: 517)

[0743] >A0A0P7ULF7_9TELE/8-l84

FIYHYKNVRWAGRHETYLCFVVKRDGPDTLFDFGHLRNRGCHVELVFLRHLGALCP GLS Y S VTWFC S WS PC YN C S RRLAHFLTRTPNLKLRIFC S RLYFCD VEDS S EGLRLLKR AG V QLS VMT YKD YF Y C W QTF V ARERGFKA WEGLHQN S VRLARKLNR (SEQ ID NO: 518)

[0744] >B 3DGZ0_D ANRE/ 15-191

FIFHYKNVRWAGRHETYLCFVVKRIGPDSLFDFGHLRNRGCHVELLFLRHLGALCPG FCYSVTWFCSWSPCSKCAQQFAHFFSQTPNFRFRIFVSRFYFCDEEDSREGFRHFKR AG V QIS VMT YKDFFY C W QTF V ARERS FKA WDGFHEN S VRFVRKFN Q (SEQ ID NO: 519)

[0745] >13 K4U3_ORENE 15-191

FLYHYKNVRWAGRNETYLCFVVKRVGPDSLFDFGHLRNRGCHVELLFLRQLGTLCP

GLSYSITWFCSWSPCANCSSRLAQFLKQTPNLRLRIFVSRLYFCDMEDSREGLRLLK K V G VHIT VMS YKDFFY C WENF V AQQS KFKA WEGLHQNT VRLARKLNR (SEQ ID NO: 520)

[0746] > AO A0F8 AS 01 _LARCR/ 15-192

FIFHYKNVRWAGRHETYLCFVVKRVGPDTLFDFGHLRNRGCHVELLFLRYLGALCP GLS Y S VTWFC S WS PC ADC S FRLS QFLNRTPNLRLRIF V S RLYFCDMEN S REGLRMLK N AG AHIT VMS YKDFF Y C W QTF V ARES NFKA WDELHRN S VRLS RKLHR (SEQ ID NO: 521)

[0747] >H2S Y A6_T AKRU/ 15- 192

FIYHYKNVRWAGRHETYLCFVVKRVGPDTLFDFGHLRNRGCHVELLFLRYLGALCP GLSYSVTWFCSWSPCVNCSIQLCQFLNNTPNLRLRIFVSRLYFCDLEDSREGLRMLTK AG VRIS VMS YKDYFY C W QKFVDC KS NFKA WEELHQN S VRLTRKLNR (SEQ ID NO: 522)

[0748] >G3P8Jl_GASAC/l5- l92

FIYHYTNMRWAGRHETYLCFVVKRVGPDSLFDFGHLRNRGCHVELLFLRHLGALCP GFS Y S ITWFC S WS PC VN C S IS LS QFLS RTPNLRLRIF V S RLYFCDMEN S RD GLRMLKK AG V QVT VMS YKDFF Y C W QTFVDRQS QFK A WKELHQN S VRLS RKLKR (SEQ ID NO: 523)

[0749] >W 5L8 S 5_AS TMX/ 15- 190

FIYH YKN VRW AGRHET YLCF V VKRIGPN S LFDF GHLRNRGCH VELS EIQS FS AFCP AL VQNDSKSCRISTDSESLHQIETSLDKTPKTGIKVFLSLSLFVTLANHSHYPQMASDLCL C VPIFLFTDFF Y C W QTF V ARES RFKA WDGLHQN S VRLS RKLKR (SEQ ID NO: 524)

[0750] >H2M862_ORYLA/l 1- 188

FIYHYKNMRWAGRHETYLCFVVKRVGPESLFDFGHLRNRGCHVELLFLRHLSALCP GLSYSITWFCSWSPCANCSFRLAQFLSQTPNLRLRIFVSRLYFCDLEDSREGLRMLKK V G VHIT VMS YKDYFY C W QTFV ARQS KFKP WDGLHQN S VRLS RKLNR (SEQ ID NO: 525) [0751]

[0752]

[0753]

[0754]

[0755]

[0756] DPGRCYRITWFTSWSPCYDCARHVADFLRTYPNLTLRIFAARLYFCEDRKPEGLRRL HKAGAQIAIMTFKDYFYCWNTFVENEKTFKAWEGLHENSVHLSRKLRR (SEQ ID NO: 531)

[0757] >A0A099 YY C6_TIN GU/9- 176

FLYNFKNMRWAGRRETYLCYVVKRNSATSCLDFGYLRNQGCHVEVLFLRYISAWD LDPGRCYRITWFTSWSPCYDCARHVADFLRAYPNLSLRIFTARLYFCEDRKPEGLRR LHRAGAQIAIMTFKDYFYCWNTFVENEKTFKAWEGLHENSVRLSRQLRR (SEQ ID NO: 532)

[0758] > AO A091 RV C 9_NES N 0/9- 176

FLYNFKNLRWAGRRETYLCYVVKRDSATSCLDFGYLRNQGCHVEVLFLRYISAWDL DPGRCYRITWFTSWSPCYDCARHVANFLRAYPNLTLRIFTARLYFCEDRKPEGLRRL HRAGAQIAIMTFKDYFYCWNTFVENEKTFKAWEGLHENSVHLSRKLRR (SEQ ID NO: 533)

[0759] >A0A091 MHE0_9PAS S/9- 176

FLYNFKNLRWAGRRETYLCYVVKRDSATSYLDFGYLRNQGCHVEVLFLRYISAWDL DPGRCYRITWFTSWSPCYDCARHVANFLHSYPNLTLRIFTARLYFCEDRKPEGLRRL HKAGAQIAIMTFKDYFYCWNTFVENEQTFKGWEGLRENSVHLSRKLRR (SEQ ID NO: 534)

[0760] >A0A093PQH0_9PAS S/9- 176

FLYNFKNLRWAGRRETYLCYVVKRDSATSCLDFGYLRNQGCHVEVLFLRYIAAWD LDPGRCYRITWFTSWSPCYDCAQHVANFLRSYPNLTLRIFTARLYFCEDRKPEGLRR LHKAGAQIAIMTFKDFFYCWNTFVENEQTFKGWEGLHENSVHLSRKLRR (SEQ ID NO: 535)

[0761] >A0A091 EUR4_CORBR/9- 176

FLYNFRNLRKAGRRETYLCYVVKRDSATSCLDFGYLRNQGCHVEVLFLRYIAAWDL

DPGRCYRITWFTSWSPCYDCAQHIANFLRSYPNLTLRIFMARLYFCEDRKPEGLRRL HKAGAQIAIMTFKDYFYCWNTFVENEQTFKGWEGLHENSVHLARKLRR (SEQ ID NO: 536)

[0762] >T0NHJ8_CAMFR/l46-317

FTHNFSNHKRTHKTYLCYEVEIHGDSGIPDKGFLCNKCHVELYLLGRIRSWKLDRKL HCRLTCFISWTPCGTCARELAEFLKENSHVSLCIFASRIYSLNDYEAGLRTLQEAGAQI AIMTFKEFRHC WETF VDHGRPF QPWDELDIN S QGLS KELQ A (SEQ ID NO: 537)

[0763] >H9GWR3_C ANLF/ 15- 197

FTQNFRNDNPSKTYLCYQVELDGSSGVLDKGSAFPGGQHAEWFLLEHIRSRNLDQK LSYKVTCFLSWTPCEKCAEEIIRFLAKNRHVSLSILASRIYTMGPYVKGLRELYDAGV HIS IMTFRDFE Y C W QTFVDHDS PFQPW ADLDRRS QQLS QQLR A (SEQ ID NO: 538)

[0764] >H0XN 38_OTOG A/ 18- 197

FTSNFTNNPAIGRRQT YLCHE V QLDGDS W VLDRGFLQS QPLHAEFCFLDRV GS W QL NPNWHYRVTCFISWSPCFSCAQKVAMFLRRNSHVKLRILAARIYDYHPGYEEGLKA LQGTGAQVAIMTHAEFEHCWDTFVDHGRPFQPWEGLDKNSQALSRRLQD (SEQ ID NO: 539)

[0765] >G3SVX3_LOXAF/l 1- 178

FRFNFIND AS VGQKQTYLC YE VELDGNS WVLDRGFLLN QRRHAELCFLDRV S S WHL DPTKH YKFT WFLS WS PCRNC AQE V V AFLGGN S H V S LS IF APRIYD Y Y S G YEEGLRS L QG AG AH V S IMTS TEFEHC WRTF VDN GCPF VPWNRLGEN S QTIS RRLQS (SEQ ID NO: 540)

[0766] >G3TLGl_LOXAF/31-201

FQLNFINDLSVGQKQAYLCYEVELDCNSRVLNRGFLCNQGCHAELCFLDQVPSWQL DLVLCYRVTWFISWSPGPDCAQEVAAFLRGNSHVSLSIFASCIYDSVEESEGGWKGK KDLGAS KKKFGHC WKTIVDN GRFFEPWNRLDES FDFLDLT (SEQ ID NO: 541)

[0767] >G 1 TLT9_RAB IT/ 18- 182

FMDHFANEDGGGLNET YLC YEV QLDGS S QGFLRNKRRHAELCFLDLVPAWRLDPA QHYRVTWFISWSPCFLCAQAVAEFLRRNAHVSLRIFAARIYTWRTDYKAGLQDLQR AG AQIAIMTP AEIQFC WNTF VDN S NPFHS HLGLGH AKP A (SEQ ID NO: 542)

[0768] >S7N2R7_MYOBR/2-l26

CFDGNNRRPRAELCFLALFQSWHLNEGKQYRLTWYSSWSPYPDCVPKLVEFLGDNS NVSLRIFAAGIHSIFTGYKRELRNLRDAGAQLAIMTLEELRWELGGEMATWRWQNE

N (SEQ ID NO: 543)

[0769] >L7Nl00_MYOLU/l5-l80

FKENFANTWENETELC YE VEVEGDTW A VEQGFLCN QPCHAELCFLCLVRS WHVDE GKQYRLTWHISWSPCPNCAQKLVKFLHDNSHVSLRIFAAGIQTTFSGHEDWLRKLRD S GAQLAIMTLKELQHC WDTFVDN GQPFEPWPNLVEHIQTES QKLKD (SEQ ID NO: 544)

[0770] >G 1 Q326_MY OLU/ 19-187

EWNFGITWAKETYLCYEVEVEGDAWVAKEGFLRNELRHAELCFLRGVSDWDLDEG KQYRLTYMSWSPCPNCAPKLVEFLDENSHVTLRIFPARIHTKSRGYQDGLRNLRDAG AQLAIMTLKEHQHC WDTFVDN GQPFRPWPNLVEHIETKS QELKD (SEQ ID NO:545)

[0771] >GlPQB2_MY OLU/4- 177

FKDNFGHNWEKTYLWYEVEFEGDAWAVEQGFLRNQLRHAELCFLHGVRSWHLDE GKQYRLTWHIS WSPCPDC AS KLVEFLGENSHV S LRIFAARIHTKYRGYEDGLRQLQD AVDHLTIMTLKELQHCWVTFVDNGQPFEPEIELLENIGAQCQKLES (SEQ ID NO:

546)

[0772] >L5M566_MYODS/4-l75

FKDNFGITW ANETYLC YEVE VEGD AW A VEQGFLRN QRSHAELCFLDRVPS WHLDE GKQYRLTCYISWSPCPDCAQKLVEFMGENSHVSLRIFAARIYTKLDGHEDGLRKLQD AGAQLAIMTLKEYEHCWDTFVDSGQLFRARDELEVHIGAQCQRLEN (SEQ ID NO: 547) [0773] >GlQB54_MYOLU/l7-l83

FKRNFRNKCKNQTYLCYEVEVEGNAWAVEQGFLRNQRRHAELCFLDRVPCWNLDG LKKYRLTCYISWSPCPDCALELVQFLGLKSNVSLRMCTAGIRTTFPGHEDGLRNLRD AGAQLTIMTRDEYEHCWDTFVDNGQPFRARDELEGHDYFLL (SEQ ID NO: 548)

[0774] >G 1 NTH0_M Y OLU/7 - 175

EVNFGHDWEKKTYLCYEVEVEGDAWVGKQGFLCNQPGHAELCFLDRVRSWHLDG GKQYRLTC YMS WTPSPDC ALELV QFLS ENSHV S LRIFAAGIRTRFHGHEDGLRQLRD AGAQLAIMTLHELQHCWDTFVDNGQPFRARDELEVHIGAQCQKLKS (SEQ ID NO: 549)

[0775] >L7NlOO_MYOLU/l94-364

KRRNFRNKCEN QTYLC YEVE VKDGKWT VEQGFLRN QPGH AELCFLDRVRS WHLDE GRQYRLTCYISWTPGPDCAQKLVEFLGENRHVSLRIFAAGIHTKYRGHEDGLRQLW D AGAQIAIMTLNELQHCWETFVDN GQPFEPWPIQVEHIQTES QKLKD (SEQ ID NO: 550)

[0776] >G 1 QB 54_M Y OLU/248-406

AEKLFFKKLLQQVYYCFGLKIEGRDQPRHAELCFQDRVRSWHLDEGKQYRLTCYIS WSPCPDCAQKLVEFLGENSHVRLRIFAARIYKKRDRYKHWLRQLRDAGAQLTIMTL NELQHCW VTFVDNGQRFEP (SEQ ID NO: 551)

[0777] >G 1 PB V 8_M Y OLU/ 144-276

FRVNFSYYRERKTYLCYEVEVEGDAWVVKQDFLRNQPRHAELCFLDGVRSWHLDE GKQYRLTCYISWSPCPVCAQELVEFLGENRHLRLRIFAARIYSIVSGYEDGLRQLWDA GAPLAIM (SEQ ID NO: 552)

[0778] >G 1 Q0Q6_MY OLU/ 140-315

FKDNFSHRRARRTYLCY QVEVEGDAWAVEY GFLCNQLRHAELCFLDRVPFWNLEE GRQYRLTCYISWSPCPDCAQRLVEFLGNNNHMRLRIFAARIYTFVSGHEDGLRQLW D AGAQLTIMTRNDLQHC WDTFVDN GDPFEPCPIQVEHIGTES QELEN (SEQ ID NO: 553) [0779] >GlQOG3_MYOLU/l3-l86

FKENFSHRRARKTYLCYEVEVEGNTWAVEQGFLHNQLRHAELCFLDRVRFWNLEE GRQYRLTC YIS WSPCPDC AQKLVEFLGQNSHV S LCIFAARIYTIV S GYKDGLCQLRD A GAQLTIMTLNDLQHC WENLVDN GEPFEPCPTLVEHIETKS QELKD (SEQ ID NO: 554)

[0780] >A0A096NK44_PAPAN/ 18-195

FTFNFNNDLSVGRHQTYLCYEVERDNGTWVMDRGFLHNKGCHAELCFLGEVPSWQ LDPAQTYRVTWFIS WSPCLRRGCAEQVRAFLQENTHVRLHIFAARIYDFDFLY QEAL RTLRD AGAQVSIMTYEEFKHC WDTFVDHGRPFQRWDGLDEHS QDLS GRLRA (SEQ ID NO: 555)

