RU2782794C2 - Constructed target-specific nucleases - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to biotechnology and medicine and is a constructed half-domain of FokI cleavage. The invention also relates to a constructed nuclease containing the specified cleavage domain, as well as is a method for cleavage of genomic cellular chromatin in an area of interest, including expression of an artificial nuclease containing the constructed domain of cleavage in a cell, wherein the nuclease site-specifically cleaves a sequence of nucleotides in the area of interest of genomic cellular chromatin.

EFFECT: invention provides for obtaining an effective cleavage system for reduction in non-target cleavage activity.

17 cl, 18 dwg, 18 tbl, 9 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority over U.S. Provisional Application No. 62/378,978, filed August 24, 2016, and U.S. Provisional Application No. 62/443,981, filed January 9, 2017, the contents of which are incorporated herein by links in full.

FEDERALLY FUNDED RESEARCH INVENTIONS

[0002] Not applicable.

FIELD OF TECHNOLOGY

[0003] The present invention relates to the field of polypeptide design and genomic engineering, and homologous recombination.

BACKGROUND OF THE INVENTION

[0004] Artificial nucleases such as engineered zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), engineered cr-RNA/tracr-RNA CRISPR/Cas system ("single guide RNA'"), also referred to as RNA-directed nucleases; and/or nucleases based on the Argonaute system ( e.g. from T. thermophilus , known as "TtAgo", (Swarts et al (2014) Nature 507(7491): 258-261) contain DNA-binding domains (nucleotide or polypeptide), associated or operably linked to cleavage domains, and these molecules have been used to target changes in genomic sequences For example, nucleases have been used to insert exogenous sequences, inactivate one or more endogenous genes, produce organisms ( e.g. crops) and cell lines with altered gene expression patterns , etc. See, for example, U.S. Patent Nos. 9,255,250; №6,689,558; №7,067,317; №7,262,054; №7,888,121; №7,972,854; №7,914,796; №7,951,925; №8,110,379; №8,409,861; патентные публикации США 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 20 1000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 and 20150056705. For example, a pair of nucleases ( eg , zinc finger nucleases, TALEN, dCas-Fok hybrids) can be used to cleave genomic sequences. Each member of said pair typically includes an engineered (non-naturally occurring) DNA-binding protein linked to one or more nuclease cleavage domains (or half-domains). When said DNA binding proteins bind to their target site, the cleavage domains that are linked to said DNA binding proteins are arranged such that they can dimerize and subsequently cleave the genome.

[0005] In general, intermolecular ion pairs (salt bridges) are essential for many DNA-protein interactions. Often the charged side chains of amino acids ( ie -NH3+, =NH2+) interact with the negatively charged phosphate groups of the DNA backbone to form a salt bridge. Said ion pairs can be quite dynamic and switch between direct pairing of two ions and pairing, which is a "solvent-separated ion pair", where a solvent ( e.g. , a water molecule) is embedded between the two ions (Chen et al (2015) J Phys Chem Lett 6:2733-2737).

[0006] In the case of zinc finger proteins, the specificity of ZFP for the target DNA sequence depends on specific, sequence-defined contacts between zinc finger domains and certain DNA bases. In addition, zinc finger domains also contain amino acid residues involved in nonspecific interactions of ion pairs with DNA backbone phosphates. Elrod-Erickson et al (1996. Structure 4:1171) demonstrated by co-crystallization of a zinc finger protein and a cognate target DNA the presence of specific amino acids capable of interacting with phosphorus groups in the DNA backbone through the formation of hydrogen bonds . Zinc finger proteins, which include the well-known Zif268 backbone, typically contain arginine as the amino-terminal residue of the second β-sheet chain, which is also located in the second position towards the carboxyl end relative to the second invariant cysteine (see Fig. 5A). This position within each zinc finger domain can be referred to as (-5) because it represents the 5th residue preceding the start of the α-helix (FIG. 5A). Arginine at this position can interact with phosphate on the DNA backbone by forming a charged hydrogen bond with the guanidine side chain group. Zinc finger proteins with a Zif268 backbone also often contain a lysine at position 4 of the residue down the amino terminus from the first invariant cysteine. This position within each "finger" can be referred to as (-14) since it is the 14th residue preceding the start of the zinc finger α-helix with two residues between the zinc-coordinating cysteine residues (FIG. 5A). Lysine can interact with phosphate on the backbone of DNA by forming a water-mediated charged hydrogen bond with the amino group of the side chain. Since phosphate groups are located along the entire length of the DNA backbone, this type of interaction between zinc fingers and the DNA molecule is usually considered non-sequence specific (J. Miller, Massachusetts Institute of Technology Ph.D. Thesis, 2002).

[0007] Recent studies have suggested that non-specific phosphate contact with side chains in some nucleases may also account for some of the non-specific cleavage activity of these nucleases (Kleinstiver et al , (2016) Nature 529(7587):490-5; Guilinger et al (2014) Nat Meth : 429-435). The researchers hypothesized that these nucleases may have "excessive DNA binding energy", meaning that they may have more affinity for their target DNA than is essentially necessary to bind and cleave the target site. Accordingly, attempts have been made to reduce the cationic charges in the DNA-binding domain of TALE (Guilinger, ibid. ) or the DNA-binding domain of Cas9 (Kleinstiver, ibid. ) to reduce the DNA binding energy of these nucleases, resulting in an increase in the specificity of in vitro cleavage. However, additional studies (Sternberg et al (2015) Nature 527(7576):110-113) also point to a role in proper folding and activation of the Cas9 nuclease domain by some cationic amino acids mutated in the Kleinstiver study of the Cas9 DNA-binding domain. . Thus, the precise role of these amino acids in Cas9 activity is unknown.

[0008] For optimum cleavage specificity by a sequence-selective (artificial) nuclease, it is desirable to create conditions that provide non-saturating target binding and activity. Under saturating conditions - by definition - an amount of nuclease is used that is in excess of what is needed to achieve full activity on the target. This excess provides no target advantage, but may nevertheless result in increased cleavage at the non-target site. In the case of monomeric nucleases, saturation conditions can be easily avoided by performing a simple dose-response study to identify and avoid a saturation plateau in the titration curve. However, in the case of a dimeric nuclease such as ZFN, TALEN or dCas-Fok, saturation conditions can be more difficult to identify and avoid if the binding affinity of the individual monomers is not the same. In such cases, a dose-response study using a simple 1:1 nuclease ratio reveals only the saturation point for the weaker-binding monomer. In such a scenario, if, for example, the affinity of the monomers differs by one order of magnitude, at the saturation point identified in the 1:1 titration study, the monomer with the higher affinity will be present at a concentration 10 times greater than desired. The resulting excess of the higher affinity monomer may in turn lead to an increase in off-target activity without providing any beneficial increase in cleavage in the intended target, potentially resulting in a decrease in overall specificity for any given pair of nucleases.

[0009] To reduce off-target cleavage, engineered obligate heterodimeric cleavage half-domains have been developed. See, for example, US Pat. Nos. 7,914,796; No. 8,034,598; #8,961,281 and #8,623,618; U.S. Patent Publications No. 20080131962 and No. 20120040398. These obligate heterodimers dimerize and cleave targets only if ZFP allows the placement of distinct engineered cleavage domains at the appropriate target site, thereby reducing and/or eliminating monomeric off-target cleavage.

[0010] However, there remains a need for additional methods and compositions based on engineered nuclease cleavage systems to reduce off-target cleaving activity.

BRIEF DESCRIPTION OF THE INVENTION

[0011] The present invention provides methods and compositions for increasing the specificity of a nuclease (for example,pairs of nucleases) in relation to the intended target in relation to other, unintended cleavage sites, also known as non-target sites. Accordingly, artificial nucleases (for example,zinc finger nucleases (ZFN), TALEN, CRISPR/Cas nucleases) containing mutations in one or more regions of the DNA-binding domain (for example,in backbone of a zinc finger protein or TALE), and/or one or more mutations in the cleavage domain or cleavage half-domain of the nucleaseFokI. In addition, this document describes ways to increase the specificity of cleaving activity using these new nucleases (for example,ZFN, TALEN, etc.) and/or independent titration of engineered partners - cleavage half-domains of the nuclease complex. When used alone or in combination, the methods and compositions of the present invention surprisingly provide an unexpected increase in target specificity by reducing off-target cleavage activity. The present invention also provides methods for using said compositions to target cleavage of cellular chromatin at a region of interest and/or integrate a transgene by targeted integration into a predetermined region of interest in cells.

[0012] Accordingly, in one aspect, provided herein is an engineered nuclease cleavage half-domain containing one or more mutations relative to the original cleavage domain ( eg, wild-type domain) from which said mutant forms are derived. In some embodiments, said one or more mutations are one or more of the mutations provided in any of the accompanying tables and drawings, including any combination of said mutants with each other and with other mutants (such as dimerization domain mutants and /or catalytic domain, as well as nickase mutations). Mutations as described herein include, but are not limited to, mutations that change the charge of the cleavage domain, for example, mutations of positively charged residues with a change to positively uncharged residues ( for example, mutations of K and R residues ( for example, mutations with a replacement for S); residues N ( e.g. → D) and Q residues ( e.g. → E), residue mutations that are predicted to be close to the DNA backbone based on molecular modeling and show variability in Fok I homologues (FIGS. 1 and 17); and/ or mutations of other residues ( eg, US Pat. No. 8,623,618 and Guo et al , (2010) J. Mol. Biol . 400(1):96-107).

[0013] The most promising mutations were found using the second criterion. Initially promising mutations were positively charged residues, which were predicted to be close to the DNA backbone when Fok I was bound to DNA. The cleavage domains described herein may include one, two, three, four, five or more of the mutations described herein and may additionally include additional known mutations. Accordingly, the mutations of the present invention do not include the specific mutations disclosed in US Pat. No. 8,623,618 ( eg N527D, S418P, K448M, Q531R, etc.) when used alone; however, the present invention provides new mutants that can be used in combination with the mutants of US Pat. No. 8,623,618. Nicase mutants, characterized in that one of the catalytic domains of the nuclease in the dimer pair contains one or more mutations that make it catalytically inactive (see US patents No. 8,703,489; No. 9,200,266; and No. 9,631,186) can also be used in combination with any of the mutants, described in this document. The nicases can be ZFN nicases, TALEN nicases, and CRISPR/dCas systems.