[0781] >A0A0D9R289_CHLSB/l98-375

FTINFNNDLSVGRRQTYLCYEVERDNGTWVMDWGFLCNQSCHVELCFLSQVSSWQ LDPAQTYRVTWFISWSPCFSGGCAEQVRAFLQENTHVRLCIFAARIYNYDPLYQEAL RMLRD AGAQVSIMT YEEFE Y CWDTFVDRGCPFQPWDGLDEHS QALS GRLRA (SEQ ID NO: 556)

[0782] >F7FXKl_MACMU/l8-l95

FTSNFNND VS VGRHQTYLC YE VERDN GTW VMDW GFLCNQGCHAELCFLGW VPS W QLDPAQTYRVTWFISWSPCFSWGCAEQVRAFLQENTHVRLHIFAARIYDYDPLYQEA LRTLRD AGAQV S IMT YDEFE Y CWDTFVDCGCPFQPWDGLDEHS Q ALSERLRA (SEQ ID NO: 557)

[0783] >GlRYY7_NOMLE/l98-375

FTFNFNNDPLV GRHQTYLC YE VERDN GTW VMDRGFLHN QGRHTELCFLGLIPYW QL DLAQTYRVTWFISWSPCFSWGCAEQVRAFLQENTHMRLRIFAARIYDYDPLYKEAL QMLRGAGAQV S IMT YDEFEHCWDTFVDHGRPFQPWDGLEEHS QALS GRLQA (SEQ ID NO: 558)

[0784] >H2P4E7_PON AB/ 198-375

FTFNFNNDPFVRRHQTYLCYEVEHDNGTWVMDRGSLHNQGRHAELRFLGLLPYWQ LDPAQIYRVTWFISWSPCFSWGCARQVRAFLQENTHVRLRIFAARIYDYDPLYKEAL QMLRD AGAQV S IMT YDEFE Y CWNTFVDHGCPFQPWDGLEEHS QALS GKLQ A (SEQ ID NO: 559)

[0785] >G 1 RYY 4_N OMLE/ 16-192

FT S NFNN GRWHKT YLC YE VERDN GTW VMDRGFLHN QGRH AELCFLDLVPS LQLDP AQTYRVTWFISWSPCFSWGCAEQVRAFLQENTHVRLRLFAARIYDYDPLYKEALQM LRG AG AQ V S IMT YHEFKHC WDTFVDHGRPF QPWDGLEEHS QALS GRLQ A (SEQ ID NO: 560)

[0786] >H2QLP4_PANTR/ 16-192

FTSNFNN GGRRKTYLC YEVERDN GTS VMDRGFLHNQGRHAELRFLDLVPS LQLDPA QIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLR D AG AQ V S IMT YDEFKHC WDTF VDHGCPF QPWDGLEEHS QALS GRLRA (SEQ ID NO: 561)

[0787] >G3 S 2 J 9_GORGO/ 18-195

FT S NFNNDLLVRRHQT YLC YE VERDN GT W VMDRGFLHN QGRH AELRFLDLVPS LQL DPAQIYRVTWFISWSPCFSWGCAGQVCEFLQENTHMRLRIFAARIYDYDPLYKKALQ MLRD AG AQ V S IMT YDEFKHC WDTF V YRGCPF QPWDGLEEHS Q ALS GRLQ A (SEQ ID NO: 562)

[0788] >H2QLP5_P ANTR/ 198-375

FTFNFNNDPLVRRHQTYLCYEVERDNGTWVMDMGFLCNEGRHAELRFLDLVPSLQ LDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEAL QMLRD AGAQV S IMT YDEFE Y CWDTFVYRGCPFQPWDGLEEHS QALS GRLRA (SEQ ID NO: 563)

[0789] >G3QVl6_GORGO/l98-375

FTFNFNNDPLVRRHQTYLCYEVERDNGTWVMDMGFLCNEGRHAELRFLDLVPSLQ

LDPAQIYRVTWFISWSPCFSWGCAGQVCEFLQENTHMRLRIFAARIYDYDPLYKKAL QMLRD AG AQ V S IMT YDEFKHC WDTF V YRGCPF QPWDGLEEHS Q ALS GRLQ A (SEQ ID NO: 564)

[0790] >A0A096N7U5_PAPAN/l78-346

FT YNFTNDPS VGQHQT YLC YKVECDNDTW VLDKGILPN QHAEQYFLYLIS FWKLDQ AQCYRVTWFISWSTCFSCAQQVATFHWENRCVSLHIFIARIYNYLPGYEGLCMLQRA GTQIS IMTS KFRHC WVTFVDHGHPFQPWDGLDEHS QALS GRLQ A (SEQ ID NO: 565)

[0791] >F6QUT3_MACMU/l71-340

FT YNFTNDPS VGQHQT YLC YKVECDNDTW VLDKGILPN QHAEQYFLYLIS FWKLDR AQCYRVTWFISWSTCFSCAQQVTTFHWENRCVSLHIFVACIYNYLPGYEGLCMLQR AGTQIS IMTS GFRHC W VTF VDHGHPF QPWDGLDEHS QALS GRLQ A (SEQ ID NO:

566)

[0792] >H9KW44_CALJA/l43-282

FTYNFTNDPSVGRHQTYLCYEVEHHNGTWVLHRGFILNEGRHAELCLLDLILFWKL DLAQRYRVTCFIS WSPCFCC AEKVAEFLQENPHVNLRIFAARIY GY QRGYKKGLRRL NRAG APIS MMKY S (SEQ ID NO: 567)

[0793] >F7CU A6_C ALJ A/202- 377

FTYNFTNDPSVGQHQTYLCYEVEHHNGTWVLHRGFILNQGRHAELCLLDLISFWKL DLAQYYTVTCFIS WSPCFSCAEKVAEFLQENPHVNLHIFAAHIY GY QRGYIKGLCRLN RAG APIS MMKY S EFS Y C WDTF VDHEHPF QPWEGLDE YTQ ALS GKLQ A (SEQ ID NO: 568)

[0794] >A0A0D9R229_CHLSB/2l3-376

FTSNFNNKPWV GQRET YLC YKVERHNDTW VLNRGFLRN QGRHAELCFLDLIPFWKL DDQQYRVTCFTSWSPCFSCAQKMAKFISKNKHVSLCIFAARIYDDQGRCQEGLRTLH RDGAKIAVMNYSEFEY CWDTFVDRGXXXXLGVKPDC (SEQ ID NO: 569)

[0795] >ABC3G_PAP AN/202-376

FTSNFYNKPWV GQHET YLC YKVERHN GTW VLNRGFLRN QGRHAELCFLDLIPFWKL DGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRCQEGLRTLH RDGAKIAMMNYSEFEYCWDTFVDRGRPFQPWDGLDEHSQDLSGRLRA (SEQ ID NO: 570)

[0796] >ABC3G_MACMU/l95-369

FTSNFNNKPWV GQHET YLC YKVERHNDTW VLNRGFLRN QGRHAELCFLDLIPFWKL DGQQYRVTCFTSWSPCFSCAQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALH RDG AKIAMMN Y S EFE Y C WDTFVDRGRPF QPWDGLDEHS Q ALS GRLRA (SEQ ID NO: 571)

[0797] >H2P4E9_PONAB/2l6-39l

FTSNFNNEPCVGRHETYLC YKVERHNDTW VLNRGFLCNQGRHAELCFLD VIPFWKL DGKQRYRVTCFTSWSPCFRCAQEMAKFISNNQHVSLCIFAARIYDDQGRCKEGLRTL DEAEAKISIMTYSEFQHCWDTFVDHGRPFQPWDGLEEHSEAWSGKLQA (SEQ ID NO: 572)

[0798] >ABC3G_GORGO/202-377

FTSNFNNEHW V GRHET YLC YE VERHNDTW VLNRGFLCNQGRHAELCFLD VIPFWKL DLHQDYRVTCFTSWSPCFSCAQEMAKFISNKKHVSLCIFAARIYDDQGRCQEGLRTL AEAGAKISIMTYSEFKHCWDTFVYHGCPFQPWDGLEEHSQALSGRLQA (SEQ ID NO: 573)

[0799] >G3 S FB 6_GORGO/202- 375

FTSNFNNEHW V GRHET YLC YE VERHNDTW VLNRGFLCNQGRHAELCFLD VIPFWKL DLHQDYRVTCFTSWSPCFSCAQEMAKFISNKKHVSLCIFAARIYDDQGRCQEGLRTL AEAGAKISIMTYSEFKHCWDTFVDHGCPFQPWDGLEEHSQSLGDRA (SEQ ID NO: 574)

[0800] >L9KA V 2_TUPCH/ 18-186

FNFHYKNLQRAGRRKTYLC YLLEEEREGAIVLD VGVLHN QEHAERRFLS SPDILQLL DGGRRYRVTWYLSWSPCSQCARAVAGFLAQHGNVSLRIFVARLYNHEDPENRQGL RTLNSTGTPIRVMTNREFALCWERFVRHQGAAFEPWAGLRENADLLLGQLED (SEQ ID NO: 575)

[0801] >Q6DIS6_XENTR/55-231

FMFQFKN VEY S GRNKTILC YT VERPEGQVFHGYLEDES AHAED AFFTS VLPQLT S GS VT VTC Y V S S S PC VN C A AS V AQCLRRNKT VRIQLA V ARLF QWEEPEIRRALKGLRS AG CQVRMMRGADYVYVWKNFVEPDHQDFIPWEDLEENARYYEEKLEE (SEQ ID NO: 576)

[0802] > AO A0P7 WIY 8_9TELE/ 108-272

FKFQFKNVEYSGRNKTFLCFLVDVQGSGGEGLRGYLEDEGAHAEEAFFQQVLPQDS ALH YM VTW Y V S S S PC A ACT AKLVEILKARKTMRLTIF S ARLFMWEEPEIQ V GLKAL AAAGCKLRMMKPTDFVYIWDTFVENDQTFTPWEDCQENYEYYQEKLAD (SEQ ID NO: 577)

[0803] >F 1 RAQ7_D ANRE/ 104-270

FKFQFKNVEYSGRNKTFLCYQVDIQGGETDGVRGYLEDEGSHAEEAFFQQVLAYDK S LRYT VTW YT S S S PC V AC A AKLVEILKARKALRLNIHC S RLFE WEEPEIQ AGLQ ALVR AGCKLRMMRPVDFVYVWSTFVENEDNFTPWEDCQDNFEYYDERLQD (SEQ ID NO: 578)

[0804] >H2LVW2_ORYLA/81-243

FKF QFRN VEY S GRNKTLLCFRVDT AGGS TEPLRG YMEDET AH AEE AFF QQ VLPN S S Q E YD VT W Y V S S S PC V AC A AKLTS ILQQRKKLRLS VFC S RLFD WEEPEIV QGLKALV Q A GCKLQMMKPADFQHVWETYVEKDQSFTLWEDCKENYEYYLEKLAD (SEQ ID NO: 579)

[0805] >AOA087X5U3_POEFO/lOl-263

FKF QFRN VE Y S GRNKTLLCFRVDT AGGN AEPLKG YMEDET AH AEE AFF QQ VLPN S S TE YD VTW Y V S S S PC V AC A AKLANILQQRKKLRLS IFC S RLFE WEEPEIVEGLKALAR A GCKLRMMKPIDFQHVWEMYVEQGESFTPWEDCQENYEYYVERLTD (SEQ ID NO: 580) [0806] >G3P582_GASAC/83-245

FKFQFRNVEYSGRNKTLLCFRVDTPGGSTEPLKGYMEDETAHAEEAFFQQVLPNTSQ EYEVTWYLSSSPCVACAAKLAHILQQRKKVRLRMFCSRLFEWEEPEIVEGLRALVSA GCKLRMMKPSDFVHVWETYVEKDQNFEAWEDCQENYDYYVEKLTD (SEQ ID NO: 581)

[0807] >H2U 6E4_T AKRU/97 -259

FKF QFRN VE Y S GRNKTLLCFRVDS PGGS TEPLKG YMEDET AH AEE AFF QQ VLPNPS L EYDITWYVSSSPCISCANTLAKMLQQRKKVRLCVFCSRLFEWDQPEVVEAIRTLVRA GCKLRMMKPSDFQHVWETYVEKGESFTPWEDCQDNYNYYKEILAD (SEQ ID NO: 582)

[0808] > AO A0P7 V 3 P5_9TELE/68-234

FKFQFKNVEYSGRNKTLLCFLVDSQKGEDELQRGYLEDEGAHAEEAFFQQVLPEDP GRR Y Q VTW Y V S S S PC A AC A AKLAEILR ARPTLRLTIF S ARLFM WEEPEIQE GLKALA A AGCKLR VMKP VDF S YTWDTF VENDQTF S PWEDC QEN YE Y Y QEKLAG (SEQ ID NO: 583)

[0809] >W5KNC9_ASTMX/26-l94

FRFQFKNVEYCGRNKTLLCYLVDQSSGADGLLRGFLEDEGLHAELAFFQLILPQDPS VNYTVTWYVSSSPCAHCCSKLLEILQERKTLRLNIFSARLLEAEEEEGQAGMKALAG AGCKLR VMKPLDFSYSWDTFVEHDERFTPWEDCQENYEYHHEKLAQ (SEQ ID NO: 584)

[0810] >H2M4W2_ORYLA/70-234

FKFQFKNVEYSGRNKTLLCYLVDTGGAGDGLLRGYLEDEGIHAEEAFFTHCLPNDPN V Q YTITW Y V S S S PCH ACS LKLAE VLKARKN VKLS IF S ARLFE WE AEE V Q AGLKALHS AGCKLRVMKPLDFSYTWDTFVETEQPLNLWEDCKDNYEYYHEKLDQ (SEQ ID NO: 585) [0811] >H2TE47_TAKRU/80-244

FQFQFKNVEYSGRNKTFLCYLVDTGKADEGLQRGYLEDEGSHAEEAFFTQCLPHDP ALKYSVTWYMSSSPCCACAAKMAEALKARRNIKLSIFAARLFEWEEAEIQAGLKAL HIAGC KIR VMKPLDF S YTWDTF VEND QPLNLW ADCKEN YE Y YHERLAD (SEQ ID NO: 586)

[0812] >I3JAF9_ORENE80-244

FKFQFKNVEYSGRNKTLLCYLVDKGNTSDGLLRGYLEDEGTHAEEAFFTQCLPHNP ALKYTVTWYVSSSPCSACAAKIAELLKDQKNLKLNIFAARLFEWEEGEIQAGLRTLH AAGCKLRVMKPLDFSYTWDTFVENDQSLNLWEDCKENYEYYHEKLSD (SEQ ID NO: 587)

[0813] >A0A0F8BFJ4_LARCR/500-664

FKFQFKNVEYSGRNKTFLCYLVDKGNTADGLLRGYLEDEGSHAEEVFFIQCLPHDPT LKYT VTW YV S S SPCS AC AAKIAEVLKARKNVKLS IFAARLFECEE VEIQAGLKALHA AGCKLRVMKPLDFSYTWDTFVENEQPLNLWEDCKENYEYYHEKLAD (SEQ ID NO: 588)

[0814] >G3 PC V 8_G AS AC/21-185

FKFQFKNVEYSGRNKTFLCYLVDKGNTDDGLLRGYLEDEGSHAEEAFFIQCIPNDPS VRYT VTW Y V S S S PC A AC A AKIAE VLKARKNIKLS IFA ARLFECEETDIQ AGLK AMHT AGCKLRVMKPLDFSYTWDTFVENEEPLNLWEDCKENYEYYQEKLAD (SEQ ID NO: 589)

[0815] > A0 A 151 P4M 1 _ALLMI/ 14-181

FKF QFKN VE Y S GRNKTFLC YIVET QGKES GTIRG YLEDE A AH AED AFFNTILPEES S LR YN VTW Y V S S S PC V AC AERIAEILKKNKNLRL AILV S RLFM WEEPEMQ A VLKKMK A A GCKLRIMKPQDFEY VW QNFVEQES KDFVPWEDIQENFLYYEEKLAE (SEQ ID NO: 590)

[0816] >K7G457_PELSE58-225

FKF QFRN VE Y S GRNKTFLC YIVET QGKES GS FRG YLEDE A AH AEG AFFNNILPTEPS L RYN VT W Y V S S S PC VPC ADQIAQILQKNKNLRLTILV S RLFM WEEPEMQ A ALKKLKT AGCKLKIMKPQDFEYIWQNFVEQEAKAFEPWEDIQENFLYYEEKLAE (SEQ ID NO: 591)

[0817] >M7B 9 Y 3_CHEM Y /50-217

FKFQFRNVEYSGRNKTFLCYVVEIQGKEPGSFQGYLEDEAAHAEDAFFNNILPTDPN LRYN VTW Y V S S S PC VPC ADRIAQ VLQKNKNLRLS ILV S RLFM WEEPEMQ A ALKKLK ASGCKLKIMKPQDFEYIWQNFAEQEAKAFEPWEDIQENFLYYEEKLSE (SEQ ID NO: 592)

[0818] >V8PlW3_OPHHA/5 l- l83

FNFQFKNVE Y S GRNKTFLC YIIEIQGKES KMLRGYLEDEAAHAED AFFNTILPKES GM CYNVTWYSSCSPCIGCADRIAKALQKNKNLHLSIAVGRLFMWEEPDMQAALKKMK TAGCKLRIMKPQDFEY (SEQ ID NO: 593)

[0819] >GlKSPl_ANOCA/l7- l85

FKFQFKNVEYSGRNKTFLCYIIEMQGKESKTLRGYLEDEAAHAEEAFFNTILPTEPGF RYE VTW YV S S SPC VSC AERIVKALKKNKNLRLSIA V GRLFMWEEPDIQAALKQMKA AGCKLRIMKPQDFEYVWKNFVEQEPKAFAPWDDIQENFQYYDEKLAE (SEQ ID NO: 594)

[0820] >R7VNAl_COLLE52-220

FKF QFRN VE Y S GRNKTFLC Y V VET QGRES VT S RG YLEDE A AH AEM AFFNTILPKES S ARYN VT W Y V S S S PC VT C ADRITETLKKNKNLRLTIM V GRLFMWEEPEMQ A ALKNM RAAGCKLRIMKPQDFEYVWQNFVEQEAKAFAPWEDIQENFQYYEEKLAE (SEQ ID NO: 595)

[0821] >HlA4Ql_TAEGU/51-219

FKF QFRN VE Y S GRNKTFLC YV VET QGKEP VT S RG YLEDE A AH AEM AFFNTILPTQ AG ARHD VT W Y V S S S PC VT C AQRICE ALRKNKGLRLTIM V GRLFMWEEPEMQ A ALRS M KEAGCKLRIMKPQDFEYVWKNFVEQEAKSFVPWEDIQENFQYYEEKLAE (SEQ ID NO: 596) [0822] >R0L7 B 9_AN APL/57-225

FKF QFRN VE Y S GRNKTFLC Y VIET QGKES VT S RG YLEDE A AH AEIAFFNTILPKES S LR YN VTW Y V S S S PC VT C ADRITETLKKNKNLRLTIM V GRLFM WEEPEMQ A ALKKMKS AGCKLRIMKPQDFEYVWQNFVEQEAKAFVPWEDIQENFQYYEEKLAE (SEQ ID NO: 597)

[0823] >A0A0Q3TEK9_AMAAE/51-219

FKF QFRN VE Y S GRNKTFLC Y V VET QGKES ATS RG YLEDE A AH AEM AFFNTILPKDS S FRYNIT W Y V S S S PC VT C AERIIETFKKNKNFRFTIM V GRFFM WEEPEMQ A AFKNMKS AGCKLRIMKPQDFEYVWQNFVEQEAKDFVPWEDIQENFNYYEEKLAE (SEQ ID NO: 598)

[0824] >FlNNK5_CHICK/51-219

FKF QFRN VE Y S GRNKTFLC YV VET QGKES KT S RG YLEDE AS H AEIAFFNTILPKES S L RYNITW YV S S SPC VTC ADRISETLRKNKNLRLTIM V GRLFMWEEPEMQAALKKMKS AGCKLRIMKPQDFEYVWQNFVEQEAKAFVPWEDIQENFQYYEEKLAE (SEQ ID NO: 599)

[0825] >F6PKH9_ORNAN/ 19- 179

FKFQFRNVEY S GRNKTFLC YVVETQGKEN QT ARGYLEDE AAHAEE AFFN S ILPADQA LKYNVTWYV S S SPC AAC ADRIADTLRRTPNLRLLLLV GRLFMWEEPEIQA ALKKLK AAGCKLRIMKPQDFEYVWQNFVEQEAKAFVPWEDIQENFLY (SEQ ID NO: 600)

[0826] >F7EXQ l_MONDO/49-218

FKF QFRN VE Y S GRNKTFLC YVVEV QGKQGQIS RG YLEDE AAHAEE AFFKTILPTDP A LRYN VTW Y V S S S PC A AC ADRIS S TLS KTKNLKMLLLV GRLFMWEEPEIKT ALKKLKE S GCKLRIMKPQDFEY VW QNFVEQES KAFVPWEDIQENFLY YEEKLAE (SEQ ID NO: 601)

[0827] >G3WZU9_SARHA/50-2l9 [0828] FKF QFRN VE Y S GRNKTFLC YVLEV QGKQGQMS RG YLEDET AH AEE AF FKTILPTDP ALRYN VTW YV S S S PC A AC ADRIS S TLS KNKNLRMLLL V GRLFMWEEPEI KAALKRLKEAGCKLRIMKPQDFEYVWQNFVEQESKAFVPWEDIQENFLYYEEKLAE

(SEQ ID NO: 602)

[0829] >I3N5I3_ICTTR/52-22l

FKF QFRN VE Y S GRNKTFLC YVVE AQGKQ V Q AS RG YLEDE A AH AEE AFFNTILPTDPS LRYN VTW Y V S S S PC A AC ADRIIKTLGKTKNLRLLILV GRLFMWEEPEIQT ALKKLKE AGCKLRIMKPQDFE YIW QNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 603)

[0830] > AO AOB 4J2Il_PONAB/52-22l

FKF QFRN VE Y S GRNKTFLC Y V VE AQGKQ V Q AS RG YLEDE A AH AEE AFFNTILP ADP ALRYN VTW Y V S S S PC A AC ADRIIKTLS KTKNLRLLILV GRLFMWEELEIQD ALKKLK EAGCKLRIMKPQDFEYVWQNFVEQESKAFQPWEDIQENFLYYEEKLAD (SEQ ID NO: 604)

[0831] >F6QZ00_CALJ A/52-221

FKF QFRN VE Y S GRNKTFLC YVVE AQGKQ V QATRG YLEDE A AH AEE AFFNTILP ADP ALRYN VTW YV S S S PC A AC ADRITKTLS KTKNLRLLILV GRLFMWEEPEIQ A ALKKLK EAGCKLRIMKPQDFEYIWQNFVEQESKAFQPWEDIQENFLYYEEKLAD (SEQ ID NO: 605)

[0832] >G3TH88_LOXAF/52-22l

FKF QFRN VE Y S GRNKTFLC YVVE AQGKQ V Q AS RG YLEDE A AH AEE AFFNTILP ADP ALRYN VTW YV S S S PC A AC ADRIIKTLNKTKNLRLLILV GRLFMWEEPEIQ A ALKKLK EAGCKLRIMKPQDFDYVWQNFVEQESKAFEPWEDIQENFQYYEEKLAD (SEQ ID NO: 606)

[0833] >HOWL56_OTOGA/52-22l

FKF QFRN VE Y S GRNKTFLC YVVE V Q AKQ V Q AS RG YLEDE A AH AEE AFFNTILP ADP ALKYNVTWYV S S SPC AAC ADHIIKTLNKTKNLRLLILV GRLFMWEEPEIQ AALKKLK EAGCKLRIMKPQDFEYIW QNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO:607)

[0834] >L9KUD0_TUPCH/52-22l

FKF QFRN VE Y S GRNKTFLC YVVE AQAKQV Q ATRG YLEDE A AH AEE AFFNTILP ADP ALRYN VTW YV S S S PC A AC ADRIIKTLS KTKNLRLLILV GRLFMWEEPEIQ A ALRKMR EAGCKLRIMKPQDFEYIW QNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 608)

[0835] >G3I9V7_CRIGR/52-22l

FKF QFRN VE Y S GRNKTFLC Y V VE AQS KQ V Q ATRG YLEDEG AH AEE AFFNTILP ADP A LKYNVTWYV S S SPC AAC ADRILKTLS KAKNLRLLILV GRLFMWEEPEV QAALKKLK EAGCKLRIMKPQDFEYIWQNFVEQESKAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 609)

[0836] > AB EC2_MOU S E/52-221

FKF QFRN VE Y S GRNKTFLC YVVE V QS KQAQATQG YLEDEG AH AEE AFFNTILP ADP ALKYN VT W Y V S S S PC A AC ADRILKTLS KTKNLRLLILV S RLFMWEEPE V QAALKKLK EAGCKLRIMKPQDFEYIW QNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 610)

[0837] >F7 AZT 6_HORS E/52-221

FKF QFRN VE Y S GRNKTFLC YVVE AQS KQ V Q AS RG YLEDE A AH AEE AFFNTIMP ADP ALRYN VTW Y V S S S PC A AC ADRIIKTLS KTKNLRLLILV GRLFMWEEPEIQ A ALKKLK EAGCRLRIMKPQDFE YVW QNFVEQES KTFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 611)

[0838] >ABEC2_BOVIN/52-22l

FKF QFRN VE Y S GRNKTFLC YVVE AQS KQ V Q AS RG YLEDETNH AEE AFFN S IMPTDP A LRYM VTW Y V S S S PC A AC ADRIVKTLNKTKNLRLLILV GRLFMWEEPEIQ A ALRKLKE AGCRLRIMKPQDFE YIW QNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 612) [0839] >S 9WK76_C AMFR/64-233

FKF QFRN VE Y S GRNKTFLC YVVE AQS KQ V Q ATRG YLEDE A AH AEE AFFNTIMPTDP ALRYIVTWYV S S SPC AAC ADRIIKTLNKTKNLRLLILV GRLFMWEEPEIQ AALRKLKE AGCKLRIMKPQDFE YVW QNFVEQES KAFEPWEDIQENFLY YEEKLAD (SEQ ID NO: 613)

[0840] >F 1 R VM 1 _PIG/52-221

FKF QFRN VE Y S GRNKTFLC Y VIE AQS KQ V Q ATRG YLEDE A AH AEE AFFNTILP ADP A VRYVVTWYV S S SPC AAC ADRIIKTLNKAKNLRLLILV GRLFMWEEPEIQAALRRLKE AGCRLRIMKPQDFE YVW QNFVEQES KAFEPWEDIQENFLY YEEKLAD (SEQ ID NO: 614)

[0841] >H0V 2E8_C A VPO/56-225

FKF QFRN VE Y S GRNKTFLC YVVEV QS KQ V Q AS RG YLEDE A AH AEE AFFN S VLP ADP ALRYN VTW YV S S S PC A AC ADRIIKTLGKTKNLRLLILV GRLFMWEEPEIQ A ALRKLK EAGCRLRIMKPQDFE YVW QNFVEQES KAFEPWEDIQENFLYYEDKLAD (SEQ ID NO: 615)

[0842] >A0A09lDJF3_FUKD A/59-228

FKF QFRN VE Y S GRNKTFLC YVVEV QS KQ V Q AS RG YLEDE A AH AEE AFFNTILP ADP A LRYS VTWYV S S SPC VAC ADRIVKTLGKTKNLRLLILV GRLFMWEEPEIQ AALRKLKE AGCRLRIMKPQDFE YVW QNFVEQES KAFEPWEDIQENFLY YEEKLAD (SEQ ID NO: 616)

[0843] >G5BNG8_HETG A/56-225

FKF QFRN VE Y S GRNKTFLC YVVEV QC KQ V Q AS RG YLEDE A AH AEE AFFNTILP ADP ALRYN VTW Y V S S S PC V AC ADRIIKTLAKTKNLRLLILV GRLFMWEEPEMQ A ALRKL KEAGCRLRIMKPQDFE YVW QNFVEHES KAFEPWEDIKENFLYYEEKLAD (SEQ ID NO: 617) [0844] >G 1 PHZ8_M Y OLU/58-227

FKF QFRN VE Y S GRNKTFLC Y VIE V QDKQ V Q AS RG YLEDET AH AEE AFFNT V VPTDP A LRYN VTW YV S S S PC V AC ADRIIKMLS KTKNLRLLILV GRLFM WEEP AMQ A ALKKLK EAGCRLRIMKPQDFEYIWQNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 618)

[0845] >GlU7Q3_RABIT/55-224

FKF QFRN VE Y S GRNKTFLC Y V VE V QGKQ V Q ATRG YLEDE A AH AEE AFFNTILP ADP ALRYN VTW Y V S S S PC A AC ADRIIRTLGKTKNLRLLILV GRLFM WEEPEIQ A ALKKLR EAGCRLRIMKPQDFEYVWQNFVEQEAKAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 619)

[0846] >M3WlK8_FELCA/61-230

FKF QFRN VE Y S GRNKTFLC YVVEAQGKQV Q AS RG YLEDE A AH AEE AFFNTILP ADP A VRYNVTWYV S S SPC AAC ADRIVRTLGKTKNLRLLILV GRLFMWEEPD V QA ALRRL KEAGCRLRIMKPQDFE YVW QNFVEQES KAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 620)