[0014] In some embodiments, the engineered cleavage half-domains are derived from Fok I or Fok I homologues and contain a mutation at one or more of amino acid residues 416, 422, 447, 448, and/or 525, with numbering corresponding to the full length wild-type Fok I, as shown in SEQ ID NO: 1 or the corresponding residues in the Fok I homologues (see Fig. 17). In other embodiments, FokI-derived cleavage half- domains contain a mutation at one or more of amino acid residues 414-426, 443-450, 467-488, 501-502, and/or 521-531, including one or more of 387, 393, 394, 398, 400, 416, 418, 422, 427, 434, 439, 441, 442, 444, 446, 448, 472, 473, 476, 478, 479, 480, 481, 487, 495 497, 506, 516, 523, 525, 527, 529, 534, 559, 569, 570 and/or 571. Mutations may include mutations of residues found in natural restriction enzymes homologous to Fok I at the appropriate positions (Fig. 17) . In some embodiments, said mutations are substitutions, e.g., substitution of a wild-type molecule residue by any other amino acid, e.g., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), histidine (H), phenylalanine (F), glycine (G), asparagine (N), serine (S), or threonine (T). Any combination of mutants is contemplated, including but not limited to those shown in the accompanying tables and drawings. In some embodiments, the Fok I nuclease domain contains a mutation at one or more of positions 416, 422, 447, 479, and/or 525 (when numbered according to the wild-type molecule, SEQ ID NO: 1). These nuclease domains may also contain one or more mutations at positions 418, 432, 441, 448, 476, 481, 483, 486, 487, 490, 496, 499, 523, 527, 537, 538 and 559, including, but not limited to ELD, KKR, ELE, KKS. See, for example, US Pat. No. 8,623,618. In other additional embodiments, said cleavage domain includes mutations of one or more of the residues shown in Table 15 ( e.g., 419, 420, 425, 446, 447, 470, 471, 472, 475, 478, 480, 492, 500, 502, 521, 523, 526, 530, 536, 540, 545, 573 and/or 574). In some embodiments, variants of the cleavage domains described herein include mutations of residues involved in nuclease dimerization (dimerization domain mutations) and one or more additional mutations; e.g., phosphate-contacting residues: e.g. , dimerization mutants (such as ELD, KKR, ELE, KKS, etc.) in combination with one, two, three, four, five, six or more mutations at amino acid positions outside the dimerization domain , for example, in amino acid residues that can participate in contacts with phosphate groups. In a preferred embodiment, the mutation at positions 416, 422, 447, 448, and/or 525 comprises a change from a positively charged amino acid to an uncharged or negatively charged amino acid. In other embodiments, mutations are introduced at positions 446, 472, and/or 478 (and optionally additional residues, eg, in dimerization or catalytic domains).

[0015] In other embodiments, the engineered cleavage half-domain contains mutations in the dimerization domain, for example, at amino acid residues 490, 537, 538, 499, 496, and 486 in addition to the mutations described herein. According to a preferred embodiment of the present invention, fusion proteins are provided wherein the engineered cleavage half-domain comprises a polypeptide in which the Gln (Q) residue present at position 486 of the wild-type molecule is replaced with a Glu (E) residue, an Ile (I) residue present in position 499 of the wild-type molecule is replaced by a Leu (L) residue and an Asn (N) residue present at position 496 of the wild-type molecule is replaced by an Asp (D) or Glu (E) (“ELD” or “ELE”) residue, in addition to one or more of the mutations described herein. In another embodiment, the engineered cleavage half-domains are derived from a wild-type Fok I cleavage half-domain or a FokI homolog, and contain mutations at amino acid residues 490, 538, and 537, numbered according to wild-type Fok I (SEQ ID NO: 1), in addition to one or more mutations of amino acid residues 416, 422, 447, 448, or 525. According to a preferred embodiment of the present invention, a fusion protein is provided wherein the engineered cleavage half-domain comprises a polypeptide in which the Glu(E) residue present at position 490 of the wild-type molecule is replaced to a Lys (K) residue, an Ile (I) residue present at position 538 of the wild-type molecule is replaced by a Lys (K) residue; and the His (H) residue present at position 537 of the wild-type molecule is replaced by a Lys (K) residue or an Arg (R) residue ("KKK" or "KKR") (see U.S. Patent No. 8,962,281, incorporated by reference herein document) in addition to one or more of the mutations described herein.

[0016] In another embodiment, the engineered cleavage half-domains are derived from the wild-type Fok I cleavage half-domain or homologues thereof, and contain mutations at amino acid residues 490 and 538, numbered according to wild-type Fok I, in addition to one or more mutations at amino acid residues 416, 422 , 447, 448, or 525. According to a preferred embodiment of the present invention, a fusion protein is provided wherein the engineered cleavage half-domain comprises a polypeptide in which the Glu (E) residue present at position 490 of the wild-type molecule is replaced by a Lys (K) residue, and an Ile (I) residue present at position 538 of the wild-type molecule is changed to a Lys (K) (“KK”) residue, in addition to one or more mutations at positions 416, 422, 447, 448, or 525. According to a preferred embodiment of the present invention a fusion protein is proposed in which the designed cleavage half-domain contains a polypeptide in which the Gln (Q) residue is present present at position 486 of the wild-type molecule is replaced by a Glu (E) residue, and the Ile (I) residue present at position 499 of the wild-type molecule is replaced by a Leu (L) (“EL”) residue (see U.S. Patent No. 8,034,598, incorporated by reference herein), in addition to one or more mutations at positions 416, 422, 447, 448, or 525.

[0017] According to one aspect of the present invention, hybrid molecules are provided wherein the engineered cleavage half-domain comprises a polypeptide wherein the amino acid residue of the wild-type molecule is at one or more of positions 387, 393, 394, 398, 400, 402, 416, 422, 427 , 434, 439, 441, 446, 447, 448, 469, 472, 478, 487, 495, 497, 506, 516, 525, 529, 534, 559, 569, 570, 571 in the Fok I catalytic domain are mutated. In some embodiments, said one or more mutations change a positively charged amino acid residue of the wild-type molecule to a neutral residue or a negatively charged residue. In any of these embodiments, the described mutants may also be introduced into a Fok I domain containing one or more additional mutations. In preferred embodiments, these additional mutations are in the dimerization domain, for example, at positions 499, 496, 486, 490, 538, and 537. Mutations include substitutions, insertions, and/or deletions of one or more amino acid residues.

[0018] In another aspect, any of the engineered cleavage half-domains described above can be incorporated into artificial nucleases, for example, by binding them to a DNA binding domain, including, but not limited to, zinc finger nucleases, TALEN, CRISPR/Cas nucleases, etc. Zinc finger proteins of zinc finger nucleases may contain non-canonical zinc coordinating residues ( e.g. CCHC rather than the canonical C2H2 configuration, see US Pat. No. 9,234,187).

[0019] In another aspect, hybrid molecules are provided comprising a DNA binding domain and an engineered FokI cleavage half-domain or homologue thereof as described herein that produce an artificial nuclease. In some embodiments, said DNA binding domain of the fusion molecule is a zinc finger binding domain ( eg, an engineered zinc finger binding domain). In other embodiments, said DNA binding domain is a TALE DNA binding domain. In other further embodiments, said DNA-binding domain comprises a DNA-binding molecule ( eg , a guide RNA) and a catalytically inactive Cas9 or Cfp1 protein (dCas9 or dCfp1). In some embodiments, the engineered fusion molecules form a nuclease complex with a catalytically inactive engineered cleavage half-domain such that the dimeric nuclease is capable of cleaving only one strand of a double-stranded DNA molecule to form a nickase (see U.S. Patent No. 9,200,266).

[0020] The methods and compositions of the present invention also encompass mutations of one or more amino acids within the DNA binding domain outside of residues that recognize nucleotides of the target sequence ( e.g., one or more mutations in the "ZFP backbone" (outside the DNA helical recognition region) or in the "TALE backbone" (outside RVD)), which can non-specifically interact with phosphates on the DNA backbone. Accordingly, in some embodiments, the present invention encompasses mutations of cationic amino acid residues in the backbone of ZFP that are not necessary for target nucleotide specificity. In some embodiments, said mutations in the ZFP backbone include mutation of a cationic amino acid residue to a neutral or anionic amino acid residue. In some embodiments, mutations in the ZFP backbone comprise a polar amino acid residue mutated to a neutral or non-polar amino acid residue. In preferred embodiments, mutations are introduced at position (-5), (-9) and/or position (-14) relative to the DNA-binding helix. In some embodiments, the zinc finger may contain one or more mutations at position (-5), (-9) and/or (-14). In additional embodiments, one or more zinc fingers in a zinc finger protein may contain mutations at position (-5), (-9) and/or (-14). In some embodiments, the amino acids at positions (-5), (-9) and/or (-14) ( e.g. , arginine (R) or lysine (K)) are mutated to alanine (A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y) and/or glutamine (Q).

[0021] According to another aspect, polynucleotides are provided that encode any of the engineered cleavage half-domains or fusion proteins as described herein.

[0022] In yet another aspect, cells are also provided that contain any of the nucleases, polypeptides ( eg, fusion molecules or fusion polypeptides), and/or polynucleotides as described herein. In one embodiment, said cells comprise a pair of fusion polypeptides, one of which contains, in addition to one or more mutations at amino acid residues 393, 394, 398, 416, 421, 422, 442, 444, 447, 448, 473, 480, 530 and /or 525, an ELD or ELE cleavage half-domain; and the other contains, in addition to one or more mutations at residues 393, 394, 398, 416, 421, 422, 442, 444, 446, 447, 448, 472, 473, 478, 480, 530 and/or 525, the KKK cleavage half-domain or KKR (see U.S. Patent No. 8,962,281).

[0023] In any of these fusion polypeptides described herein, the ZFP partners may additionally contain mutations in the zinc finger DNA binding domain at positions (-5), (-9) and/or (-14). In some embodiments, the Arg (R) at position -5 is replaced with Tyr (Y), Asp (N), Glu (E), Leu (L), Gln (Q), or Ala (A). In other embodiments, Arg (R) at position (-9) is replaced by Ser (S), Asp (N), or Glu (E). According to additional implementation options, Arg (R) at position (-14) is replaced by Ser (S) or Gln (Q). In other embodiments, said fusion polypeptides may contain mutations in the zinc finger DNA binding domain wherein the amino acids at positions (-5), (-9) and/or (-14) are substituted for any of the above amino acids in any combination.

[0024] The present invention also provides cells that have been modified with the polypeptides and/or polynucleotides of the present invention. In some embodiments, nuclease-mediated transgene insertion or nuclease-mediated gene knockout occurs in said cells. Said modified cells, and any cells derived from said modified cells, do not necessarily contain the nucleases of the present invention for more than some time, however, genomic modifications mediated by such nucleases persist.

[0025] According to another aspect, methods for targeted cleavage of cellular chromatin in a region of interest are provided; methods for stimulating homologous recombination in a cell; ways to treat the infection; and/or methods of treating the disease. These methods can be implementedin vitro, ex vivoorin vivo, or a combination of the above can be implemented. These methods involve cleaving cellular chromatin at a predetermined region of interest in cells by expressing a pair of fusion polypeptides as described herein (those.a pair of hybrid polypeptides, in which one or both of the hybrid polypeptides contains engineered cleavage half-domains as described herein). In some embodiments, targeted cleavage of the target site is increased by at least 50%-200% (or any value in between) or more, including 50-60% (or any value in-between), 60%-70% ( or any value in between), 70% - 80% (or any value in between), 80% - 90% (or any value in between, 90% - 200% (or any value in between), compared to cleavage domains without mutations as described herein.Similarly, when using methods and compositions as described herein, cleavage of the off-target site is reduced by 1-100-fold or more, including, but not limited to, 1-50-fold (or any intermediate value).

[0026] The engineered cleavage half-domains described herein can be used in methods for targeted cleavage of cellular chromatin at a region of interest and/or homologous recombination at a predetermined region of interest in cells. Cells include cultured cells, cell lines, cells in the body, cells removed from the body for treatment/treatment where said cells and/or their progeny are returned to the body after treatment, and cells removed from the body modified using hybrid molecules according to the present invention, which are then returned to the body according to the method of treatment (cell therapy). The region of interest in the cellular chromatin may be, for example, a genomic sequence or a portion thereof. The compositions include fusion molecules or polynucleotides encoding fusion molecules that contain a DNA binding molecule ( e.g., an engineered zinc finger binding domain, or a TALE binding domain, or an engineered CRISPR guide RNA) and a cleavage half-domain as described.

[0027] A fusion molecule can be expressed in a cell, for example, by delivering said fusion molecule to said cell as a polypeptide, or by delivering to the cell a polynucleotide encoding said fusion molecule, said polynucleotide, if DNA, being transcribed and translated to produce a hybrid molecule. In turn, if the polynucleotide is an mRNA encoding a fusion molecule, upon delivery of said mRNA to a cell, said mRNA is translated, thereby producing a fusion molecule.

[0028] According to other aspects of the present invention, methods and compositions are provided for increasing the specificity of engineered nucleases. In one aspect, methods are provided for increasing the overall target specificity of a cleavage by reducing off-target cleavage activity. In some embodiments, engineered partners, the cleavage half-domains of the engineered nuclease complex, are used to contact the cell, with each partner of said complex being used in a ratio other than 1:1 with the other partner. In some embodiments, the ratio of the two partners (cleavage half-domains) used is 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10, or 1:20, or any intermediate option. In other embodiments, the ratio of the two partners is greater than 1:30. In other embodiments, the two partners are involved in a selected ratio other than 1:1. In some aspects, each partner is delivered to said cell as mRNA, or delivered in a viral or non-viral vector, wherein different amounts of mRNA or vector encoding each of the partners are delivered. In additional embodiments, both partners in the nuclease complex can be contained in the same viral or non-viral vector, but are intentionally expressed in such a way that one partner is expressed at a higher or lower level than the other, which ultimately allows the delivery of cleavage half-domains to the cell in ratio other than 1:1. In some embodiments, all cleavage half-domains are expressed using different promoters that provide different expression efficiencies. In other embodiments, said two cleavage domains are delivered to said cell using a viral or non-viral vector, both of which are expressed from the same open reading frame, however, the genes encoding said two partners are separated by a sequence ( e.g. , 2A self-cleaving sequence or IRES) resulting in to a lower level of expression of the 3'-partner, so that the ratio of these two partners is 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10 or 1:20, or any in between. In other embodiments, the two partners are involved in a selected ratio other than 1:1.

[0029] Also provided are methods for reducing off-target nuclease activity using complexes of two or more nucleases. For example, the present invention provides methods for varying the ratio of DNA-binding molecules using complexes of two or more nucleases. In some embodiments, said DNA binding molecules are polypeptide DNA binding domains ( e.g. , ZFN, TALEN, dCas-Fok, mega-TAL, meganucleases), while in other embodiments, said DNA binding molecules are guide RNAs for use. with RNA-directed nucleases. In preferred embodiments, the ratio of said two or more DNA binding molecules is 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10, or 1:20, or matches any intermediate option. In other embodiments, the two DNA binding molecules are involved in a selected ratio other than 1:1. In some aspects, the non-1:1 ratio is achieved by changing the ratio of guide RNAs used to transfect the cell. In other aspects, this ratio is changed by changing the ratio of each Cas9 protein/guide RNA complex used to treat the cells of interest. In yet another aspect, the altered ratio is achieved by using different ratios of DNA encoding guide RNAs (viral or non-viral) to treat cells, or by using promoters that provide different expression strengths for differential expression of DNA binding molecules in cells. The occurrence of non-target events can be reduced by a factor of 2-1000 (or any amount in between) or more, including, but not limited to, at least 10, 50, 60, 70, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 times (or any amount in between) or more.

[0030] Accordingly, according to another aspect, a method for cleavage of cellular chromatin in a region of interest may include: (a) selecting a first sequence in the region of interest; (b) constructing a first DNA binding molecule for specific binding to the first sequence; (c) cell expression of a first fusion molecule containing a first DNA binding molecule ( eg , zinc finger molecule, TALE, sRNA) and a cleavage domain (or half-domain); and (d) cellular expression of a second fusion protein, wherein the second fusion molecule comprises a second DNA binding domain and a second cleavage domain (or half-domain), wherein at least one of the fusion molecules comprises a linker as described herein; also, the first fusion molecule binds to the first sequence, and the second fusion molecule binds to a second sequence located 2-50 nucleotides from the first sequence, so that the formation of an active nuclease complex and cleavage of cellular chromatin in the region of interest can occur. In some embodiments, both hybrid molecules contain a linker as described herein between the DNA binding domain and the catalytic nuclease domain.

[0031] Also provided are methods for altering a region of cellular chromatin, for example, to introduce targeted mutations. In some embodiments, methods for altering cellular chromatin include introducing into said cell one or more targeted nucleases to provide a double-strand break in the cellular chromatin at a predetermined site, and a donor polynucleotide homologous to the nucleotide sequence of the cellular chromatin at the region of the break. Cellular DNA repair processes are activated in the presence of a double-strand break; the donor polynucleotide is used as a template for rupture repair, which results in the introduction of all or part of the donor's nucleotide sequence into the cellular chromatin. Accordingly, the sequence in the cellular chromatin can be changed and, in some embodiments, can be converted to the sequence present in the donor polynucleotide.

[0032] Targeted changes include, but are not limited to, point mutations ( i.e. , the conversion of one base pair to another base pair), substitutions ( i.e. , the conversion of multiple base pairs to another sequence of identical length), insertions of one or more pairs bases, deletions of one or more base pairs; and any combination of the aforementioned sequence changes. Changes may also include the conversion of base pairs that are part of the coding sequence, so that the encoded amino acid is changed.

[0033] The donor polynucleotide may be DNA or RNA, may be linear or circular, and may be single or double stranded. It can be delivered to the cell as a deproteinized nucleic acid, complexed with one or more delivery agents ( e.g. , liposomes, nanoparticles, poloxamers), or in a viral delivery vehicle such as, for example, adenovirus, lentivirus, or adeno-associated virus ( AAV). The length of the donor sequences can vary from 10 to 1000 nucleotides (or any intermediate integer number of nucleotides) or more. In some embodiments, said donor molecule contains the full length gene flanked by regions of homology to the target cleavage site. In some embodiments, the donor sequence lacks homologous regions and is inserted into the target locus by a homology-independent mechanism ( ie , NHEJ). In other embodiments, said donor molecule comprises a smaller stretch of nucleic acid flanked by homologous regions for use in the cell (ie , for gene editing). In some embodiments, said donor molecule contains a gene encoding a functional or structural component, such as shRNA, RNAi, miRNA, or the like. In other embodiments, said donor molecule contains sequences encoding a regulatory element that binds to and/or modulates the expression of a gene of interest. In other embodiments, said donor is a regulatory protein of interest ( eg, ZFP TF, TALE TF, or CRISPR/Cas TF) that binds to and/or modulates expression of a gene of interest.

[0034] In any of the above methods, cellular chromatin may be located in the genome of a chromosome, episome, or organelle. Cellular chromatin can be found in any type of cell, including, but not limited to, prokaryotic and eukaryotic cells, fungal cells, plant cells, animal cells, mammalian cells, primate cells, and human cells.

[0035] According to another aspect of the invention, cells are also provided that contain any of the polypeptides ( eg, hybrid molecules) and/or polynucleotides as described herein. In one embodiment, said cells comprise a pair of fusion molecules, each containing a cleavage domain as described herein. Cells include cultured cells, cells in the body, and cells that have been removed from the body for treatment/treatment, in cases where these cells and/or their progeny are returned to the body after treatment. The region of interest in the cellular chromatin may be, for example, a genomic sequence or a portion thereof.

[0036] In another aspect, provided herein is a kit comprising a fusion protein as described herein, or a polynucleotide encoding one or more zinc finger proteins, cleavage domains, and/or fusion proteins as described herein; auxiliary reagents; and, optionally, instructions and suitable containers. Said set may also include one or more nucleases or polynucleotides encoding such nucleases.

[0037] These and other aspects will be well understood by a person skilled in the art in light of the above description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In FIG. 1 shows the amino acid sequence (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2) of the wild type Fok I nuclease portion. The sequence reflects the Fok I catalytic nuclease domain and the numbering corresponds to the wild-type Fok I protein used to generate the 1FOK.pdb and 2FOK.pdb crystal structures (Wah, ibid. ) (the Q amino acid of the nuclease domain starts at position 384). Boxed positions indicate possible mutation sites.

[0039] In FIG. 2A-2C are schematic representations of models for the interaction of the Fok I domain with the DNA molecule. On FIG. 2A shows the localization of amino acids R422, R416 and K525. On FIG. 2B shows the localization of the amino acids R447, K448 and R422. Fig. 2C illustrates a subset of different types of ZFNs that can be generated by incorporating 1, 2, or 3 (1×, 2×, or 3×, respectively) mutations (either R→Q or R→L) into the zinc finger backbone. Black arrows indicate the positions of mutations.

[0040] In FIG. 3A and 3B show the activity of BCL11A-specific ZFNs carrying the novel Fok I mutations described herein. On FIG. 3A shows targeted modification in CD34+ cells for the BCL11A-specific ZFN SBS#51857-ELD/SBS#51949-KKR against the BCL11A cognate target (identified by the unique identifier 'registration mark' PRJIYLFN, SEQ ID NO: 13) and two non-target sites, also designated by identifiers - "registration marks" NIFMAEVG (SEQ ID NO: 14) and PEVYOHIU (SEQ ID NO: 20). ZFPs are described in PCT/US 2016/032049. All experiments were performed with 2 µg mRNA of each ZFN for nuclease delivery; the values reflect the percentage of sequence reads that contain insertions and deletions (% indels) corresponding to nuclease activity. On FIG. 3A shows the results of a serine substitution at positions 416, 422, 447, 448, and 525 of the Fok I domain in one or both ZFNs. On FIG. 3B shows a similar data set, except that the backbones of the heterodimeric Fok I dimerization domain are reversed ; in FIG. 3A shows the results using mutations in the SBS#51857-ELD/SBS#51949-KKR pair, while FIG. 3B shows the results using mutations in the SBS#51857-KKR/SBS#51949-ELD pair.

: серый цвет; либо >10

: черный цвет). Каждая точка данных соответствует целевой активности и объединенной нецелевой активности для разных пар ZFN, несущих мутации FokI на одной из ZFN в паре. Указаны точки данных, соответствующие исходной паре.[0041] In FIG. 4 is a graph of target and non-target activity for a number of TCRA-specific (targeting the constant region, also known as TRAC) ZFN Fok I variants (PCT Publication WO 2017106528). With the exception of two replicates of the original ZFN pair, the Fok I domain in one of the two ZFNs carries a positively charged residue mutation. The distance between the alpha carbon of the mutated Fok I residue and the nearest phosphate oxygen in the DNA backbone was calculated in the ZFN-DNA molecular model (Miller et al (2007) Nat Biotech 25(7):778-785); data points are color-coded based on the specified calculated distance (or <10

: grey colour; or >10

: black color). Each data point corresponds to target activity and pooled non-target activity for different pairs of ZFNs carrying Fok I mutations on one of the ZFNs in the pair. The data points corresponding to the original pair are indicated.