[0847] >GlLCS0_AILME/80-249

FKF QFRN VE Y S GRNKTFLC YVVEAQGKQV Q ATRG YLEDE A AH AEE AFFNTILP ADP ALRYT VTWYV S S SPC AAC ADRIARTLGQTKNLRLLILAGRLLLWEEPD VRA ALRRLA EAGCRLRVMKPQDFEYVWQQFVEQEPKAFEPWEDIQENFLYYEEKLAD (SEQ ID NO: 621)

[0848] >M3YQH9_MUSPF/61-230

FKF QFRN VE Y S GRNKTFLC YVVETQAKQV Q ATRG YLEDETNH AEE AFFNTILPS DP A LRYN VTW Y V S S S PC A AC ADRILRTLGKTKNLRLLILV GRLFMWEEPE V Q A ALRKLK EAGCRLRIMKPQDFE YVW QNFVEQES KAFEPW GDIQENFLYYEEKLAD (SEQ ID NO: 622)

[0849] >E2RDL7_CANLF/52-22l

FKF QFRN VE Y S GRNKTFLC YVVEAQGKQV Q AS RG YLEDE A AH AEE AFFNTILPTDP A LRYN VTW Y V S S S PC A AC ADRIIRTLGKTKNLRLLILV GRLFM WEEPE V Q A ALRKLKE AGCRLRIMKPQDFE YVW QNFVEQES KAFQPWEDIQENFLYYEEKLAD (SEQ ID NO: 623)

[0850] >S7PKW6_MYOBR/l 15-291

FYFHFKNCPDHGRNGCYLCYEVKRQRGLPLVGTGVFENEPKHTEICFLNWFKTQQN LS REEKYH VTWFMS WS PCF QC ARQ V VEFLKDHE Y V QLS IF V ARL Y Y S S RPE Y QQGL RS LQG AG AQ V AIMTPDDFA Y CRKVFVHDHKPFRYWKGIYIN S CS LS KTLED (SEQ ID NO: 624)

[0851] >S7NDM8_MYOBR/6- l77

FNSNFKNLDGGCKSTFLCFEVEREDGSVLYQNGVFRNQHAELCFIEWFHEKVLCPDA QYHVTWYISWSPCFECAEQVADFLNENENVDLSISAARLYLCEDEDEQGLQDLVAT G AKV AMM APEDFKY C WDNFV YN GW QFT Y WKN VRRN Y GRLQEKLDE (SEQ ID NO: 625)

[0852] >S7PKW6_MY OBR/281 -406

NSCSLSKTEDILRHAELCFLDWFREKVLCPDAQYHVTWYISWSPCFECAEQVADFLN ENENVDLSISAARLYLCEDEDEQGLQDLVATGAKVAMMAPEDLTAKMVPDETPMF DPS (SEQ ID NO: 626)

[0853] >T0NHJ8_CAMFR/406-573

FSFHFKNLMFAGRNCTYLCYQVKREHCSPVPDKGVFQNEPCHAELCFLSWFNKRLS PDECYHITWFMSWSPCFACTEQVAKFLEKNRNVRLSIFAARLYYFWQPAVQQGLRR LHGV GAC VGIMS Y QDFKY C WENFVYNRMPFKPWEKQCEN S KILVTKLEE (SEQ ID NO: 627)

[0854] >F7DDEl_HORSE/l2- l79

FSFHFRNLEFAGRNCSYICYRVEGLSGSPGSEQGVFLNECRHAELCFLHWFRGRLSPD EYYHVTWFISWSPCSNCAREVAEFLKRHRNVELSIFAARLYYWQRNKPDLRNLCSS GAQLAIMFYQDFRYCWDNFVHNGREFIPWEKINVNSRLLATNLEE (SEQ ID NO: 628) [0855] >F7B644_HORSE/l90-346

FSFHFRNLKFAGRKCSYLCYRVEGLSGSPGSEQGVFLNERRHAELCFLDWFRVRLSP DEYYRVTWFISWSPCSYCAREVADFLKQYRNVKLSIFAARLYYCRDHAQGLRSLCSS GAQLAIMFFWDFRY CWDNFVHN GRDFIPW (SEQ ID NO: 629)

[0856] >FlMP6l_BOVIN/29-l99

FYFQFCNLLYARRNCSYICYKVERKYHSRAFDWGVFHNQRCHTELRFLSWFHAEKL RPNERYHITWFMSWSPCMKCAKEVADFLGRHQNVTLSIFTARLYNFQEEGSRQGLL RLSDQGAHVDIMSYQEFKYCWKKFVNSRRPFRPWKKLYRNYQRLVEELED (SEQ ID NO: 630)

[0857] >L5K8J0_PTEAL/256-426

FFFEFQNLLY AGRKS S YLCFQ VERQHS S P VS D W G VFEN QP YH AELCFLNWFRAEKLS P YEH YD VT WFLS W S PC S TC AEEIAIFLS NHKN VRLS IF V S RIY YFWKP AFRQGLQELD HLGVQLDAMSFDDFKYCWENFVDNGMPFRCWKKVHRNYKFVLRKLNE (SEQ ID NO: 631)

[0858] >M3W3RO_FELC A/20- 189

FRFHFPNLLYAGRKLCYLCFQVETDYFSCDSDRGVFRNKRCHAEQCFLSWFRDQYP CRDEYYNVTWFLSWSPCPTCAEEVVEFLEEYRNLTLSIFTSRLYYFYHPNYQQGLRK LWDAGVQLDIMSCDDFEHCWDNFVDHGMRFQRRNLLKDYDFLAAELQE (SEQ ID NO: 632)

[0859] >G 1 LWD3_AILME/20- 186

FFFQFPSLCYAGRKFCYLCFQVGRGHPSDWGVFRNKPYHAESCFLSWFRAQNLSPDE DYHVTWFSSWSPCHTCADEVVEFLGQYRHVTLSIFAARLYYFWDPPFQNGLRRLQS AGVRLDIMSFADYKRCWENFVDHGMRFQSRNLLRHRDLLASRLEN (SEQ ID NO: 633)

[0860] >L9KTG4_TUPCH/9-l77

FYFHFQNLLFAGRNTTFLCCRVDKERHGTVLVSGVFTHQYHAESHFLLWFQKNFLSL DKDF Q VTW YLS WS PCP AC AKQ V ADFLA VHRN V S LTIFS ARLY YFWDPEFRD GLHRL FEKGARVAIMSPKDFENCWEGFVFNGRDFRPWDNMVENYQSLRITLQE (SEQ ID NO: 634)

[0861] >I3M955_ICTTR/l4- l86

FLFHFRNLRWAGRNNTFLCYQVDRERDSTVIHRGVFKTQRLHAELCFLYWLHDYPL FPDQHFHITWFIS WS PC S DC AQQ V A AFLAS HS NLS LT V YT ARLY YFWKHS Y QEGLRA LQREGARVEIMSIREFEHCWENFVYPGRPFRPWKNLFRNYYFQVKKLQK (SEQ ID NO: 635)

[0862] >G312 J 2_CRIGR/40-210

FYFHFKNLRYALRKNTFLCYEVNRECNELVLCQGVFRKENLHAEVCFLYWFHTQVL PPDEKYKITW Y VS WS PCNEC AEKV AS FLDTHRNLS LAIF S S RLY YFWDPD Y QDKLRR LNQAGAQIAAMDFPEFEKCWNKFVDNGKSFRPWKRLKINFRFQDNKLRD (SEQ ID NO: 636)

[0863] >F7EWS7_RAT/40-2l0

FYFHFKNVRY AGRKNNFLC YE VN GDC ALPVLRQGVFRKQHIHAELCFIYWFHDKVL S PMEEFKVTW YMS WS PC S KC AEQ V ARFLA AHRNLS LAIF S S RLY Y YLNPN Y QQKLC RLIQEGVHVAAMDLPEFKKCWNKFVDNGQPFRPWMRLRINFSFYDCKLQE (SEQ ID NO: 637)

[0864] >ABEC3_RAT/26-l95

FKFHFKNLRYADRKDTFLCYEVTRDCDSPVLHHGVFKNKNIHAEICFLYWFHDKVL SPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRL V QEGAQV AAMDLYEFKKC WKKFVDN GRRFRPWKKLLTNFRY QDS KLQE (SEQ ID NO: 638)

[0865] >ABEC3_MOUSE/26- 195

FKFHFKNLGYAGRKDTFLCYEVTRDCDSPVLHHGVFKNKNIHAEICFLYWFHDKVL SPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRL V QEGAQV AAMDLYEFKKC WKKFVDN GRRFRPWKRLLTNFR Y QD S KLQE (SEQ ID NO: 639) [0866] >F6XHA6_MACMU/2l3-383

FYFHFENLQKAGRNETWLCFAVEIKQHSTVWKTGVFRNQHCHAERCFLSWFCDNTL SPKKNY QVTWYISWSPCPECAGEVAEFLATHSNVKLTIYTARLYYFWDTDY QEGLR SLSEEGASMEIMGYEDFKYCWENFVYNGEPFKPWKGINTNFRFLERRLWK (SEQ ID NO: 640)

[0867] > AO A096NK50_P AP AN/213-383

FYFHFKNLRTADRNETWLCFAVEIKQRSTVWRTGVFRNQHCHAERCFLSWFRDNPL S PKKN Y Q VTW YTS WS PCPEC AGE V AEFLARY S N V QLTIYT ARLY YFWDTD Y QEGLR SLSEEGASVEIMGYEDFKYCWENFVCDGEPFKPWKGINTNFRFLERHLRK (SEQ ID NO: 641)

[0868] >G3 RD21 _GORGO/200- 370

FYFHFKNLRKAGRNESWLCFTMEVKHHSPVWKRGVFRNQHCHAERCFLSWFCDDI LSPNTNYQVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGL RSLNQEGASVKIMGYKDFKYCWENFVYNDEPFKPWKGLKYNFLFLDSKLQE (SEQ ID NO: 642)

[0869] >A0A096NK49_PAPAN/ 167-338

FYFHFKNLRKAGRNESLLCFTMEIKQCSTVWKRGVFRNQHCHAERCFLSWFCEDILS PNTDY QVTWYTSWSPCLDCAGEVAEFLARHSNVKLAIFAARLYYFWDTDY QQGLR SLSEEGASVQIMGYEDFKYCWENFVYNDEPFKPWKGLKYNFLFLDSKLQE (SEQ ID NO: 643)

[0870] >A0A0D9R238_CHLSB/ 197-367

FYFHFKNLRKAGRNESWLCFTMEIKQCSTVWKRGVFRNQRCHAERCFLSWFCEDIL S PNTDY Q VT W YTS WS PCLDC AGE V AEFLARHS N VKLTIYT ARLY YFWHTD Y QQGL RSLSEEGASVEIMGYEDFKCCWENFVYNDEPFKPWKGLKYNFLFLDSKLQE (SEQ ID NO: 644) [0871] >G3RRB4_GORGO/39-209

FYFQFKNLWEADRNETWLCFTVEGKRRSVVWKTGVFRNQHCHAERCFLSWFCDDI LSPNTNYQVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGL RSLNQEGASVKIMGYKDFKYCWENFVYNDEPFKPWKGLKYNFLFLDSKLQE (SEQ ID NO: 645)

[0872] >G3RUE8_GORGO/l7- l87

FYFQFKNLWEADRNETWLCFTVEGKRRSVVWKTGVFRNQHCHAERCFLSWFCDDI LSPNTNYEVTWYTSWSPCPECAGKVAEFLARHSNVNLTIFTARLCYFWDTDYQEGL RSLNQEGASVKIMGYKDFVSCWKNFVYSDEPFKPWKGLQTNFRLLKRRLRE (SEQ ID NO: 646)

[0873] >Q6ICH2_HUMAN/l7- l99

FYDNFENEPILGRSYTWLCYEVKIRGRSNLWDTGVFRGEHCHAERCFLSWFCDDILS PNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCS LSQEGASVKIMGYKDFVSCWKNFVYSDEPFKPWKGLQTNFRLLKRRLRE (SEQ ID NO: 647)

[0874] >ABC3D_HUMAN/2l3-383

FYFHFKNLLKAGRNESWLCFTMEVKHHSAVRKRGVFRNQHCHAERCFLSWFCDDIL SPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCS LSQEGASVKIMGYKDFVSCWKNFVYSDEPFKPWKGLQTNFRLLKRRLRE (SEQ ID NO: 648)

[0875] >ABC3C_GORGO/l7- l87

FYFQFKNLWEADRNETWLCFTVEGKRRSVVWKTGVFRNQHCHAERCFLSWFCDDI LS PNTN Y Q VTW YTS W S PCPEC AGE V AEFLARHS N VNLTIFT ARLY YF QDTD Y QEGLR SLSQEGVAVKIMDYKDFKYCWENFVYNDEPFKPWKGLKYNFRFLKRRLQE (SEQ ID NO: 649)

[0876] >ABC3C_HUMAN/l7-l 87

FYFQFKNLWEADRNETWLCFTVEGKRRSVVWKTGVFRNQHCHAERCFLSWFCDDI LSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGL RSFSQEGVAVEIMDYEDFKYCWENFVYNNEPFKPWKGFKTNFRFFKRRFRE (SEQ ID NO: 650)

[0877] >H2P4E8_PONAB/ 17-187

FYFQFKNLWEADRNETWLCFTVEVKHHTVVWKRGVFRNQHCHAESCFLSWFCNNI LSPNTNYRVTWYASWSPCPECAGEVAKFLARHSNVKLTIFTARLYYFQNPYYQQGL RRLSQEGVAVGIMDYEDFKDCWENFVYSDEPFKPWKGINTNFRLLKKRLRE (SEQ ID NO: 651)

[0878] >A0A096NK46_PAPAN/ 17-187

FYFQFKNLWEANRNETWLCFTVEVKQRSTVWERGVFQNQHCHAERCFLSWFCEDIL SPNTDYQVTWYTSWSPCPECAGEVAEFLARHNNVMLTIYTARLYYSQDPNYQQGLR SLSEKGVSVKIMDYEDFKYCWENFVYNDEPFKPWKGLKYNFLFLDSKLQE (SEQ ID NO: 652)

[0879] >A0A0D9R255_CHLSB/ 17-187

FYFQFKNLREANRNETWLCFTVEGRHHLTVWKTGVFRNQHCHAEKCFLSWFCKNIL SPNTDYQVTWYTSWSPCPECAREVAKFLARHNNVMLTIYTARLYYSQCPNYQQGLR S LS EKG V S VEIMD YEDFKY C W QNF V YDGEPFKPWKGLKT S FRFLKRCLRE (SEQ ID NO: 653)