[0042] In FIG. 5A and 5B schematically depict the core region of a zinc finger. On FIG. 5A (SEQ ID NO: 3) shows the second finger amino acids of the Zif268 protein, with beta-fold and alpha-helical structures marked. The localization of amino acids involved in the recognition of specific DNA bases (-1-6) is also shown. Positively charged residues that have the potential to interact with the DNA phosphate backbone are marked with boxes. Invariant cysteine residues involved in zinc coordination are underlined. On FIG. 5B is an enlarged 3D image of a single finger (the solid sphere represents a coordinated zinc ion) and shows trends in interaction with DNA from different regions of each zinc finger. DNA is represented in the diagram, where phosphates are indicated by the symbol "P" and DNA bases are enclosed in rounded boxes. Gray arrows indicate the approximate positions of the boxed residues, and black arrows indicate interactions between the zinc finger protein and DNA.

[0043] In FIG. 6 (SEQ ID NO: 4-6) shows the retention of amino acids at each position in the zinc finger. The first rows show the amino acid sequence alignment in the well-known Zif268 and Sp1 zinc fingers (Zif268 finger 2 (SEQ ID NO: 4), Zif268 finger 3 (SEQ ID NO: 5) and Sp1 finger 2 (SEQ ID NO: 6). )). The zinc-coordinating cysteine and histidine residues are framed, as are the recognition helices. The positively charged arginine (R) and lysine (K) residues that are in contact with DNA backbone phosphates are also boxed. The numbers below the first three rows are the frequencies of each amino acid at each position in the 4867 different naturally occurring zinc fingers analyzed. The symbols to the left of the diagram are single-letter codes corresponding to amino acid residues, the frequencies of which are given in the table. At the phosphate contact position, three uncharged amino acids, alanine, leucine, and glutamine (enclosed in ovals) were identified with a low but non-zero frequency of occurrence.

[0044] In FIG. 7A and 7B (SEQ ID NOs: 7 and 8) are images of ZFP backbones, including protein modules with either six zinc fingers (FIG. 7A, SEQ ID NO: 7) or five zinc fingers (FIG. 7B, SEQ ID NO: 8). The symbols above some of the boxed positions indicate mutations that were tested at the indicated position. Each finger is identified by labels F1-F6. Each of these proteins is assembled from three different "modules" designated "Module A", "Module B", and "Module C". Mutations at positions -14, -9 and -5 of the N-terminal finger in each module can be introduced by changing the sequence of the PCR primer used in the assembly process.

[0045] In FIG. 8A-8C are graphs showing the targeted and non-targeted cleavage activity of TCRA (TRAC)-specific ZFN (PCT Publication WO 2017106528) containing a novel backbone with zinc finger mutations. according to the present invention. Both TCRA (TRAC)-specific ZFNs contain 6 zinc finger repeats; for simplicity of the experiment, mutations at position -5 were introduced only into the N-terminal finger of each module (for example, F1, F3 or F5 in a full size ZFN). Accordingly, each individual ZFN may contain 0, 1, 2, or 3 mutations, and a pair of ZFNs as a whole may contain up to a total of 6 mutations (eg, 0, 1, 2, 3, 4, 5, or 6 mutations). The values in the graph represent the average values for all ZFN pairs tested with a given number and type of mutations at position -5. The standard error of the mean is indicated by error bars. For each ZFN pair, activity was measured against the three non-targets listed in Table 3; non-target values averaged to obtain plotted values include the relative activity of the original TCRA (TRAC) ZFN against each of these three non-target targets for each design. On FIG. 8A shows the relative activity of the parent TCRA (TRAC) ZFN, where the datasets reflect changes in either target (black bars) or non-target (grey bars) activity resulting from substitution of the indicated amino acids at position -5 in one or more zinc finger repeats in only one of the two ZFNs in a pair. On FIG. 8B shows the relative level of activity when said arginine-alanine substitution is administered to one or both ZFN partners simultaneously. Left half of Fig. 8B corresponds to ZFN pairs where the indicated number of mutations occurs in only one ZFN in the pair (and corresponds to the left third of FIG. 8A) and the right half of FIG. 8B corresponds to pairs of ZFNs where the same number of mutations were introduced into both ZFNs in a pair (for example, 2 corresponds to one mutation in each ZFN in a pair, 4 corresponds to two mutations in each ZFN in a pair, and 6 corresponds to three mutations in each ZFN in a pair. The experiments shown in Fig. 8A and 8B were performed in CD34+ cells at a dose of 6 μg per experiment. On FIG. 8C shows data similar to the right two thirds of FIG. 8A, with the RNA dosage being 2 μg per experiment.

[0046] In FIG. 9 is a graph showing target (black bars) and non-target (grey bars) cleavage activity at the NIFMAEVG non-target site of BCL11A-specific ZFNs containing the novel zinc finger backbone mutations of the present invention (in this case, use the three-letter abbreviation and indicate the number of arginine residues in position -5, mutated with a change to the specified residue; for example , "6 Gln" means that 6 arginines were mutated in the ZFN pair (3 per ZFN) with a replacement for 6 glutamines). The standard error is indicated by error bars. Experiments were performed on CD34+ cells at a dose of 2 μg mRNA for ZFN per experiment.

[0047] In FIG. 10 shows a Western blot image for detection of BCL11A-specific ZFN expression in CD34+ cells after transfection with either mRNA encoding both ZFN partners on the same polynucleotide linked by the 2A sequence (51857-2a- 51949 ) or dosing of mRNA encoding ZFN separately; or a mixture of two mRNAs encoding each partner, at the indicated doses. Proteins were detected with an anti-Flag antibody and the amount of protein expressed after mRNA transfection was shown to be consistent with the amount of mRNA used. As expected, the 2a construct provided more 5' ZFN SBS#51857 compared to 3'ZFN, SBS#51949.

[0048] In FIG. 11 shows dose titration for the two partners of BCL11A-specific ZFN 51949 and 51857 either at target (BCL11A, left panel) or at non-target NIFMAEVG (right panel). The results demonstrate that changing the ratios of ZFN partners can maintain the target activity along with a decrease in off-target activity (comparison of activity against the target target BCL11A when treated with 60 μg of each mRNA (target activity - 85.92% indels, vs. target activity 86.42% at treated with 60 µg 51949, 6.6 µg 51857) with reduced off-target activity (off-target activity 27.34% with 60 µg of each mRNA; vs. 4.21% indels with 60 µg 51949 and 6.6 µg 51857)).

[0049] In FIG. 12 is a table showing the target and non-target cleaving activity of BCL11A-specific ZFNs when treated with CD34+ cells with either a single mRNA encoding both ZFN partners as described above (51857/51949 2a) or titrated doses of ZFN partners where one partner (51949) contains the Fok I R→S mutation at position 416. The identifying "registration marks" are shown in Table 1 of Example 2 (SEQ ID NOs: 13-53). Data corresponding to PRJIYLFN reflect the relative level of sequence reads in the intended BCL11A target containing indels consistent with ZFN activity. Data matching all other identifying "registration marks" listed in the left column correspond to confirmed or suspected non-target loci for the ZFN 51857/51949 pair. The ratios in the right column correspond to the activity in the sample treated with 51857/51949 2a divided by the activity in the sample treated with titrated 51857/51949 R416S at the indicated locus.

[0050] In FIG. 13 shows the results of an unbiased capture analysis comparing two pairs of ZFNs. The left panel (“original ZFN pair”) shows the results of using the SBS51857 and SBS51949 pair, and the right panel (“variant ZFN pair”) shows the result of applying SBS63014 and SBS65721) containing the original pair and, additionally, ZFP backbone mutations as described in herein, as well as the Fok I R416S mutation in the SBS65721 construct. In particular, in a variant of the pair, each ZFN of the pair contains three R→Q mutations in the fingers, and the SBS65721 construct additionally contains the Fok I R416S mutation. The data demonstrate that these mutations reduce the number of unique captures from 21 locations for the original pair to 4 for the specified variant. In addition, if partners in ZFN pairs are administered in unequal amounts, the number of captures is also reduced. In the case of the original pair, the number of captures is reduced from 21 (equal doses) to 13 (unequal doses) localizations (from 28% to 3.4% of non-target aggregates, respectively), and in the case of the variant pair, the number of captures is reduced from 4 to 2 (from 0 .26% to 0.08% total untargeted cleavage, respectively). The combination of these two approaches results in an overall reduction from 21 sites for the original couple to 2 for the option when dosed by partners at unequal concentrations, with a decrease from 28% non-target events in the pool for the original pair to 0.08% of non-target events in the pool for the option.

[0051] In FIG. 14 shows results demonstrating the reduction in off-target digestion using ZFN as described herein when produced under large scale production conditions. The ZFN pair used included SBS63014 and SBS65722.

[0052] In FIG. 15A-15D show results demonstrating a reduction in off-target cleavage using ZFN mutants (targeting AAVS1) as described herein. On FIG. 15A shows the activity results of the parent ZFN 30035/30054. On FIG. 15B shows target activity and target/non-target cleavage activity ratio for three sets of Fok I mutants: Fok I ELD mutants containing additional single mutations (left dataset); Fok I KKR mutants containing additional single mutations (data set in the middle); and Fok I ELD and KKR mutants containing the same additional single mutations (right dataset). On FIG. 15C shows a target activity matrix where the Fok I ELD or KKR domains contain two mutations, and FIG. 15D shows target/non-target activity ratios for the data shown in FIG. 15C.

[0053] In FIG. 16A and 16B are results demonstrating a reduction in off-target cleavage using examples of AAVS1-targeted ZFN mutants as described herein. On FIG. 16A shows mutants in the context of ELD and KKR, and FIG. 16B shows mutants in the context of ELD-KKR.

[0054] In FIG. 17 shows an alignment of Fok I and Fok I homologues (SEQ ID NOs: 54-64). Hatching shows the degree of conservation. The numbering corresponds to the wild-type Fok I domain (SEQ ID NO: 1).

[0055] In FIG. 18 shows examples of mutations whose positions are mapped to Fok I or Fok I homologues shown in FIG. 17.

DETAILED DESCRIPTION OF THE INVENTION

[0056] The present invention provides methods and compositions for increasing the specificity of targeted cleavage by engineered nucleases by differentially reducing non-targeted cleavage. These methods include reducing non-specific interactions between the Fok I cleavage domain and DNA, reducing non-specific interactions between the zinc finger backbone and DNA, and changing the ratios of each cleavage half-domain partner from the default ratio of 1:1.

general description

[0057] In the practice of the methods, as well as in the preparation and use of compositions as described herein, unless otherwise indicated, standard techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell cultures, recombinant DNA and related fields are used. known from the prior art. These techniques are described in detail in published sources. See for example Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, 1989; and third edition, 2001; Ausubel et al. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and updates from time to time; series: METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (PB Becker, ed.) Humana Press, Totowa, 1999.

Definitions

[0058] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, in single-stranded or double-stranded form. For the purposes of the present invention, these terms are not limiting in relation to the length of the polymer. The terms may include known analogs of naturally occurring nucleotides, as well as nucleotides modified with base, sugar, and/or phosphate moieties ( eg , phosphothioate backbones). In general, an analogue of a particular nucleotide has the same specificity for base pairing; those. the analog of A will pair with the base of T.

[0059] The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. This term also applies to polymers of amino acids in which one or more amino acids are chemical analogues or modified derivatives of the corresponding naturally occurring amino acids.

[0060] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules ( eg , between a protein and a nucleic acid). Not all components of a binding interaction need to be sequence-specific ( eg , contacts with phosphate residues in the DNA backbone), provided that the interaction as a whole remains sequence-specific. Such interactions are typically characterized by a dissociation constant (K _d ) of 10 ^-6 M ^-1 or less. "Affinity" refers to the strength of binding: increased binding affinity correlates with lower K _d . "Non-specific binding" refers to non-covalent interactions that occur between any molecule of interest ( e.g. an engineered nuclease) and a macromolecule ( e.g. DNA), and are independent of the target sequence.

[0061] A "binding protein" is a protein that is capable of non-covalently binding to another molecule. The binding protein may, for example, bind to a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein) and/or a protein molecule (protein binding protein). In the case of a protein-binding protein, the latter may bind to itself (to form homodimers, homotrimers, etc.) and/or may bind to one or more molecules of another protein or proteins. A binding protein may have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activities. In the case of an RNA-targeted nuclease system, the target RNA is heterologous to the nuclease component (Cas9 or Cfp1); both can be constructed.

[0062] A "DNA binding molecule" is a molecule that can bind to DNA. Such a DNA binding molecule may be a polypeptide, a protein domain, a domain within a larger protein, or a polynucleotide. In some embodiments, said polynucleotide is DNA, while in other embodiments, said polynucleotide is RNA. In some embodiments, said DNA-binding molecule is a nuclease protein domain ( e.g. , Fok I domain), while in other embodiments, said DNA-binding molecule is a guide RNA component of an RNA-directed nuclease ( e.g. , Cas9 or Cfp1) .

[0063] A “DNA-binding protein” (or binding domain) is a protein, or domain within a larger protein, that binds DNA in a sequence-specific manner, such as through one or more zinc fingers or through interaction with one or more more RVD in zinc finger protein or TALE, respectively. The term Zinc Finger DNA Binding Protein is often abbreviated to Zinc Finger Protein or ZFP.

[0064] A "zinc finger DNA-binding protein" (or binding domain) is a protein or domain within a larger protein that binds DNA in a sequence-specific manner via one or more zinc fingers, which are amino acid sequence regions in composition of the binding domain, the structure of which is stabilized due to the coordination of the zinc ion. The term Zinc Finger DNA Binding Protein is often abbreviated to Zinc Finger Protein or ZFP.

[0065] A "TALE DNA binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains/units. The repeat domains are involved in the binding of TALE to a cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology to other TALE repeat sequences within the naturally occurring TALE protein. See, for example, US Patent No. 8,586,526, incorporated herein in its entirety by reference.

[0066] Zinc finger and TALE DNA binding domains can be "engineered" to bind to a predetermined sequence of nucleotides, for example, by constructing (altering one or more amino acids) a helical recognition region of a naturally occurring zinc finger protein or by constructing amino acids involved in DNA binding ("double variable repeat residue", or RVD region). Accordingly, engineered zinc finger proteins or TALE proteins are non-naturally occurring proteins. Non-limiting examples of zinc finger and TALE protein engineering methods are represented by design and choice. A designed protein is a non-naturally occurring protein whose design/composition is the result of predominantly rational criteria. Rational design criteria include the use of substitution rules and computer algorithms to process information from a database that stores existing ZFP or TALE design information and binding data. See, for example, US Pat. Nos. 8,586,526; No. 6,140,081; No. 6,453,242; and #6,534,261; see also WO 98/53058; W098/53059; W098/53060; WO 02/016536 and WO 03/016496.

[0067] "Selected"("selected") is a zinc finger protein, TALE protein, or CRISPR/Cas system that is not naturally occurring, obtained mainly by applying an empirical method, such as phage display, two-hybrid "interaction trap" system , rational design or hybrid selection. See for example US 5,789,538; US 5,925,523; US 6,007,988; US 6,013,453; US 6,200,759; WO 95/19431; W096/06166; W098/53057; W098/54311; WO00/27878; WO 01/60970; WO 01/88197 and WO 02/099084.

[0068] "TtAgo" is a prokaryotic Argonaute protein thought to be involved in gene silencing. TtAgo originates from Thermus thermophilus bacteria. See, for example, Swarts et al, ibid .; G. Sheng et al ., (2013) Proc. Natl. Acad. Sci . USA 111, 652). The "TtAgo System" includes all necessary components, including, for example, guide DNA for cleavage by the TtAgo enzyme.

[0069] "Recombination" refers to the process of exchanging genetic information between two polynucleotides, including but not limited to non-homologous end joining (NHEJ) and homologous recombination. For the purposes of the present invention, "homologous recombination (HR)" refers to a specialized form of such exchange occurring, for example, during the repair of double-strand breaks in cells by homology-directed repair mechanisms. This process requires nucleotide sequence homology and uses a "donor" molecule to repair the "target" molecule ( i.e. , the molecule in which the double-strand break has occurred) from the template; and is also known as "non-crossover gene conversion" or "short-path gene conversion" because it results in the transfer of genetic information from the donor to the target. Without wishing to be bound by a particular theory, such transfer may involve the correction of mismatches in the heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis-dependent strand annulation", which uses the donor to resynthesize the genetic information that becomes part of the target, and/ or related processes. Such specialized HR often results in a change in the sequence of the target molecule such that part or all of the donor polynucleotide sequence is incorporated into the target polynucleotide.

[0070] In certain methods as described herein, one or more targeted nucleases as described herein create a double strand break (DSB) in a target sequence (e.g., cellular chromatin) at a predetermined site ( e.g., a gene or locus of interest). LCB mediates the integration of a construct ( e.g. donor) as described in this document. Said construct is not necessarily homologous to the nucleotide sequence at the break. The expression construct may be physically integrated; or, alternatively, the expression cassette is used as a template to repair the gap by homologous recombination, which results in the introduction of all or part of the nucleotide sequence from the expression cassette into the cell chromatin. Accordingly, the first sequence in the cellular chromatin may be altered and, in some embodiments, converted to a sequence present in the expression cassette. Accordingly, the use of the terms "substitute" or "substitution" may refer to the substitution of one nucleotide sequence for another (ie, a sequence substitution in the informational sense), and does not necessarily require a physical or chemical substitution of one polynucleotide for another.

[0071] In any of the methods described herein, additional engineered nucleases can be used to further cleave two strands (double-strand cleavage) at an additional target site within the cell.

[0072] According to certain embodiments of methods for targeted recombination and/or replacement and/or sequence alteration in a region of interest in cellular chromatin, a chromosomal sequence is altered by homologous recombination with an exogenous "donor" nucleotide sequence. Such homologous recombination is stimulated by a double-strand break in the cellular chromatin in the presence of sequences homologous to the break region.

[0073] In any of the methods described herein, the first nucleotide sequence ("donor sequence") may contain sequences that are homologous, but not identical, to genomic sequences in the region of interest, which stimulates homologous recombination with the insertion of a non-identical sequence in the region of interest . Accordingly, in some embodiments, portions of the donor sequence that are homologous to sequences in the region of interest exhibit approximately 80-99% (or corresponding to any intermediate integer value) sequence identity of the genomic sequence to be replaced. In other embodiments, the homology between the donor and genomic sequences is greater than 99%, such as when the donor and genomic sequences differ by only 1 nucleotide over 100 contiguous base pairs. In certain cases, the non-homologous part of the donor sequence may contain sequences not present in the region of interest, respectively, new sequences are introduced into the region of interest. In these cases, the non-homologous sequence is usually flanked by sequences having a length of 50-1000 base pairs (or a length corresponding to any intermediate integer value) or a length corresponding to any number of base pairs in excess of 1000, homologous or identical to sequences in the region of interest. In other embodiments, the donor sequence is not homologous to the first sequence and is incorporated into the genome through non-homologous recombination mechanisms.

[0074] Any of the methods described herein can be used to partially or completely inactivate one or more target sequences in a cell by targeted integration of a donor sequence, or by cleavage of a target sequence or sequences followed by an error-prone NHEJ-mediated repair that destroys expression the gene or genes of interest. Cell lines with partially or completely inactivated genes are also proposed.

[0075] In addition, the methods of targeted integration as described herein can also be used to integrate one or more exogenous sequences. Said exogenous nucleic acid sequence may contain, for example, one or more genes, or one or more cDNA molecules, or any type of coding or non-coding sequence, as well as one or more control elements ( eg, promoters). In addition, the exogenous nucleic acid sequence can produce one or more RNA molecules ( eg , small hairpin RNAs (shRNAs), inhibitory RNAs (RNAi), microRNAs (miRNAs), etc.).

[0076] "Cleavage" refers to a break in the covalent backbone of a DNA molecule. Cleavage can be initiated in a variety of ways, including, but not limited to, enzymatic or chemical hydrolysis of the phosphodiester bond. Both single strand and double strand cleavage is possible, and two strand cleavage may result from two separate single strand cleavages. Cleavage of DNA can lead to the formation of either "blunt" ends or stepped ends. In some embodiments, hybrid polypeptides are used to target double-stranded DNA cleavage.

[0077] A "cleavage half-domain" is a polypeptide sequence which, when combined with a second polypeptide (identical or different), forms a complex having cleaving activity (preferably double-stranded (cleaving both strands) cleaving activity). The terms "first and second splitting half-domains";"+ and - cleavage half-domains" and "right and left cleavage half-domains" are used interchangeably to refer to pairs of cleavage half-domains that dimerize. The term "cleavage domain" is used interchangeably with the term "cleavage half-domain". The term " FokI cleavage domain" includes the FokI sequence shown in SEQ ID NO: 1, as well as any FokI homologues, including, but not limited to, the sequences shown in FIG. 17.

[0078] An "engineered cleavage half-domain" is a cleavage half-domain modified to form obligate heterodimers with another cleavage half-domain ( eg, another engineered cleavage half-domain).

[0079] The term "sequence" refers to a sequence of nucleotides of any length, which can be represented by DNA or RNA; may be linear, circular or branched, and may be either single or double stranded. The term "transgene" refers to a sequence of nucleotides that is integrated into the genome. The transgene may be of any length, for example, from 2 to 100,000,000 nucleotides (or a length corresponding to any intermediate or greater integer value), preferably from about 100 to 100,000 nucleotides (or a length corresponding to any intermediate integer value), more preferably - a length of about 2,000 to 20,000 nucleotides (or any intermediate integer value); even more preferably, a length of from about 5 to 15 kB (or a length corresponding to any intermediate integer value).

[0080] "Chromosome" is a complex of chromatin containing the entire genome or part of the genome of the cell. The genome of a cell is often characterized by its karyotype, which is the totality of all the chromosomes that are contained in the cell's genome. The genome of a cell may contain one or more chromosomes.

[0081] "Episome" is a replicating nucleic acid, nucleoprotein complex, or other structure containing a nucleic acid that is not part of the cell's chromosomal karyotype. Examples of episomes include plasmids, minicircles, and certain viral genomes. The liver-specific constructs described herein can be maintained in episomal form, or alternatively can be stably integrated into a cell.

[0082] An "exogenous" molecule is a molecule that is not normally present in a cell, but can be introduced into a cell using one or more genetic, biochemical, or other means. "Normal presence in a cell" is defined for a specified cell in relation to a particular stage of development and specific environmental conditions. Accordingly, for example, a molecule that is present only during embryonic muscle development is an exogenous molecule when applied to an adult muscle cell. Similarly, a heat shock induced molecule is an exogenous molecule when applied to a cell that has not been subjected to heat shock. The exogenous molecule may include, for example, a functional variant of a misbehaving endogenous molecule or a misbehaving variant of a normally functioning endogenous molecule.