[0880] >F6XHB 3_MACMU/37-207

FYFQFKNLREANRNETWLCFAVEIKQRSTVLKTGVFRNQHCHAERCFLSWFCEDILS PNTDYQVTWYTSWSPCLDCAGEVAKFLARHNNVMLTIYTARLYYSQYPNYQQGLR S LS EKG V S VKIMD YEDFKY C WE KFV YDGEPFKPWKGLKT S FRFLKRRLRE (SEQ ID NO: 654)

[0881] >H0VE76_C A VPO/ 1-121

LHAELSFLSWFHDTELSFDENYKVTWYMSWSPCPECAKEIVTFLDNHHNVTLTIYVA RLY YHWNPT YKEGLR ALV QGGTRLYTM AFPEFEDC W S LFVNETFRPWENFHKCC S L QDKTLQK (SEQ ID NO: 655) [0882] >G5 A YU 4_HETG A/ 17-183

FYFHFENLPYAGRNKTFLCYEVKRRDNKLHKGVVQNQLSRTELSFISCFHATELCLD ETYKVTWYISWSPCVECAEEIVKFLANHRNVFLTVFIARLYYYREHTFKEGLQALDN GG V QMHMMCLQDFKDC W S LFV S ETFRPWKGLRK Y YLF QNKTLKQ (SEQ ID NO: 656)

[0883] >H0 VZF7_C A VPO/ 17-183

FYFHFENLPNAGRHKTFLCFEVKNNELHKGFFLNQLHAELRFLSWLHDTCLCPYEYY QVTWYMSWSPCVECAEELTTFLAGHRNVTLTIYVARLYYHQFPVYKNRLQALIKKG AT VKVMFFRDFLY C WRRFV YNFKRF YD WPNLHKN S LH Y YKTLQH (SEQ ID NO: 657)

[0884] >G5 A YU3_HETGA/ 12-181

FYFHFENLPDPGWNKTFLCYEVKRQDQKLRKGVFQNQPLHAELRFLSWFHDTLLCP LGSYQVTLYVSWSPCSECAEELTTFLAGHRNVTMTIYVAQLYYCNKSPNREGLKILI AED ARLRVMF YDEFLY C WRNFVKN YNNFDPW S LLDEN S RYHNRILQN (SEQ ID NO: 658)

[0885] >G5 A YU 5_HETG A/20- 189

FRFYFRNLRCAGRNKTFLCYEVKRRDNKLHKGVVLNQPLHAELRFLSWFHDTLLCP LGSYQVTLYVSWSPCSECAEELTTFLAGHRNVTMTIYVAQLYYCNKSPNREGLKILI AED ARLRVMF YDEFLY C WRNFVKN YNNFDPW S LLDEN S RYHNRILQN (SEQ ID NO: 659)

[0886] > A0 A09lEM42_FUKD A/42-211

FKFHFENLPNPGWHKTFLC YTIES QKNRLRN GVFQN QRLHAELRFLS WFHDNWLCP GNSYRVTFYMSWSPCSECAEELTTFLAGHRNVTLTIFFSKLYYCDDSSNREGLKTLA AGGARLFVMFDTDFS Y CWTNFVNC YNYFEPWPLLDDN S KY CNRILQK (SEQ ID NO: 660) [0887] >A0A09lEM42_FUKD A/225-394

FYFHFNNLCFAGRNKTFLC YTIES KKNRLRN GVFQN QRLHAELRFLS WFHDNWLCP GNSYQVTLYMSWSPCSECAEELTTFLAGHRNVTLTIFFSKLYCCDDSPHREGLKTLA AGGARLFVMFDTDFSYCWTNFVNCYNYFEPWPLLDDNSKYYKSILQK (SEQ ID NO: 661)

[0888] >H0X YD2_OTOG A/ 17- 183

FNCHFNNRP YLRRNDTWLCFE VKT S S NS PGFY S G VFQN QPWHTELCILTW ARPMLS HHHFYQITWYMSWSPCANCAWQVAAFLATHENVSLTIYTAHIYYMWRQDYRQGM LRMIEEGTR V YIMF S KEF QHC WENF VDHGMC WNR VKKN YEFLVT QLNE (SEQ ID NO: 662)

[0889] >H0X YD2_OTOG A/ 192-348

FYN QFNNTP VPGRTDT WLCFE VKNN S NS PGFHRGS S ENRH AE V GFLT WF QKEMPPN HHYEVTWYIS WSPC VHC AWHVVNFLTSNPNMTLTIFAARLYYIYHPEEGTKVHIV S L KEFKY CW AKLVYN GMRFMPW Y QFNENY QLLVTQLKK (SEQ ID NO: 663)

[0890] >A0A096N7U5_PAPAN/ 17- 167

FY YNFENRS VLGWNTT WLCHKVKTKDPS KLLDTRIF GGQHHPEMRFLD WFC KYIS R S PCPE Y AEKV AEFLVKN GKVTLTIF V AHLY YFWEPD Y QN G VRPH AS MTIMN YDEF Q HCWDKFVYNRMLFDPWKQLNTNYALLHSMLGE (SEQ ID NO: 664)

[0891] >H9KW44_CALJA/2-l32

QHHPEMRFLHWFRKWKLHSDQEYEVTWFVSWSPCPVCARNVAEFLTEDGKVTLTIF VARLYYFWIPHYREELRRLCQPRATMKIMSYGEFQHCWDKFVDNRLYKPWNKLPK HYTLLHITLGE (SEQ ID NO: 665)

[0892] >ABC3 G_PAPAN/ 17- 191

FFYNFNNRPILRRNTVWLCYEVKTRGPSMPWDAKIFRGQKYHPEMRFLHWFRKWR QLHRDQEYEVTWYVSWSPCTGCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQEA LRVLCQPHATMKIMNYNEFQHCWNKFVRGREPFEPWENLPKHYTLLHATLGE (SEQ ID NO: 666) [0893] >G7PFK4_MACFA/l3-l87

FFYNFNNRPILRRNTVWLCYEVKTRGPSVPWGTKIFRGQKYHPEMRFLRWFHKWRQ LHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQA LRILCQPHATMKIMNYNEFQDCWNKFVDGGKPFKPRNNLPKHYTLLQATLGE (SEQ ID NO: 667)

[0894] >A0A0D9R229_CHLSB/28-202

FVYYFNNRPILGRNIVWLCCEVKTKDPSGPLDANIFQGEKDHPEMKFLHWFRKWRQ LHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPHYQEAL RILCQPHATMKIMNYNEFQHCWNEFVDGGKPFKPRKNLPKHYTLLHATLGE (SEQ ID NO: 668)

[0895] >ABC3 G_M ACMU/ 10-184

FVSNFNNRPILGLNTVWLCCEVKTKDPSGPLDAKIFQGKKYHPEMRFLRWFHKWRQ LHHDQEYKVTWYVSWSPCTRCANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQA LRILCQPHATMKIMNYNEFQDCWNKFVDGGKPFKPRNNLPKHYTLLQATLGE (SEQ ID NO: 669)

[0896] >H2P4E9_PONAB/31-202

FS YNFKNRPILRRNT VWLC YE VKTKGPS RPLD AKIFRGQKNHPEMRFFHWF S KWRT LHRDQECEVTWYMSWSPCTKCTRNVATFLAEDPKVTLTIFVARLYYFWDPDYQEAL RSLCRPRANMKIMNYDEFQHCWNKFVYSRELFEPWNNLPKYYIPLHKV (SEQ ID NO: 670)

[0897] >ABC3 G_HUMAN/ 17-190

FS YNFYNRPILRRNT VWLC YE VKTKGPS RPLD AKIFRGQKYHPEMRFFHWF S KWRK LHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEAL RSLCQPRATMKIMNYDEFQHCWSKFVYSRELFEPWNNLPKYYILLHIMLG (SEQ ID NO: 671) [0898] >ABC3 G_GORGO/ 17-190

FS YNFNNRPILRRNT VWLC YE VKTKDPS RPLD AKIFRGQKYHPEMRFFHWF S KWRK LHRDQEYEVTWYISWSPCTKCTRNVATFLAEDPKVTLTIFVARLYYFWDQDYQEAL RSLCQPRATMKIMNYDEFQHCWSKFVYSRELFEPWNNLPKYYMLLHIMLG (SEQ ID NO: 672)

[0899] >G3RD2l_GORGO/l7-l86

FSYNFNNRPILRRNTVWLCYEVKTKGPSRPLDAKIFRGQQYHAEMCFLSWFCGNQLP AYKCFQITCFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALRRL RQAGARVKIMDDEEFA Y C WENFVY S GQPFMPWHKFDDNY AFLHRTLKE (SEQ ID NO: 673)

[0900] >ABC3F_HUMAN/l7-l86

FSYNFYNRPILRRNTVWLCYEVKTKGPSRPLDAKIFRGQEHHAEMCFLSWFCGNQLP AYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCR LS Q AG AR VKIMDDEEFA Y C WENFVY S GQPFMPW YKFDDN Y AFLHRTLKE (SEQ ID NO: 674)

[0901] >A0A096NK49_PAPAN/ 1 - 153

WFCFEVKTRGPSMPWDAKIFRGQQYHAEMCFLSRFCGNQLPAYKRFQITWFVSWTP CPDCVVKVAEFLAEHPNVTLTISAARLYYYWETDYRRALCRLRQAGAHVKIMDNEE FAY CWENFVYNGQSFMPWDKFDDNY AFLHCKLKE (SEQ ID NO: 675)

[0902] >A0A096NK45_PAPAN/ 17-187

FYYHFENKPILGRSYTWLCYEVKIKDPSKLWYTGVFRGQEHHAEMCFLSRFCGNQL PAYKRFQITWFVSWNPCPDCVAKVIEFLAEHPNVTLTISTARLYYYWGRDWQRALC RLRQAGARVKIMDYEEFAYCWENFVYNGQSFMPWDKFDDNY AFLHCKLKE (SEQ ID NO: 676)

[0903] >F7 FX J 2_M ACMU/2- 130

EHHAEMCFLSRFCGNQLPAYKRFQITWFVSWNPCPDCVAKVIEFLAEHPNVTLTIST ARLYYYWGRDWQRALCRLRQAGARVKIMDYEEFAYCWENFVYNDQSFMPWYKF NDNY AFLHRMLKE (SEQ ID NO: 677)

[0904] > AO A0D9R238_CHLS B/17-183

FYYNFENEPILGRRYTWLC YE VKIKDPS KLWDTGVFPGQQYHAEM YFLS WFCGN QL PAYKHFQITWFVSWNPCPDCVAKVTEFLAEHRNVTLTISAARLYYYWGKDWRRAL CRLHQAGARVKIMDDEEFAYCWENCVYNGQPFMPWDKFDDNYAFLHLKLKE (SEQ ID NO: 678)

[0905] >A0A096NK50_PAPAN/ 17-199

FDYNFENEPILGRSYTWLCYEVKIEDPSKLWDTGVFQGQQYHAEMCFLSRFCGNQLP AYKRFQITWFVSWNPCPDCVAKVTEFLAEHPNVTLTISAARLYYYWGRDWRRALRR LHQ AG AR VKIMD YEEFA Y C WENFV YN GQS FMPWDKFDDN Y AFLHC KLKD (SEQ ID NO: 679)

[0906] >F6XH A6_MACMU/ 17-199

FNYNFENEPILGRSYTWLCYEVKIKDPSKLWDTGVFRGQQYHAEMCFLSWFCGNQL PAYKRFQITWFVSWNPCPDCVAKVTEFLAEHPNVTLTISAARLYYYWGKDWRRALR RLHQAGALVKIMD YEEFA YCWENFVYNGQSFMPWDKFDDNYASLHCKLKE (SEQ ID NO: 680)

[0907] >A0A0D9R289_CHLSB/l7-l87

FYYHFENEPILGRSYTWLCYEVKIKDPSKLWDTGVFRGQEHHAEMCFLSWFCGNQL PAHKRFQITWFVSWTPCPDCVAKVAEFLAEYPNVTLTISAARLYYYWETDYRRALC RLRQAGARVKIMD YEEFA YCWENCVYNGQPFMPWYKFDDNYAFLHHKLKE (SEQ ID NO: 681)

[0908] >GlRYY7_NOMLE/l7-l87

FYYNFENEPILRRSYTWLCYEVKIKDPSKLWDTGVFRGQEYHAEMCFLSWFCGNQL PAYKRFQITWFVSWTPCPDCVAKVAVFLAEHPNVTLTISAARLYYYWEKDWQRALC RLS Q AG AR VKIMD YEEFEY C WENFV YN GEPFMPW YKFDDN Y AFLHHTLKE (SEQ ID NO: 682) [0909] >H2P4E7_PONAB/ 17-187

FYYDFENEPILRRNYTWLC YE VKIKDPS KLWDTGVFRGQEHHAEMCFLS WFCGN QL SAYERFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTVSAARLYYYWERGYRRALR RLRQAGAHVKIMDYEEFAYCWENFVYNGQPFMPWYKFDDNYAFLHHTLKE (SEQ ID NO: 683)

[0910] >H2QLP5_PANTR/ 17-187

FYYNFENEPILGRSYTWLCYEVKIRGHSNLWDTGVFRGQEHHAEMCFLSWFCGNQL SAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLYYYWERDYRRALC RLS Q AG AR VKIMDDEEFA Y C WENFV YN GQPFMPW YKFDDN Y AFLHRTLKE (SEQ ID NO: 684)

[0911] >ABC3D_HUMAN/l7-l99

FYDNFENEPILGRSYTWLCYEVKIRGRSNLWDTGVFRGPENHAEMCFLSWFCGNRL PANRRFQITWFVS WNPCLPC VVKVTKFLAEHPNVTLTIS AARLYYYRDRDWRW VLL RLHKAGARVKIMDYEDFAYCWENFVCNGQPFMPWYKFDDNYASLHRTLKE (SEQ ID NO: 685)

[0912] >ABC3 B_HUMAN/ 17-187

FYDNFENEPILGRSYTWLCYEVKIRGRSNLWDTGVFRGQQYHAEMCFLSWFCGNQL PAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCR LS Q AG AR VTIMD YEEFA Y C WENFV YN GQQFMPW YKFDEN Y AFLHRTLKE (SEQ ID NO: 686)

[0913] >G3 SFT2_GORGO/ 17-187

FYDNFENEPILGRSYNWLCYEVKIRGRSNLWNTGVFRGQEHHAEMCFLSWFCGNQL PAYKCFQITWFVSWTPCPDCVAKLAEFLAEYPNVTLTISTARLYYYWERDYRRALCR LS QAGARMKIMD YEEC A Y C WENFVYKGQQFMPW YKFDENY AFLHHTLKE (SEQ ID NO: 687)

[0914] Sequences 6. Sequences of Claims: [0915] NLS sequences:

KRT ADGS EFES PKKKRKV (SEQ ID NO: 1)

KRT ADGS EFEPKKKRKV (SEQ ID NO: 2)

[0916] Cas9 for BE4max, AncBE4max and ABEmax:

[0917] DKKY S IGLAIGTN S V GW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLV EEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKAL VRQQLPEKYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNRE DLLRKQRTFDN GS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYV GPLA RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSL LYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDR EMIEERLKTY AHLFDDKVMKQLKRRRYTGW GRLSRKLIN GIRDKQSGKTILDFLKSD GFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKV VDELVKVMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRS DKNRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYK VREINN YHH AHD A YLN A V V GT ALIKK YPKLES EF V Y GD YKV YD VRKMIAKS E QEIGKAT AKYFFYSNIMNFFKTEITLAN GEIRKRPLIETN GETGEIVWDKGRDFATVR KVLS MPQ VNIVKKTE V QT GGF S KES ILPKRN S DKLIARKKD WDPKKY GGFD SPT V AY S VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLP KY S LFELEN GRKRML AS AGELQKGNELALPS KY VNFLYLAS H YEKLKGS PEDNEQK QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT LTNLGAPAAFKYFDTTIDRKRYTSTKE VLD ATLIHQS ITGLYETRIDLS QLGGD (SEQ ID NO: 3)

[0918] rAPOBEC:

[0919] S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKETCLLYEINW GGRHS I WRHT S QNTNKH VE VNFIEKFTTERYFCPNTRC S GG WFLS W S PCGECS RAITEFLS R YP HVTLFIYIARLYHHADPRNRQGLRDLIS S GVTIQIMTEQES GY C WRNFVNY SPSNE AH WPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWA TGLK (SEQ ID NO: 4)

[0920] Anc689 APOBEC:

[0921] S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKETCLL YE IKW GT S HKI WRHSSKNTTKHVEVNFIEKFTSERHFCPSTSCSITWFLSWSPCGECSKAITEFLSQHPN VTLVIY V ARLYHHMD QQNRQGLRDLVN S G VTIQIMT APE YD Y C WRNF VN YPPGKE AHWPRYPPLWMKLYALELHAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI LWATGLK (SEQ ID NO: 5) [0922] Anc687 APOBEC:

[0923] S S ET GP V A VDPTLRRRIEPHEFE VFFDPRELRKE ACLLYEIKW GT S HKI WRN S GKNTTKH VE VNFIEKFT S ERHFCPS IS C S ITWFLS W S PC WECS KAIREFLS QHPN VTLVIYVARLFQHMDQQNRQGLRDLVNSGVTIQIMTASEYDHCWRNFVNYPPGKEA HWPRYPPLWMKLY ALELHAGILGLPPCLNILRRKQPQLTFFTIALQSCHY QRLPPHIL WATGLK (SEQ ID NO: 6)

[0924] Anc686 APOBEC:

[0925] S S ET GP V A VDPTLRRRIEPEFFNRN YDPRELRKET YLLYEIKW GKES KI WRHTSNNRTQHAEVNFLENFFNELYFNPSTHCSITWFLSWSPCGECSKAIVEFLKEHP N VNLEIY V ARLYLCEDERNRQGLRDLVN S G VTIRIMNLPD YN Y C WRTF V S HQGGDE DYWPRHFAPWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL WATGLK (SEQ ID NO: 7)

[0926] Anc655 APOBEC:

[0927] S S ET GP V A VDPTLRRRIEPF YF QFNNDPR ACRRKT YLC YELKQDGS T W VWKRTLHNKGRHAEICFLEKISSLEKLDPAQHYRITWYMSWSPCSNCAQKIVDFLKE HPHVNLRIYVARLY YHEEERY QEGLRNLRRS GV S IRVMDLPDFEHCWETFVDN GGG PFQPWPGLEELNSKQLSRRLQAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPH ILWATGLK (SEQ ID NO: 8)

[0928] Anc733 APOBEC:

[0929] S S ET GP V A VDPTLRRRIEPFHF QFNNDPR A YRRKT YLC YELKQDGS TW VLDRTLRNKGRHAEICFLDKINSWERLDPAQHYRVTWYMSWSPCSNCAQQVVDFL KEHPHVNLRIFAARLYYHEQRRYQEGLRSLRGSGVPVAVMTLPDFEHCWETFVDHG GRPFQPWDGLEELNSRSLSRRLQAGILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLP PHILWATGLK (SEQ ID NO: 9)

[0930] ETGI domain:

[0931] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES TDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 10)

[0932] 32 a.a. linker:

[0933] SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 11)

[0934] BE4max:

[0935] MKRT ADGS EFES PKKKRKV S S ET GP V A VDPTLRRRIEPHEFE VFFDPRE LRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLS WSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQE SGY CWRNFVNYSPSNEAHWPRYPHLWVRLYVLELY CIILGLPPCLNILRRKQPQLTF FTIALQSCHYQRLPPHILWATGLKSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK Y S IGLAIGTN S V GW A VITDE YK VPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET AE AT RLKRT ARRR YTRRKNRIC YLQEIF S NEM AKVDDS FFHRLEES FLVEEDKKHERHPIF G NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPD N S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLIAQLPGEKKN GLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN G

SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWM TR

KS EETITPWNFEE V VD KG AS AQS FIERMTNFDKNLPNEKVLPKHS LLYE YFT V YNELT

KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AH

LFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH

DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK

PENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYL

YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN

VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETR

QITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYH

H AHD A YLN A V V GT ALIKKYPKLES EF V Y GD YKV YD VRKMIAKS EQEIGKAT AKYFF

YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIV K

KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG

KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG RK

RMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLD

EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF KYF

DTTIDRKRYTSTKE VLD ATLIHQS ITGLYETRIDLS QLGGDS GGS GGS GGS TNLSDIIEK

ET GKQLVIQES ILMLPEE VEE VIGNKPES DIL VHT A YDES TDEN VMLLT S D APE YKPW

ALVIQDS N GENKIKMLS GGSGGSGGS TNLS DIIEKETGKQLVIQES ILMLPEE VEE VIG

NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRT

ADGSEFEPKKKRKV (SEQ ID NO: 12)

[0936] AncBE4max 689:

[0937] MKRT ADGS EFES PKKKRKV S S ET GP V A VDPTLRRRIEPHEFE VFFDPRE LRKETCLLYEIKWGTSHKIWRHSSKNTTKHVEVNFIEKFTSERHFCPSTSCSITWFLS W SPCGECS KAITEFLS QHPNVTLVIY VARLYHHMDQQNRQGLRDLVN S GVTIQIMT A PEYD Y C WRNFVNYPPGKE AHWPRYPPLWMKLY ALELHAGILGLPPCLNILRRKQPQ LTFFTIALQS CH Y QRLPPHILW ATGLKS GGSSGGSSGS ETPGTS ES ATPES SGGSSGGS DKKY S IGLAIGTN S V GW A VETDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET A EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK VMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKY S LFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGST NLS DIIEKET GKQLVIQES ILMLPEE VEE VIGNKPES DILVHT A YDES TDEN VMLLT S D APE YKPW ALVIQDS N GENKIKMLS GGSGGSGGS TNLS DIIEKETGKQLVIQES ILMLPE E VEE VIGNKPES DILVHT A YDES TDEN VMLLT S D APE YKPW ALVIQDS N GENKIKML S GGS KRT ADGS EFEPKKKRKV (SEQ ID NO: 13)

[0938] AncBE4max687:

[0939] MKRT ADGS EFES PKKKRKV S S ET GP V A VDPTLRRRIEPHEFE VFFDPRE LRKE ACLLYEIKW GT S HKIWRN S GKNTTKH VE VNFIEKFT S ERHFCPS IS C S GG WFLS W SPCWECS KAIREFLS QHPNVTLVIYVARLFQHMDQQNRQGLRDLVN S GVTIQIMT A SEYDHCWRNFVNYPPGKEAHWPRYPPLWMKLYALELHAGILGLPPCLNILRRKQPQ LTFFTIALQS CH Y QRLPPHILW ATGLKS GGSSGGSSGS ETPGTS ES ATPES SGGSSGGS DKKY S IGLAIGTN S V GW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET A EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL NPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKY KEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK VMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQL QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKY S LFE LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGST NLS DIIEKET GKQLVIQES ILMLPEE VEE VIGNKPES DILVHT A YDES TDEN VMLLT S D APE YKPW ALVIQDS N GENKIKMLS GGSGGSGGS TNLS DIIEKETGKQLVIQES ILMLPE E VEE VIGNKPES DILVHT A YDES TDEN VMLLT S D APE YKPW ALVIQDS N GENKIKML S GGS KRT ADGS EFEPKKKRKV (SEQ ID NO: 14) [0940] TadA WT:

[0941] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGW NRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGR VVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIK AQKKAQSSTD (SEQ ID NO: 15)

[0942] Staphylococcus aureus TadA:

[0943] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLR ETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYG ADDPKGGCS GS LMNLLQQS NFNHRAIVDKG VLKE AC S TLLTTFFKNLR ANKKS TN

(SEQ ID NO: 16)

[0944] Bacillus subtilis TadA:

[0945] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQ RS IAH AEMLVID

[0946] E AC KALGTWRLEG ATLY VTLEPCPMC AG AWLS R VEK V VF G AFDPK GGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

(SEQ ID NO: 17)

[0947] Salmonella typhimurium (S. typhimurium) TadA:

[0948] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLV HNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCA GAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDF FRMRRQEIKALKKADRAEGAGPAV (SEQ ID NO: 18)

[0949] Shewanella putrefaciens (S. putrefaciens) TadA:

[0950] MDE YWMQVAMQM AEKAEAAGE VPV GA VLVKDGQQIATGYNLS IS Q HDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGAR DEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQ RAQQGIE (SEQ ID NO: 19)

[0951] Haemophilus influenzae F3031 (H. influenzae) TadA:

[0952] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGE GWNLS IVQS DPT AH AEIIALRN G AKNIQN YRLLN S TLY VTLEPCTMC AG AILHS RIKR LVF GAS D YKT G AIGS RFHFFDD YKMNHTLEIT S G VLAEECS QKLS TFF QKRREEKKIE KALLKSLSDK (SEQ ID NO: 20)

[0953] Caulobacter crescentus (C. crescentus) TadA:

[0954] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIAT AGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARI GRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRK AKI (SEQ ID NO: 21)

[0955] Geobacter sulfurreducens (G. sulfurreducens) TadA:

[0956] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGR GHNLREGS NDPS AH AEMIAIRQ A ARRS ANWRLT G ATLY VTLEPCLMCMG AIILARLE RV VF GC YDPKGG A AGS LYDLS ADPRLNHQ VRLS PG V C QEECGTMLS DFFRDLRRRK KAKATPALFIDERKVPPEP (SEQ ID NO: 22)

[0957] TadA 7.10:

[0958] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR AIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVF GVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ KKAQSSTD (SEQ ID NO: 23)

[0959] ABEmax:

[0960] MKRT ADGS EFES PKKKRKVMS E VEF S HE Y WMRH ALTLAKR A WDERE VPV GA VLVHNNRVIGEGWNRPIGRHDPT AHAEIM ALRQGGLVMQNYRLID ATLY VT LEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILAD ECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSG GSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVP S KKFKVLGNTDRHS IKKNLIG ALLFDS GET AE ATRLKRT ARRR YTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI NAS G VD AKAILS ARLS KS RRLENLIAQLPGEKKN GLF GNLIALS LGLTPNFKS NFDLA EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPL SASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAI VDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF LDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGW GRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLH EHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD YDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI ARKKD WDPKKY GGFDS PT V AY S VLV V AKVEKGKS KKLKS VKELLGITIMERS S FEK NPIDFLE AKG YKE VKKDLIIKLPKY S LFELEN GRKRMLAS AGELQKGNELALPS KY V NFLYLAS H YEKLKGS PEDNEQKQLF VEQHKH YLDEIIEQIS EF S KRVILAD ANLD KVL SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ SITGLYETRIDLSQLGGDKRTADGSEFEPKKKRKV (SEQ ID NO: 24)

[0961] lO a.a. linker:

[0962] SGGSGGSGGS (SEQ ID NO: 25)

[0963] 4 a. a. linker: [0964] SGGS (SEQ ID NO: 26)

[0965] N-term NLS:

[0966] AAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAG CGGAAAGTC (SEQ ID NO: 27)

[0967] C-term NLS:

[0968] AAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAG GAAAGTC (SEQ ID NO: 28)

[0969] Cas9 BE4max:

[0970] GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTG GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTG CTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTG TTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC GAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG AAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCC AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAG GACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC GCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACA TCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCA AGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGC AGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCT ACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCA AGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACC AGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCC ATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCC CTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAG AAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGG GCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCC CAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTAT AACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC CTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGG AAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTC GACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAA ACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAG AGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGA TGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAG

CTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTG

AAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGC

CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGC

CTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATC

CTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAG

CCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGG

ACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGC

TGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACG

AGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGG

AACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAG

CTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAA

CCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGA

ACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACA

ATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCA

TCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCC

TGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAG

TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCA

GTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCT

GAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGA

GTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAG

CGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCAT

GAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCC

TCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGG

ATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAA

AGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGA

ACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGC

GGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAA

AGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCA

TGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCT

ACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCG

AGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGG

GAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCA

CTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGT

GGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC

CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAA

CAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTT

TACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATC

GACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCAC

CAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGT

GAC (SEQ ID NO: 29)

[0971] Cas9 ABEmax:

[0972] GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTG GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTG CTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTG TTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA

CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT

GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA

CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT

GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA

CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA

CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC

GAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC

AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG

AAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCC

AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAG

GACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC

GCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACA

TCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCA

AGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGC

AGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCT

ACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCA

AGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACA

GAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACC

AGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCC

ATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCC

CTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAG

AAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGG

GCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCC

CAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTAT

AACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTC

CTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGG

AAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTC

GACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACAT

ACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAA

ACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAG

AGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGA

TGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAG

CTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTG

AAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGC

CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGC

CTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATC

CTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAG

CCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGG

ACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGC

TGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACG

AGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGG

AACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAG

CTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAA

CCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGA

ACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACA

ATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCA TCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCC

TGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAG

TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCA

GTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCT

GAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGA

GTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAG

CGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCAT

GAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCC

TCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGG

ATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAA

AGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGA

ACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGC

GGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAA

AGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCA

TGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCT

ACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCG

AGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGG

GAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCA

CTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGT

GGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC

CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAA

CAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTT

TACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATC

GACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCAC

CAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGT

GAC (SEQ ID NO: 30)

[0973] BE4max XTEN linker:

[0974] TCTGGAGGATCTAGCGGAGGATCCTCTGGCAGCGAGACACCAGGA ACAAGCGAGTCAGCAACACCAGAGAGCAGTGGCGGCAGCAGCGGCGGCAGC

(SEQ ID NO: 31)

[0975] ABEmax XTEN linker first:

[0976] TCTGGAGGATCTAGCGGAGGATCCTCTGGAAGCGAGACACCAGGC ACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCCGGAGGATCC

(SEQ ID NO: 32)