[0083] The exogenous molecule may be, including, a small molecule, for example, obtained using a combinatorial chemistry method, or a macromolecule, such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules , or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, may be single or double stranded; linear, branched or ring; and can be of any length. Nucleic acids include duplex-forming as well as triplex-forming nucleic acids. See, for example, US Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, ligases, deubiquitinases, integrases, recombinases, ligases, topoisomerases, gyrase and helicase.

[0084] An exogenous molecule may refer to the same type of molecule as an endogenous molecule, such as an exogenous protein or nucleic acid. For example, the exogenous nucleic acid may contain an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in said cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes, including those with neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. Also, an exogenous molecule may be of the same type of molecule as an endogenous molecule, but come from a species different from that of the cell. For example, a human nucleic acid sequence can be introduced into a cell line originally derived from a mouse or hamster. Methods for introducing exogenous molecules into plant cells are known to those skilled in the art and include, but are not limited to, protoplast transformation, silicon carbide ( e.g. , WHISKERS™), Agrobacterium -mediated transformation, lipid-mediated transfer ( i.e. , liposomes, including with neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment ( e.g. using a "gene gun"), calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0085] In contrast, an "endogenous" molecule is a molecule normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid may include a chromosome, mitochondrial, chloroplast, or other organelle genome, or a naturally occurring episomal nucleic acid. Additional endogenous molecules may include proteins, such as transcription factors and enzymes.

[0086] As used herein, the term "exogenous nucleic acid product" includes both polynucleotide and polypeptide products, such as transcription products (polynucleotides such as RNA) and translation products (polypeptides).

[0087] A "hybrid" molecule is one in which two or more subunit molecules are linked, preferably covalently linked. Subunit molecules may refer to molecules of the same chemical type, or may refer to molecules of different chemical types. Examples of fusion molecules include, but are not limited to, fusion proteins (e.g., a protein DNA binding domain fusion with a cleavage domain), a fusion polynucleotide DNA binding domain ( e.g., sRNA) operably linked to a cleavage domain, and fusion nucleic acids (e.g., , the nucleic acid encoding the fusion protein).

[0088] Expression of a fusion protein in a cell can result from delivery of said fusion protein to said cell, or delivery of a polynucleotide encoding said fusion protein into a cell, where said polynucleotide is transcribed and the transcript is translated to produce said fusion protein. Protein expression in a cell may also involve trans-splicing, polypeptide cleavage, and polypeptide ligation. Methods for delivering polynucleotides and polypeptides to cells are described in various sections of this document.

[0089] "Gene" for the purposes of the present invention includes the region of DNA encoding the gene product (see below), as well as all DNA regions that regulate the production of the specified gene product, while such regulatory sequences can be either contiguous or non-contiguous coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulation sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and a locus. - control elements.

[0090] "Gene expression" refers to the conversion of information contained in a gene into a gene product. A gene product may be a direct transcription product of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation and editing, as well as proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation and glycosylation.

[0091] "Modulation" of gene expression refers to a change in the activity of a gene. Modulation of expression may include, but is not limited to, gene activation and gene repression. Genome editing (eg, cleavage, alteration, inactivation, random mutation) can be used to modulate expression. Gene inactivation refers to any decrease in gene expression compared to expression in a cell that contains ZFP, TALE, or the CRISPR/Cas system as described herein. Accordingly, gene inactivation can be partial or complete.

[0092] A "region of interest" is any region of cellular chromatin, such as, for example, a gene or a non-coding sequence within or adjacent to a gene, to which an exogenous molecule is desired to be linked. Linking may be for targeted DNA cleavage and/or targeted recombination. The region of interest may be, for example, in a chromosome, in an episome, in the genome of an organelle (eg, mitochondria, chloroplast) or the infecting viral genome. The region of interest may be within the coding region of a gene, within transcribed non-coding regions such as, for example, leader sequences, trailer sequences, or introns, or within non-transcribed regions, 5' or 3' from the coding region. . The region of interest can be as short as one base pair, or as long as 2000 base pairs, or any integer number of base pairs.

[0093] A safe harbor locus is a locus within the genome where a gene can be inserted without any detrimental effects to the host cell. Most preferred is a safe harbor locus in which expression of the inserted gene sequence is not disturbed by any pass-through expression of adjacent genes. Non-limiting examples of safe harbor loci for targeting a nuclease or nucleases include CCR5, HPRT, AAVS1, Rosa , and albumin. See, for example, US Pat. Nos. 7,951,925; No. 8,771,985; #8,110,379; No. 7,951,925; U.S. Publication No. 20100218264; No. 20110265198; No. 20130137104; No. 20130122591; No. 20130177983; No. 20130177960; No. 20150056705 and No. 20150159172.

[0094] "Reporter gene" or "reporter sequence" refers to any sequence that produces a protein product that is easy to measure, preferably, although not necessarily, using routine analysis. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance ( e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding stained or fluorescent or luminescent proteins ( eg , green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins mediating enhanced cell growth and/or gene amplification ( eg , dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. "Expression tags" include sequences encoding reporters that can be operably linked to a desired gene sequence to monitor expression of a gene of interest.

[0095] "Eukaryotic" cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T cells), including stem cells (pluripotent and multipotent) .

[0096] The terms "operably linked" and "operably linked" are used interchangeably in relation to the relative positioning of two or more components (such as elements of a sequence), in which these components are organized in such a way as to ensure the normal functioning of both components and the possibility of mediating at least one component of a function performed on at least one other component. For example, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if said transcriptional regulatory sequence controls the level of transcription of said coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional control sequence is typically cis-operably linked to a coding sequence, but need not be directly adjacent to said coding sequence. For example, an enhancer is a transcriptional regulation sequence that is operably linked to a coding sequence even though they are not contiguous.

[0097] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid with a sequence that is not identical to the full-length protein, polypeptide, or full-length nucleic acid, but retains the same function as the full-length protein, polypeptide or nucleic acid. A functional fragment may contain more, fewer, or the same number of residues as the corresponding natural molecule, and/or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid or protein (eg, analysis of coding function, ability to hybridize with another nucleic acid, enzymatic activity) are well known in the art.

[0098] A polynucleotide "vector" or "construct" is capable of transferring gene sequences to target cells. Generally, "vector construct", "expression vector", "expression construct", "expression cassette", and "gene transfer vector" means any nucleic acid construct capable of directing expression of a gene of interest and transferring gene sequences to target cells. Accordingly, the term includes cloning and expression bases as well as integrating vectors.

[0099] The terms "subject" and "patient" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice; and other animals. Accordingly, the term "subject" or "patient" as used herein means any patient or mammalian subject to whom the expression cassettes of the present invention may be administered. Subjects according to the present invention include subjects suffering from a disorder.

[0100] The terms "treat" (or "treat") and "treatment" (or "treatment") as used herein refer to reducing the severity and/or frequency of symptoms, eliminating symptoms and/or the underlying cause, preventing the onset of symptoms and/or their root cause, and alleviate or repair the damage. Cancer, monogenic diseases, and graft-versus-host disease are non-limiting examples of conditions that can be treated using the compositions and methods described herein.

часть хроматина эукариотических клеток находится в форме нуклеосом, при этом нуклеосомный кор содержит приблизительно 150 пар оснований ДНК, ассоциированных с октамером, содержащим по две копии каждого из гистонов H2A, H2B, H3 и H4; и линкерную ДНК (вариабельной длины в зависимости от организма), расположенную между нуклеосомными корами. Молекула гистона H1 обычно ассоциирована с линкерной ДНК. Подразумевается, что для целей настоящего изобретения термин «хроматин» охватывает все типы клеточных нуклеопротеинов, как прокариотических, так и эукариотических. Клеточный хроматин включает как хромосомный, так и эписомный хроматин.[0101] "Chromatin" is a nucleoprotein structure containing the cellular genome. Cellular chromatin contains nucleic acid, primarily DNA, and protein, including histones and nonhistone chromosomal proteins.

part of the chromatin of eukaryotic cells is in the form of nucleosomes, while the nucleosome core contains approximately 150 base pairs of DNA associated with an octamer containing two copies of each of the histones H2A, H2B, H3 and H4; and linker DNA (of variable length depending on the organism) located between the nucleosomal cores. The H1 histone molecule is usually associated with linker DNA. For the purposes of the present invention, the term "chromatin" is intended to encompass all types of cellular nucleoproteins, both prokaryotic and eukaryotic. Cellular chromatin includes both chromosomal and episomal chromatin.

[0102] An "accessible region" is a site in cellular chromatin where a target site present in a nucleic acid can be bound by an exogenous molecule that recognizes said target site. Without wishing to be bound by a particular theory, it is believed that the accessible region is the region not packaged in a nucleosomal structure. The distinctive structure of the accessible region can often be detected by sensitivity to chemical and enzymatic probes, such as nucleases.

[0103] A "target site" or "target sequence" is a nucleic acid sequence that defines the portion of the nucleic acid to which the binding molecule binds, if there are sufficient conditions for binding. For example, the sequence 5'GAATTC3' is the target site of the Eco RI restriction endonuclease. A "intended" or "target" sequence is a sequence to which a binding molecule is expected to bind, and a "non-intended" or "non-target" sequence includes any sequence bound by a binding molecule that is not the intended target.

DNA binding molecules/domains

[0104] This document describes compositions containing a DNA-binding molecule/domain that specifically binds to a target site at any gene or locus of interest. Any DNA binding molecule/domain may be used in the compositions and methods as described herein, including, but not limited to, zinc finger DNA binding domain, DNA binding domain TALE, DNA-binding portion of CRISPR/Cas nuclease (guides or sRNA) or DNA-binding domain from meganuclease.

[0105] In some embodiments, said DNA binding domain comprises a zinc finger protein. Preferably, said zinc finger protein is non-naturally occurring, in the sense that it is designed to bind to the selected target site.See for example, sources: Beerliet al. (2002)nature biotechnol. 20:135-141; Paboet al. (2001)Ann. Rev. Biochem. 70:313-340; Isalanet al. (2001)nature biotechnol. 19:656-660; Segalet al. (2001)Curr. Opin. Biotechnol. 12:632-637; Chooet al. (2000)Curr. Opin. Struct. Biol. 10:411-416; US Pat. Nos. 6,453,242; #6,534,261; #6,599,692; No. 6,503,717; No. 6,689,558; No. 7,030,215; No. 6,794,136; No. 7,067,317; No. 7,262,054; No. 7,070,934; No. 7,361,635; No. 7,253,273; and US Patent Publications No. 2005/0064474; No. 2007/0218528; No. 2005/0267061, each of which is incorporated herein in its entirety by reference. In some embodiments, said DNA binding domain comprises the zinc finger protein described in US Patent Publication No. 2012/0060230 (for example,in Table 1), incorporated herein in its entirety by reference.

[0106] The engineered zinc finger binding domain may have novel binding specificity compared to a naturally occurring zinc finger protein. Design methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of databases containing triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more zinc finger amino acid sequences that bind a particular triplet or quadruplet sequence. See, for example, US Pat. Nos. 6,453,242 and 6,534,261, incorporated herein by reference in their entirety.

[0107] Examples of selection methods, including using phage display and two-hybrid systems, are described in US patents 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as WO 98/37186; W098/53057; WO00/27878; WO 01/88197 and GB 2,338,237. In addition, increased binding specificity for zinc finger binding domains has been described, for example, in US Pat. No. 6,794,136.

[0108] In addition, as described in this and other references, zinc finger domains and/or multiple zinc finger proteins can be connected to each other using any suitable linker sequences, including, for example, linkers of length 5 or more amino acids. See also examples of linker sequences of 6 or more amino acids in US Pat. Nos. 6,479,626; No. 6,903,185; and #7,153,949. The proteins described herein may contain any combination of suitable linkers between the individual zinc fingers of said protein. In addition, increased binding specificity for zinc finger binding domains has been described, for example, in US Pat. No. 6,794,136.

[0109] Selecting a target site; ZFPs and methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art and are detailed in US Pat. Nos. 6,140,081; No. 5,789,538; No. 6,453,242; #6,534,261; No. 5,925,523; No. 6,007,988; No. 6,013,453; No. 6,200,759; WO 95/19431; W096/06166; W098/53057; W098/54311; WO00/27878; WO 01/60970 WO 01/88197; WO 02/099084; W098/53058; W098/53059; W098/53060; WO 02/016536 and WO 03/016496.

[0110] In addition, as described in this and other references, zinc finger domains and/or multiple zinc finger proteins can be connected to each other using any suitable linker sequences, including, for example, linkers of 5 or more amino acids in length. . See also examples of linker sequences of 6 or more amino acids in US Pat. Nos. 6,479,626; No. 6,903,185; and #7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of said protein.

[0111] Typically, ZFPs include at least three fingers. Certain ZFPs include four, five, or six fingers. ZFPs that include three fingers typically recognize a target site that is 9 or 10 nucleotides long; ZFPs that include four fingers typically recognize a target site that is 12 to 14 nucleotides long; while ZFP containing six fingers is able to recognize target sites that include 18 to 21 nucleotides. ZFPs may also be fusion proteins that include one or more regulatory domains, which may be transcriptional activation or repression domains.

[0112] In some embodiments, said DNA-binding domain may be derived from a nuclease. For example, recognition sequences by homing endonucleases and meganucleases are known, such as I-Sce I, I- Ceu I, PI - Psp I, PI- Sce , I -Sce IV, I- Csm I, I- Pan I, I -Sce II, I -Ppo I, I -Sce III, I- Cre I, I -Tev I, I -Tev II and I -Tev III. See also U.S. Patent No. 5,420,032; US Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al . (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to an unnatural target site. See, for example , Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 20070117128.

[0113] In some embodiments, the zinc finger protein used with the mutant cleavage domains described herein contains one or more mutations (substitutions, deletions, and/or insertions) in regions of the backbone ( e.g., regions outside the helical recognition region of size 7 amino acids numbered from -1 to 6), for example, at one or more of positions -14, -9 and/or -5 ( see, for example, Fig . 5A). The remainder of the wild-type molecule at one or more of these positions may be deleted, replaced by any amino acid residue, and/or include one or more additional residues. In some embodiments, the Arg (R) at position -5 is replaced with Tyr (Y), Asp (N), Glu (E), Leu (L), Gln (Q), or Ala (A). In other embodiments, Arg (R) at position (-9) is replaced by Ser (S), Asp (N), or Glu (E). According to additional implementation options, Arg (R) at position (-14) is replaced by Ser (S) or Gln (Q). In other embodiments, the fusion polypeptides may contain mutations in the zinc finger DNA binding domain, wherein the amino acids at positions (-5), (-9) and/or (-14) are substituted for any of the above amino acids in any combination.

[0114] In other embodiments, the DNA binding domain comprises an engineered domain from a transcription activator (TAL)-like effector (TALE) similar to those derived from plant pathogens Xanthomonas (see Boch et al , (2009) Science 326: 1509-1512; and Moscou and Bogdanove, (2009) Science 326: 1501) and Ralstonia (see Heuer et al (2007) Applied and Environmental Microbiology 73(13): 4379-4384); U.S. Patent Publications No. 20110301073 and No. 20110145940. Plant pathogenic bacteria from the genus Xanthomonas are known to cause many diseases in important agricultural plants. The pathogenicity of Xanthomonas depends on a conserved type III secretion system (T3S), which introduces more than 25 different effector proteins into the plant cell. Among these introduced proteins are transcription activator-like effectors (TALE) mimicking plant transcription activators and manipulating the plant transcriptome (see Kay et al (2007) Science 318:648-651). These proteins contain a DNA-binding domain and a transcriptional activation domain. One of the best described TALEs is AvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 and WO 2010079430). TALE contain a centrally located domain of tandem repeats, where each repeat contains approximately 34 amino acids, which play a key role in the DNA-binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcription activation domain (for a review see Schornack S, et al (2006) J Plant Physiol 163(3): 256-272). In addition, the phytopathogenic bacteria Ralstonia solanacearum , biovar 1 strain GMI1000 and R. solanacearum biovar 4 strain RS1000, have two genes homologous to the AvrBs3 Xanthomonas family, called brg11 and hpx17 (see Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). These genes are 98.9% identical to each other in nucleotide sequence, but differ in the deletion of 1575 base pairs in the hpx17 repeat domain. However, both gene products share less than 40% sequence identity with the AvrBs3 Xanthomonas protein family.

[0115] The specificity of these TAL effectors depends on the sequences found in the tandem repeats. The repeat sequence contains approximately 102 base pairs, and the repeats are typically 91-100% homologous to each other (Bonas et al, ibid.). The repeat polymorphism is typically located at positions 12 and 13, and there appears to be a one-to-one correspondence between the identity of the hypervariable double residues (variable repeat double residue, or RVD region) at positions 12 and 13, and the identity of the contiguous nucleotides in the target TAL- effector (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al (2009) Science 326:1509-1512). Under experimental conditions, it was determined that in the natural code for DNA recognition of these TAL effectors, the HD sequence at positions 12 and 13 (double variable repeat residue, or RVD) leads to binding to cytosine (C), NG binds to T, NI to A , C, G, or T, NN binds to A or G, and ING binds to T. These DNA-binding repeats have been assembled into proteins with novel combinations and repeat numbers, yielding artificial transcription factors capable of interacting with novel sequences and activating expression non-endogenous reporter gene in plant cells (Boch et al, ibid.). The engineered TAL proteins were fused to the FokI cleavage half-domain, resulting in hybridization with the TAL effector nuclease domain (TALEN), including TALEN with atypical RVDs. See, for example, US Pat. No. 8,586,526.

[0116] In some embodiments, TALEN contains a cleavage domain or a cleavage half-domain of an endonuclease ( eg, Fok I). In other embodiments, the TALE nuclease is mega-TAL. These mega-TAL nucleases are fusion proteins containing the TALE DNA-binding domain and the meganuclease cleavage domain. The meganuclease cleavage domain is active in monomer form and does not require dimerization to be active. (See Boissel et al ., (2013) Nucl Acid Res : 1-13, doi: 10.1093/nar/gkt1224).

[0117] In other additional embodiments, the nuclease contains a compact TALEN. These are single-stranded fusion proteins where the TALE DNA-binding domain is linked to the TevI nuclease domain. This fusion protein can either function as a nickase with a localization defined by the TALE region, or can create a double-strand break, depending on where the TALE DNA-binding domain is located relative to the TevI nuclease domain (see Beurdeleyet al (2013)NatComm: 1-8 DOI: 10.1038/ncomms2782). In addition, the nuclease domain may also exhibit DNA-binding functions. Any TALEN can be used in combination with additional TALENs (for example,With one or more TALEN (cTALEN orFoxI-TALEN) with one or more mega-TALE.

[0118] In addition, as described in these and other references, zinc finger domains and/or multiple zinc finger proteins or TALE can be linked together using any suitable linker sequences, including, for example, linkers of length 5 or more amino acids. See also examples of linker sequences of 6 or more amino acids in US Pat. Nos. 6,479,626; No. 6,903,185; and #7,153,949. The proteins described herein may contain any combination of suitable linkers between the individual zinc fingers of the protein. In addition, increased binding specificity for zinc finger binding domains has been described, for example, in US Pat. No. 6,794,136. In some embodiments, the DNA binding domain is part of a CRISPR/Cas nuclease system comprising a single guide RNA (sRNA) DNA binding molecule that binds to DNA. See, for example, U.S. Patent No. 8,697,359 and U.S. Patent Publications No. 20150056705 and No. 20150159172. The CRISPR locus (clustered regularly distributed short palindromic repeats), which encodes the RNA components of this system, and the Cas (CRISPR-associated) locus, which encodes proteins (Jansen et al ., 2002. Mol. Microbiol . 43: 1565-1575; Makarova et al ., 2002. Nucleic Acids Res. 30: 482-496; Makarova et al ., 2006. Biol. Direct 1: 7; Haft et al ., 2005. PLoS Comput. Biol . 1: e60) constitute the gene sequences of the CRISPR system. /nuclease Cas. Microorganism host CRISPR loci contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage.

[0119] In some embodiments, said DNA-binding domain is part of the TtAgo system (see Swarts et al , ibid ; Sheng et al , ibid ). In eukaryotes, gene silencing is mediated by proteins of the Argonaute (Ago) family. According to this paradigm, Ago is associated with small (19-31 nucleotides) RNA. This protein-RNA silencer recognizes target RNAs through Watson-Crick base pairing between small RNA and target, and cleaves the target RNA endonucleolytically (Vogel (2014) Science 344:972-973). In contrast, prokaryotic Ago proteins bind to small single-stranded DNA fragments; their function is presumably to detect and remove foreign (often viral) DNA (Yuan et al ., (2005) Mol. Cell 19, 405; Olovnikov, et al . (2013) Mol. Cell 51, 594; Swarts et al . ., ibid. ). Examples of prokaryotic Ago proteins include those from Aquifex aeolicus , Rhodobacter sphaeroides , and Thermus thermophilus .

[0120] One of the best characterized prokaryotic Ago proteins is from T. thermophilus (TtAgo; Swarts et al . ibid. ). TtAgo binds to single-stranded DNA fragments of 15 nucleotides or 13-25 nucleotides with 5' phosphate groups. This TtAgo-linked "guide DNA" serves to direct the protein-DNA complex to bind the Watson-Crick complementary DNA sequence in a foreign DNA molecule. Once the sequence information in these guide DNAs has provided identification of the target DNA, the TtAgo/guide DNA complex cleaves the target DNA. Such a mechanism is also supported by the structure of the TtAgo/guide DNA complex when bound to the target DNA (G. Sheng et al., ibid. ). Ago from Rhodobacter sphaeroides (RsAgo) has similar properties (Olivnikov et al. , ibid. ).

[0121] Exogenous DNA guides of an arbitrary DNA sequence can be loaded with the TtAgo protein (Swarts et al. ibid. ). Since the specificity of TtAgo cleavage is controlled by the guide DNA, the TtAgo/DNA complex formed with the exogenous examiner-selected guide DNA accordingly directs the targeted TtAgo DNA cleavage to a target DNA complementary to the examiner-selected one. Thus, a targeted double-strand break in DNA can be created. The use of the TtAgo/guide DNA system (or orthologous Ago/guide DNA systems from other organisms) allows targeted cleavage of genomic DNA in cells. This cleavage may be single or double stranded. For cleavage of mammalian genomic DNA, it is preferable to use a TtAgo variant codon-optimized for expression in mammalian cells. In addition, it may be preferable to treat the cell with an in vitro TtAgo-DNA complex in which the TtAgo protein is hybridized to a cell-penetrating peptide. In addition, it may be preferable to use a TtAgo protein variant that has been altered by mutagenesis to provide increased activity at 37°C. Ago/RNA-mediated DNA cleavage can be used to influence a variety of outcomes, including gene knockout, targeted gene addition, gene editing, targeted gene deletion using standard DNA break techniques in the art.