[0977] ABEmax XTEN linker second:

[0978] TCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGAGACACCTGGCA CAAGCGAGAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGGGGGGTCA

(SEQ ID NO: 33)

[0979] BE4max 10 a.a. linker 1:

[0980] AGCGGCGGGAGCGGCGGGAGCGGGGGGAGC (SEQ ID NO: 34)

[0981] BE4max 10 a.a. linker 2:

[0982] AGCGGAGGATCCGGAGGATCTGGAGGCAGC (SEQ ID NO: 35) [0983] BE4max 4 a.a. linker:

[0984] TCTGGCGGCTCA (SEQ ID NO: 36)

[0985] Rat APOBEC1:

[0986] TCCTCAGAGACTGGGCCTGTCGCCGTCGATCCAACCCTGCGCCGCC GGATTGAACCTCACGAGTTTGAAGTGTTCTTTGACCCCCGGGAGCTGAGAAAGG AGACATGCCTGCTGTACGAGATCAACTGGGGAGGCAGGCACTCCATCTGGAGGC ACACCTCTCAGAACACAAATAAGCACGTGGAGGTGAACTTCATCGAGAAGTTTA CCACAGAGCGGTACTTCTGCCCCAATACCAGATGTAGCATCACATGGTTTCTGAG CTGGTCCCCTTGCGGAGAGTGTAGCAGGGCCATCACCGAGTTCCTGTCCAGATAT CCACACGTGACACTGTTTATCTACATCGCCAGGCTGTATCACCACGCAGACCCAA GGAATAGGCAGGGCCTGCGCGATCTGATCAGCTCCGGCGTGACCATCCAGATCA TGACAGAGCAGGAGTCCGGCTACTGCTGGCGGAACTTCGTGAATTATTCTCCTAG CAACGAGGCCCACTGGCCTAGGTACCCACACCTGTGGGTGCGCCTGTACGTGCTG GAGCTGTATTGCATCATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGCGGAGAA AGCAGCCCCAGCTGACCTTCTTTACAATCGCCCTGCAGTCTTGTCACTATCAGAG GCTGCCACCCCACATCCTGTGGGCCACAGGCCTGAAG (SEQ ID NO: 37)

[0987] Anc689 APOBEC:

[0988] AGCAGTGAAACCGGACCAGTGGCAGTGGACCCAACCCTGAGGAGA CGGATTGAGCCCCATGAATTTGAAGTGTTCTTTGACCCAAGGGAGCTGAGGAAG GAGACATGCCTGCTGTACGAGATCAAGTGGGGCACAAGCCACAAGATCTGGCGC CACAGCTCCAAGAACACCACAAAGCACGTGGAAGTGAATTTCATCGAGAAGTTT ACCTCCGAGCGGCACTTCTGCCCCTCTACCAGCTGTTCCATCACATGGTTTCTGTC TTGGAGCCCTTGCGGCGAGTGTTCCAAGGCCATCACCGAGTTCCTGTCTCAGCAC CCTAACGTGACCCTGGTCATCTACGTGGCCCGGCTGTATCACCACATGGACCAGC AGAACAGGCAGGGCCTGCGCGATCTGGTGAATTCTGGCGTGACCATCCAGATCA TGACAGCCCCAGAGTACGACTATTGCTGGCGGAACTTCGTGAATTATCCACCTGG CAAGGAGGCACACTGGCCAAGATACCCACCCCTGTGGATGAAGCTGTATGCACT GGAGCTGCACGCAGGAATCCTGGGCCTGCCTCCATGTCTGAATATCCTGCGGAGA AAGCAGCCCCAGCTGACATTTTTCACCATTGCTCTGCAGTCTTGTCACTATCAGC GGCTGCCTCCTCATATTCTGTGGGCTACAGGCCTTAAA (SEQ ID NO: 38)

[0989] Anc687 APOBEC:

[0990] TCATCAGAAACAGGACCAGTCGCCGTGGACCCAACACTGAGGAGA AGGATTGAGCCCCATGAATTTGAAGTCTTTTTCGACCCCAGGGAGCTGAGGAAG GAGGCATGCCTGCTGTACGAGATCAAGTGGGGCACAAGCCACAAGATCTGGCGC AACAGCGGCAAGAACACCACAAAGCACGTGGAAGTGAATTTCATCGAGAAGTTT ACCTCCGAGCGGCACTTCTGCCCCTCTATCAGCTGTTCCATCACATGGTTTCTGTC TTGGAGCCCTTGCTGGGAGTGTTCCAAGGCCATCCGCGAGTTCCTGTCTCAGCAC CCTAACGTGACCCTGGTCATCTACGTGGCCCGGCTGTTTCAACACATGGACCAGC AGAACAGGCAGGGCCTGCGCGATCTGGTGAATTCTGGCGTGACCATCCAGATCA TGACAGCCTCAGAGTACGACCATTGCTGGCGGAACTTCGTGAATTATCCACCTGG CAAGGAGGCACACTGGCCAAGATACCCACCCCTGTGGATGAAGCTGTATGCACT GGAGCTGCACGCAGGAATCCTGGGCCTGCCTCCATGTCTGAATATCCTGCGGAGA AAGCAGCCCCAGCTGACATTTTTCACTATCGCACTGCAGAGCTGTCATTACCAGA GACTGCCTCCTCATATCCTGTGGGCTACAGGCCTTAAA (SEQ ID NO: 39)

[0991] Anc686 APOBEC:

[0992] AGCAGCGAGACAGGACCCGTGGCAGTGGACCCTACACTGAGGAGG AGGATTGAGCCCGAATTTTTCAACAGGAACTACGACCCCAGAGAGCTGCGGAAG GAGACATACCTGCTGTATGAGATCAAGTGGGGCAAGGAGTCCAAGATCTGGCGG CACACCTCTAACAATAGAACACAGCACGCCGAGGTGAACTTCCTGGAGAACTTC TTTAATGAGCTGTACTTTAATCCTTCTACCCACTGCAGCATCACATGGTTCCTGAG CTGGTCCCCATGCGGCGAGTGTTCTAAGGCCATCGTGGAGTTTCTGAAGGAGCAC CCCAACGTGAATCTGGAGATCTACGTGGCCAGGCTGTATCTGTGCGAGGACGAG AGGAACAGGCAGGGCCTGCGGGATCTGGTGAATAGCGGCGTGACCATCAGAATC ATGAACCTGCCTGACTACAATTATTGTTGGCGCACATTCGTGTCCCACCAGGGAG GCGACGAGGATTATTGGCCAAGGCACTTTGCACCATGGGTGCGCCTGTACGTGCT GGAGCTGTATTGCATCATCCTGGGCCTGCCCCCTTGTCTGAACATCCTGCGGAGA AAGCAGCCCCAGCTGACATTCTTCACCATCGCACTGCAGAGTTGTCATTACCAGC GACTGCCTCCTCATATCCTGTGGGCTACAGGCCTTAAA (SEQ ID NO: 40)

[0993] Anc655 APOBEC:

[0994] TCATCAGAGACCGGACCTGTGGCAGTGGACCCAACCCTGCGACGG AGAATCGAGCCCTTTTACTTTCAGTTCAACAACGACCCAAGAGCCTGCCGGAGAA AGACCTACCTGTGCTATGAGCTGAAGCAGGACGGCTCTACCTGGGTGTGGAAGC GGACACTGCACAACAAGGGCAGACACGCCGAGATCTGCTTCCTGGAGAAGATCA GCTCCCTGGAGAAGCTGGACCCTGCCCAGCACTACAGGATCACATGGTATATGTC TTGGAGCCCATGCTCCAACTGTGCCCAGAAGATCGTGGATTTTCTGAAGGAGCAC CCACACGTGAATCTGCGGATCTACGTGGCCAGACTGTACTATCACGAGGAGGAG AGGTATCAGGAGGGCCTGAGGAACCTGAGGCGCTCCGGCGTGTCTATCAGAGTG ATGGACCTGCCCGATTTCGAGCACTGCTGGGAGACATTCGTGGATAACGGAGGA GGACCTTTCCAGCCATGGCCCGGCCTGGAGGAGCTGAATAGCAAGCAGCTGTCC CGGAGACTGCAGGCAGGAATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGAGGC GCAAGCAGCCCCAGCTGACATTTTTCACCATCGCACTGCAGAGTTGTCATTATCA GCGACTGCCTCCTCATATCCTGTGGGCTACAGGCCTTAAA (SEQ ID NO: 41)

[0995] Anc733 APOBEC:

[0996] AGCAGCGAGACCGGACCTGTGGCAGTGGACCCAACCCTGAGAAGA CGCATTGAGCCATTTCATTTTCAGTTTAACAACGACCCCAGAGCCTACCGGAGAA AGACCTACCTGTGCTATGAGCTGAAGCAGGACGGCTCCACCTGGGTGCTGGATC GGACACTGAGAAACAAGGGCCGGCACGCCGAGATCTGTTTCCTGGACAAGATCA ATTCCTGGGAGAGGCTGGATCCCGCCCAGCACTACCGCGTGACATGGTATATGA GCTGGTCCCCTTGCTCTAACTGTGCCCAGCAGGTGGTGGATTTCCTGAAGGAGCA CCCACACGTGAATCTGCGGATCTTTGCCGCCAGACTGTACTATCACGAGCAGAGG CGCTATCAGGAGGGCCTGCGGAGCCTGAGGGGAAGCGGAGTGCCTGTGGCCGTG ATGACCCTGCCAGACTTCGAGCACTGCTGGGAGACATTTGTGGATCACGGCGGCC GGCCATTCCAGCCATGGGACGGCCTGGAGGAGCTGAACTCTAGGAGCCTGTCCC GGAGACTGCAGGCAGGAATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGAGGCG CAAGCAGCCCCAGCTGACCTTTTTTACCATCGCACTGCAGAGTTGTCACTACCAG AGACTGCCTCCTCATATCCTGTGGGCTACAGGCCTTAAA (SEQ ID NO: 42) [0997] TadA WT:

[0998] TCTGAAGTCGAGTTTAGCCACGAGTATTGGATGAGGCACGCACTGA CCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGTGCTGG TGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGCCACG ACCCTACCGCACACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTCATGC AGAATTACCGCCTGATCGATGCCACCCTGTATGTGACACTGGAGCCATGCGTGAT GTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGTGTTCGGAGCACG GGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACCCCGG CATGAACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCGCCGC CCTGCTGAGCGATTTCTTTAGAATGCGGAGACAGGAGATCAAGGCCCAGAAGAA GGCACAGAGCTCCACCGAC (SEQ ID NO: 43)

[0999] TadA 7.10:

[1000] TCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCTGA CCCTGGCCAAGAGGGCACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCTGG TGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCACG ACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCATGC AGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTGAT GTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGAGG AACGCAAAAACCGGCGCCGCAGGCTCCCTGATGGACGTGCTGCACTACCCCGGC ATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCGCC CTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGG CCCAGAGCTCCACCGAC (SEQ ID NO: 44)

[1001] BE4max:

[1002] ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAG AAGCGGAAAGTCTCCTCAGAGACTGGGCCTGTCGCCGTCGATCCAACCCTGCGC CGCCGGATTGAACCTCACGAGTTTGAAGTGTTCTTTGACCCCCGGGAGCTGAGAA AGGAGACATGCCTGCTGTACGAGATCAACTGGGGAGGCAGGCACTCCATCTGGA GGCACACCTCTCAGAACACAAATAAGCACGTGGAGGTGAACTTCATCGAGAAGT TTACCACAGAGCGGTACTTCTGCCCCAATACCAGATGTAGCATCACATGGTTTCT GAGCTGGTCCCCTTGCGGAGAGTGTAGCAGGGCCATCACCGAGTTCCTGTCCAGA TATCCACACGTGACACTGTTTATCTACATCGCCAGGCTGTATCACCACGCAGACC CAAGGAATAGGCAGGGCCTGCGCGATCTGATCAGCTCCGGCGTGACCATCCAGA TCATGACAGAGCAGGAGTCCGGCTACTGCTGGCGGAACTTCGTGAATTATTCTCC TAGCAACGAGGCCCACTGGCCTAGGTACCCACACCTGTGGGTGCGCCTGTACGT GCTGGAGCTGTATTGCATCATCCTGGGCCTGCCCCCTTGTCTGAATATCCTGCGG AGAAAGCAGCCCCAGCTGACCTTCTTTACAATCGCCCTGCAGTCTTGTCACTATC AGAGGCTGCCACCCCACATCCTGTGGGCCACAGGCCTGAAGTCTGGAGGATCTA GCGGAGGATCCTCTGGCAGCGAGACACCAGGAACAAGCGAGTCAGCAACACCA GAGAGCAGTGGCGGCAGCAGCGGCGGCAGCGACAAGAAGTACAGCATCGGCCT GGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGT GCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAA GAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCG GCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCT ATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC ACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACC

CCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCA

TCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGC

TGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGA

GGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT

GCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGA

CGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCT

GATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGC

CCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA

TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCT

GGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCC

GACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCC

CCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACC

CTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCT

TCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGG

AAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGG

AACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG

ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGC

GGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGA

AGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAG

CAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTT

CGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGAC

CAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCT

GTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGA

GGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGA

CCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTA

CTTC A AG A A A ATC G AGTGCTTC G ACTCC GTGG A A ATCTCC GGC GTGG A AG ATC G

GTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAG

GACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACC

CTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC

CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGC

TGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGC

AAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGC

AGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGG

TGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCC

CCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGA

AAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAG

AACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGAT

CGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGG

AAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGC

GGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATG

TGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGT

GCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG

AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA

TTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCG

AACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCA

CAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGA ATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT

CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCA

CCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAA

GTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGT

GCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGT

ACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAA

CGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGA

TCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC

CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAG

AGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACT

GGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT

GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAG

AGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCG

ACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGC

TGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCT

CTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA

ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATA

ATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCA

TCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGG

ACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGG

CCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTT

CAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGT

GCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGAT

CGACCTGTCTCAGCTGGGAGGTGACAGCGGCGGGAGCGGCGGGAGCGGGGGGA

GCACTAATCTGAGCGACATCATTGAGAAGGAGACTGGGAAACAGCTGGTCATTC

AGGAGTCCATCCTGATGCTGCCTGAGGAGGTGGAGGAAGTGATCGGCAACAAGC

CAGAGTCTGACATCCTGGTGCACACCGCCTACGACGAGTCCACAGATGAGAATG

TGATGCTGCTGACCTCTGACGCCCCCGAGTATAAGCCTTGGGCCCTGGTCATCCA

GGATTCTAACGGCGAGAATAAGATCAAGATGCTGAGCGGAGGATCCGGAGGATC

TGGAGGCAGCACCAACCTGTCTGACATCATCGAGAAGGAGACAGGCAAGCAGCT

GGTCATCCAGGAGAGCATCCTGATGCTGCCCGAAGAAGTCGAAGAAGTGATCGG

AAACAAGCCTGAGAGCGATATCCTGGTCCATACCGCCTACGACGAGAGTACCGA

CGAAAATGTGATGCTGCTGACATCCGACGCCCCAGAGTATAAGCCCTGGGCTCTG

GTCATCCAGGATTCCAACGGAGAGAACAAAATCAAAATGCTGTCTGGCGGCTCA

AAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTC

(SEQ ID NO: 45)

[1003] AncBE4max689:

[1004] ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAG AAGCGGAAAGTCAGCAGTGAAACCGGACCAGTGGCAGTGGACCCAACCCTGAG GAGACGGATTGAGCCCCATGAATTTGAAGTGTTCTTTGACCCAAGGGAGCTGAG GAAGGAGACATGCCTGCTGTACGAGATCAAGTGGGGCACAAGCCACAAGATCTG GCGCCACAGCTCCAAGAACACCACAAAGCACGTGGAAGTGAATTTCATCGAGAA GTTTACCTCCGAGCGGCACTTCTGCCCCTCTACCAGCTGTTCCATCACATGGTTTC TGTCTTGGAGCCCTTGCGGCGAGTGTTCCAAGGCCATCACCGAGTTCCTGTCTCA GCACCCTAACGTGACCCTGGTCATCTACGTGGCCCGGCTGTATCACCACATGGAC CAGCAGAACAGGCAGGGCCTGCGCGATCTGGTGAATTCTGGCGTGACCATCCAG ATCATGACAGCCCCAGAGTACGACTATTGCTGGCGGAACTTCGTGAATTATCCAC

CTGGCAAGGAGGCACACTGGCCAAGATACCCACCCCTGTGGATGAAGCTGTATG

CACTGGAGCTGCACGCAGGAATCCTGGGCCTGCCTCCATGTCTGAATATCCTGCG

GAGAAAGCAGCCCCAGCTGACATTTTTCACCATTGCTCTGCAGTCTTGTCACTAT

CAGCGGCTGCCTCCTCATATTCTGTGGGCTACAGGCCTTAAATCTGGAGGATCTA

GCGGAGGATCCTCTGGCAGCGAGACACCAGGAACAAGCGAGTCAGCAACACCA

GAGAGCAGTGGCGGCAGCAGCGGCGGCAGCGACAAGAAGTACAGCATCGGCCT

GGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGT

GCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAA

GAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCG

GCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCT

ATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCC

ACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACC

CCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCA

TCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGC

TGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGA

GGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGT

GCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGA

CGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCT

GATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGC

CCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGA

TGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCT

GGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCC

GACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCC

CCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACC

CTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCT

TCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGG

AAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGG

AACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG

ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGC

GGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGA

AGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAG

CAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTT

CGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGAC

CAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCT

GTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGA

GGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGA

CCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTA

CTTC A AG A A A ATC G AGTGCTTC G ACTCC GTGG A A ATCTCC GGC GTGG A AG ATC G

GTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAG

GACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACC

CTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCC

CACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGC

TGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGC

AAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGC

AGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGG

TGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCC CCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGA

AAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAG

AACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGAT

CGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGG

AAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGC

GGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATG

TGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGT

GCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG

AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA

TTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCG

AACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCA

CAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGA

ATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT

CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCA

CCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAA

GTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGT

GCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGT

ACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAA

CGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGA

TCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC

CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAG

AGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACT

GGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT

GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAG

AGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCG

ACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGC

TGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCT

CTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA

ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATA

ATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCA

TCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGG

ACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGG

CCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTT

CAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGT

GCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGAT

CGACCTGTCTCAGCTGGGAGGTGACAGCGGCGGGAGCGGCGGGAGCGGGGGGA

GCACTAATCTGAGCGACATCATTGAGAAGGAGACTGGGAAACAGCTGGTCATTC

AGGAGTCCATCCTGATGCTGCCTGAGGAGGTGGAGGAAGTGATCGGCAACAAGC

CAGAGTCTGACATCCTGGTGCACACCGCCTACGACGAGTCCACAGATGAGAATG

TGATGCTGCTGACCTCTGACGCCCCCGAGTATAAGCCTTGGGCCCTGGTCATCCA

GGATTCTAACGGCGAGAATAAGATCAAGATGCTGAGCGGAGGATCCGGAGGATC

TGGAGGCAGCACCAACCTGTCTGACATCATCGAGAAGGAGACAGGCAAGCAGCT

GGTCATCCAGGAGAGCATCCTGATGCTGCCCGAAGAAGTCGAAGAAGTGATCGG

AAACAAGCCTGAGAGCGATATCCTGGTCCATACCGCCTACGACGAGAGTACCGA

CGAAAATGTGATGCTGCTGACATCCGACGCCCCAGAGTATAAGCCCTGGGCTCTG

GTCATCCAGGATTCCAACGGAGAGAACAAAATCAAAATGCTGTCTGGCGGCTCA AAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTC (SEQ ID NO: 46)

[1005] AncBE4max687:

[1006] ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAG AAGCGGAAAGTCTCATCAGAAACAGGACCAGTCGCCGTGGACCCAACACTGAGG AGAAGGATTGAGCCCCATGAATTTGAAGTCTTTTTCGACCCCAGGGAGCTGAGG AAGGAGGCATGCCTGCTGTACGAGATCAAGTGGGGCACAAGCCACAAGATCTGG CGCAACAGCGGCAAGAACACCACAAAGCACGTGGAAGTGAATTTCATCGAGAAG TTTACCTCCGAGCGGCACTTCTGCCCCTCTATCAGCTGTTCCATCACATGGTTTCT GTCTTGGAGCCCTTGCTGGGAGTGTTCCAAGGCCATCCGCGAGTTCCTGTCTCAG CACCCTAACGTGACCCTGGTCATCTACGTGGCCCGGCTGTTTCAACACATGGACC AGCAGAACAGGCAGGGCCTGCGCGATCTGGTGAATTCTGGCGTGACCATCCAGA TCATGACAGCCTCAGAGTACGACCATTGCTGGCGGAACTTCGTGAATTATCCACC TGGCAAGGAGGCACACTGGCCAAGATACCCACCCCTGTGGATGAAGCTGTATGC ACTGGAGCTGCACGCAGGAATCCTGGGCCTGCCTCCATGTCTGAATATCCTGCGG AGAAAGCAGCCCCAGCTGACATTTTTCACTATCGCACTGCAGAGCTGTCATTACC AGAGACTGCCTCCTCATATCCTGTGGGCTACAGGCCTTAAATCTGGAGGATCTAG CGGAGGATCCTCTGGCAGCGAGACACCAGGAACAAGCGAGTCAGCAACACCAG AGAGCAGTGGCGGCAGCAGCGGCGGCAGCGACAAGAAGTACAGCATCGGCCTG GACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTG CCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAG AACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGG CTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTAT CTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCAC AGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCC ATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATC TACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTG ATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGG GCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC AGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACG CCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGA TCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCC TGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATG CCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGG CCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGA CGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCC CCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCT GCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTC GACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGA AGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGA ACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGA CAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCG GCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAA GATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGC AGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTC GAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACC AACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTG

TACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAG

GGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGAC

CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTAC

TTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGT

TCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGG

ACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCC

TGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCC

ACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCT

GGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGC

AAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGC

AGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGG

TGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCC

CCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGA

AAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAG

AACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGAT

CGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGG

AAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGC

GGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATG

TGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGT

GCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAG

AGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGA

TTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCG

AACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCA

CAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGA

ATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGT

CCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCA

CCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAA

GTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGT

GCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGT

ACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAA

CGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGA

TCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGC

CCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAG

AGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACT

GGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCT

GGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAG

AGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCG

ACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGC

TGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCT

CTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGA

ACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATA

ATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCA

TCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGG

ACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGG

CCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTT

CAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGT GCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGAT

CGACCTGTCTCAGCTGGGAGGTGACAGCGGCGGGAGCGGCGGGAGCGGGGGGA

GCACTAATCTGAGCGACATCATTGAGAAGGAGACTGGGAAACAGCTGGTCATTC

AGGAGTCCATCCTGATGCTGCCTGAGGAGGTGGAGGAAGTGATCGGCAACAAGC

CAGAGTCTGACATCCTGGTGCACACCGCCTACGACGAGTCCACAGATGAGAATG

TGATGCTGCTGACCTCTGACGCCCCCGAGTATAAGCCTTGGGCCCTGGTCATCCA

GGATTCTAACGGCGAGAATAAGATCAAGATGCTGAGCGGAGGATCCGGAGGATC

TGGAGGCAGCACCAACCTGTCTGACATCATCGAGAAGGAGACAGGCAAGCAGCT

GGTCATCCAGGAGAGCATCCTGATGCTGCCCGAAGAAGTCGAAGAAGTGATCGG

AAACAAGCCTGAGAGCGATATCCTGGTCCATACCGCCTACGACGAGAGTACCGA

CGAAAATGTGATGCTGCTGACATCCGACGCCCCAGAGTATAAGCCCTGGGCTCTG

GTCATCCAGGATTCCAACGGAGAGAACAAAATCAAAATGCTGTCTGGCGGCTCA

AAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGGAAAGTC

(SEQ ID NO: 47)

[1007] ABEmax

[1008] ATGAAACGGACAGCCGACGGAAGCGAGTTCGAGTCACCAAAGAAG AAGCGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGTATTGGATGAGGCACGCA CTGACCCTGGCAAAGCGAGCATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGTG CTGGTGCACAACAATAGAGTGATCGGAGAGGGATGGAACAGGCCAATCGGCCGC CACGACCCTACCGCACACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTGGTC ATGCAGAATTACCGCCTGATCGATGCCACCCTGTATGTGACACTGGAGCCATGCG TGATGTGCGCAGGAGCAATGATCCACAGCAGGATCGGAAGAGTGGTGTTCGGAG CACGGGACGCCAAGACCGGCGCAGCAGGCTCCCTGATGGATGTGCTGCACCACC CCGGCATGAACCACCGGGTGGAGATCACAGAGGGAATCCTGGCAGACGAGTGCG CCGCCCTGCTGAGCGATTTCTTTAGAATGCGGAGACAGGAGATCAAGGCCCAGA AGAAGGCACAGAGCTCCACCGACTCTGGAGGATCTAGCGGAGGATCCTCTGGAA GCGAGACACCAGGCACAAGCGAGTCCGCCACACCAGAGAGCTCCGGCGGCTCCT CCGGAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTACTGGATGAGACATGCCCT GACCCTGGCCAAGAGGGCACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGCT GGTGCTGAACAATAGAGTGATCGGCGAGGGCTGGAACAGAGCCATCGGCCTGCA CGACCCAACAGCCCATGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGGTCAT GCAGAACTACAGACTGATTGACGCCACCCTGTACGTGACATTCGAGCCTTGCGTG ATGTGCGCCGGCGCCATGATCCACTCTAGGATCGGCCGCGTGGTGTTTGGCGTGA GGAACGCAAAAACCGGCGCCGCAGGCTCCCTGATGGACGTGCTGCACTACCCCG GCATGAATCACCGCGTCGAAATTACCGAGGGAATCCTGGCAGATGAATGTGCCG CCCTGCTGTGCTATTTCTTTCGGATGCCTAGACAGGTGTTCAATGCTCAGAAGAA GGCCCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAGGCTCCTCTGGCTCTGA GACACCTGGCACAAGCGAGAGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCG GGGGGTCAGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACTCTGTGG GCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGC TGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGT TCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGA AGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAAC GAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTG GTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGAC GAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTG GTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCAC

ATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAAC

AGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCG

AGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCA

GACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGA

AGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCA

ACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGG

ACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG

CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACAT

CCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA

GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA

GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTA

CGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA

GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAG

AGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCA

GATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCA

TTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCC

TACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGA

AAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC

GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA

ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATA

ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC

TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGA

AAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCG

ACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA

CCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA

CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGA

GATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGAT

GAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGC

TGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGA

AGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC

TGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC

TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCC

TGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGC

CCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGA

CAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCT

GGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACG

AGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGG

AACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAG

CTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAA

CCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGA

ACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACA

ATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCA

TCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCC

TGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAG

TGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCA

GTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCT GAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGA

GTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAG

CGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCAT

GAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCC

TCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGG

ATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAA

AGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGA

ACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGC

GGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAA

AGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCA

TGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCT

ACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCG

AGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGG

GAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCA

CTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGT

GGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTC

CAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAA

CAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTT

TACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATC

GACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCAC

CAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGT

GACTCTGGCGGCTCAAAAAGAACCGCCGACGGCAGCGAATTCGAGCCCAAGAAG

AAGAGGAAAGTC (SEQ ID NO: 48)

[1009] SCN9a RNA:

[1010] GUU AGU CCUU A A A AU GU AGGG (SEQ ID NO: 49)

[1011] MPDU1 RNA:

[1012] GUUCCCGGUCAUGCACUACAG (SEQ ID NO: 50)

[1013] HBG site 1 RNA:

[1014] CUU GACC AAUAGCCUU GAC A (SEQ ID NO: 51)

[1015] HBG site 2 RNA:

[1016] AUAUUUGCAUUGAGAUAGUG (SEQ ID NO: 52)

REFERENCES

[1017] 1. Landrum, M.J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic acids research 44, D862-868 (2016).

[1018] 2. Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. & Liu, D.R.

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-+ (2016). [1019] 3. Gaudelli, N.M. et al. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage. Nature 551, 464-+ (2017).

[1020] 4. Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nature biotechnology 35, 441-443 (2017).

[1021] 5. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353 (2016).

[1022] 6. Komor, A.C. et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv 3, eaao4774 (2017).

[1023] 7. Komor, A.C., Badran, A.H. & Cell, L.-D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell (2017).

[1024] 8. Satomura, A. et al. Precise genome-wide base editing by the CRISPR Nickase system in yeast. Sci Rep 7, 2095 (2017).

[1025] 9. Zhang, Y. et al. Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nat Commun 8, 118 (2017).

[1026] 10. Lu, Y.M. & Zhu, J.K. Precise Editing of a Target Base in the Rice Genome Using a Modified CRISPR/Cas9 System. Molecular Plant 10, 523-525 (2017).

[1027] 11. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion. Nature biotechnology 35, 438-440 (2017).

[1028] 12. Billon, P. et al. CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons. Molecular Cell 67, 1068-+ (2017).

[1029] 13. Kuscu, C. et al. CRISPR-STOP: gene silencing through base-editing- induced nonsense mutations. Nature methods 14, 710-712 (2017).

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OTHER EMBODIMENTS

[1075] The foregoing has been a description of certain non-limiting embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

EQUIVALENTS AND SCOPE

[1076] In the claims articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[1077] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms“comprising” and“containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[1078] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[1079] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.