[0122] Accordingly, any DNA binding molecule/any DNA binding domain can be used.

hybrid molecules

[0123] Also provided are hybrid molecules containing DNA-binding domains ( e.g. , ZFP or TALE, CRISPR/Cas components such as a single guide RNA) as described herein, and a heterologous regulatory (functional) domain (or a functional fragment thereof) . Common domains include, for example, transcription factor domains (activators, repressors, coactivators, corepressors), silencers, oncogenes ( e.g. myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos, etc.) .P.); DNA repair enzymes and their associated factors and modifiers; DNA rearrangement enzymes and associated factors and modifiers; chromatin-associated proteins and their modifiers ( eg kinases, acetylases and deacetylases); as well as DNA-modifying enzymes ( eg, methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases) and their associated factors and modifiers. See U.S. Patent Publications No. 20050064474 for a detailed description of hybrid DNA binding domains and nuclease cleavage domains; No. 20060188987 and No. 2007/0218528, incorporated herein in their entirety by reference.

[0124] Suitable domains for providing activation include the HSV VP16 activation domain (see, for example, Hagmann et al., J. Virol . 71, 5952-5962 (1997)), nuclear hormone receptors (see, for example, Torchia et al ., Curr Opin Cell Biol 10:373-383 (1998)); nuclear factor kappa-B p65 subunit (Bitko & Barik, J. Virol . 72:5610-5618 (1998) and Doyle & Hunt, Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther . 5:3-28 (1998)); or artificial chimeric functional domains, for example VP64 (Beerli et al ., (1998) Proc. Natl. Acad. Sci. USA 95:14623-33); and degron (Molinari et al ., (1999) EMBO J. 18, 6439-6447). Additional examples of activation domains include Oct 1, Oct-2A, Sp1, AP-2 and CTF1 (Seipel et al., EMBO J . 11, 4961-4968 (1992) as well as p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2 See, for example, Robyr et al (2000) Mol Endocrinol 14:329-347 Collingwood et al (1999) J Mol Endocrinol 23:255-275 Leo et al ( 2000) Gene 245:1-11 Manteuffel-Cymborowska (1999) Acta Biochim Pol 46:77-89 McKenna et al (1999) J Steroid Biochem Mol Biol 69:3-12 Malik et al (2000) Trends Biochem Sci 25:277-283 and Lemon et al (1999) Curr Opin Genet Dev 9:499-504 Additional examples of activation domains include, but are not limited to, OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7 and -8, CPRF1, CPRF4, MYC-RP/GP and TRAB1 See for example Ogawa et al (2000) Gene 245:21- 29 Okanami et al (1996) Genes Cells 1:87-99 Goff et al (1991) Genes Dev 5:298-309 Cho et al (1999) Plant Mol Biol 40:419-429 ; Ulmason et al . (1999) Proc Natl Acad Sci USA 96:5844-5849; Sprenger- Haussels et al . (2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. biol . 41:33-44; and Hobo et al . (1999) Proc. Natl. Acad. sci. USA 96:15,348-15,353.

[0125] Those skilled in the art will appreciate that when a fusion protein (or nucleic acid encoding it) is formed between a DNA binding domain and a functional domain, either an activation domain or a molecule that interacts with an activation domain is suitable for use as a functional domain. . Substantially any molecule capable of recruiting an activating complex and/or activating activity (such as, for example, histone acetylation) to a target gene is suitable for use as the activating domain of the fusion protein. Insulator domains, localization domains, and chromatin remodeling proteins such as ISWI-containing domains and/or proteins containing methyl-binding domains suitable for use as functional domains in hybrid molecules are described, for example, in US Patent Publications 2002/0115215 and 2003/0082552 and also WO 02/44376.

[0126] Examples of repressor domains include, but are not limited to, KRAB A/B, KOX, TGF-beta inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B ), Rb and MeCP2. See, for example, Bird et al . (1999) Cell 99:451-454; Tyler et al . (1999) Cell 99:443-446; Knoepfler et al . (1999) Cell 99:447-450; and Robertson et al . (2000) Nature Genet . 25:338-342. Additional examples of repressor domains include, but are not limited to, ROM2 and AtHD2A. See, for example, Chem et al . (1996) Plant Cell 8:305-321; and Wu et al . (2000) Plant J. 22:19-27.

[0127] Fusion molecules are constructed using cloning and biochemical conjugation methods well known to those skilled in the art. Fusion molecules contain a DNA-binding domain and a functional domain ( for example , a transcriptional activation or repression domain). The fusion molecules also optionally contain nuclear localization signals (such as, for example, SV40 mid-T antigen signals) and epitope tags (such as, for example, FLAG and hemagglutinin). Fusion proteins (and the nucleic acids encoding them) are produced by design so that all fusion components retain the translational reading frame.

[0128] Fusion products of a functional domain polypeptide component (or a functional fragment thereof) on the one hand and a non-protein DNA binding domain (e.g., antibiotic, intercalator, minor groove binding component, nucleic acid) on the other hand are constructed using biochemical conjugation methods known to those skilled in the art. See, for example, the catalog of the Pierce Chemical Company (Rockford, Illinois). Methods and compositions have been described for producing a minor groove-binding component hybridized to a polypeptide. Mapp et al . (2000) Proc. Natl. Acad. sci. USA 97:3930-3935. In addition, the single guide RNA of the CRISPR/Cas system binds to functional domains to form active transcriptional regulators and nucleases.

[0129] In some embodiments, the target site is located in an accessible region of cellular chromatin. Accessible areas can be defined as described in, for example, US Pat. Nos. 7,217,509 and 7,923,542. In cases where the target site is not in an accessible region of the cellular chromatin, one or more accessible regions can be obtained as described in US Pat. Nos. 7,785,792 and 8,071,370. In additional embodiments, the DNA binding domain of the fusion molecule is capable of binding to cellular chromatin whether its target site is in an accessible region or not. For example, such DNA binding domains are capable of binding to linker DNA and/or nucleosomal DNA. Examples of this type of "pioneer" DNA binding domains can be found in certain steroid receptors and in hepatocyte nuclear factor 3 (HNF3) (Cordingley et al . (1987) Cell 48:261-270; Pina et al . (1990) Cell 60: 719-731 and Cirillo et al (1998) EMBO J 17:244-254).

[0130] The hybrid molecule may be formulated with a pharmaceutically acceptable carrier, as is known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 17th ed., 1985; and US Pat. Nos. 6,453,242 and 6,534,261.

[0131] The functional component/domain of the fusion molecule can be selected from any of a variety of different components capable of influencing gene transcription after the fusion molecule has bound to a target sequence via its DNA-binding domain. Thus, said functional component may include, but is not limited to, domains of various transcription factors such as activators, repressors, coactivators, corepressors, and silencers.

[0132] Additional examples of functional domains are described in, for example, US Pat. Nos. 6,534,261 and 6,933,113.

[0133] Functional domains regulated by exogenous small molecules or ligands can also be selected. For example, RheoSwitch® technology can be used, according to which the functional domain changes into an active conformation only in the presence of an external RheoChem™ ligand (see, for example, US 20090136465). Accordingly, ZFP can be operably linked to a regulable functional domain, with the resulting ZFP-TF activity controlled by an external ligand.

Nucleases

[0134] In some embodiments, the fusion protein contains a DNA-binding domain and a cleavage domain (nuclease domain). Accordingly, gene modification can be achieved using a nuclease, such as an engineered nuclease. Engineered nuclease technology is based on the construction of naturally occurring DNA-binding proteins. For example, the construction of homing endonucleases with individualized DNA binding specificity has been described. Chames et al . (2005) Nucleic Acids Res 33(20):e178; Arnould et al . (2006) J. Mol. biol . 355:443-458. In addition, the construction of ZFP has also been described. See, for example, US Pat. Nos. 6,534,261; No. 6607882; No. 6824978; No. 6979539; No. 6933113; No. 7163824; and No. 7013219.

[0135] In addition, ZFP and/or TALE were fused to nuclease domains to produce ZFN and TALEN, a functional entity capable of recognizing the intended target nucleic acid by the engineered DNA binding domain (ZFP or TALE) and leading to DNA cleavage near the binding site DNA through nuclease activity. See, for example, Kim et al . (1996) Proc Nat'l Acad Sci USA 93(3):1156-1160. Recently, such nucleases have been used to modify the genome in various organisms. See, for example, US Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and international publication WO 07/014275.

[0136] Accordingly, the methods and compositions described herein are broadly applicable and may involve any nuclease of interest. Non-limiting examples of nucleases include meganucleases, TALEN, and zinc finger nucleases. Said nuclease may contain a heterologous DNA binding domain and a cleavage domain (eg, zinc finger nucleases; meganuclease DNA binding domains with heterologous cleavage domains); or alternatively, the DNA-binding domain of a naturally occurring nuclease can be altered to bind to a chosen target site ( eg , a meganuclease engineered to bind to a site other than the cognate binding site).

[0137] Any of the nucleases described herein may contain an engineered TALE DNA binding domain and a nuclease domain ( eg , an endonuclease and/or meganuclease domain), also referred to as TALEN. Methods and compositions for constructing these TALEN proteins for stable site-specific interaction with a user-selected target sequence have been described in published sources (see US patent No. 8,586,526). In some embodiments, said TALEN contains a cleavage domain or cleavage half-domain of an endonuclease ( eg , Fok I). In other embodiments, said TALE nuclease is mega-TAL. These mega-TAL nucleases are fusion proteins containing the TALE DNA-binding domain and the meganuclease cleavage domain. This meganuclease cleavage domain is active as a monomer and does not require dimerization for its activity. (See Boissel et al ., (2013) Nucl Acid Res : 1-13, doi: 10.1093/nar/gkt1224.) In addition, the nuclease domain may also exhibit DNA-binding functions.

[0138] In other additional embodiments, the nuclease contains a compact TALEN (cTALEN). These are single-stranded fusion proteins where the TALE DNA-binding domain is linked to the TevI nuclease domain. This fusion protein can either function as nicases with localization determined by the TALE region or can produce a double-strand break, depending on where the TALE DNA-binding domain is located relative to the Tev I nuclease domain (see Beurdeley et al (2013) Nat Comm : 1-8 DOI: 10.1038/ncomms2782). Any TALEN can be used in combination with additional TALENs ( eg one or more TALENs (cTALEN or Fok I-TALEN) with one or more mega-TALs) or other DNA-cleaving enzymes.

[0139] In some embodiments, the nuclease contains a meganuclease (homing endonuclease) or a portion thereof that exhibits cleavage activity. Naturally occurring meganucleases recognize cleavage sites of 15-40 bp in length and are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst-box family, and the HNH family. Examples of homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I -TevI, I-TevII and I-TevIII. The sequences of their recognition are known. See also US Pat. No. 5,420,032; US Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al . (1989) Gene 82:115-118; Perler et al . (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet . 12:224-228; Gimble et al . (1996) J. Mol. Biol. 263:163-180; Argast et al . (1998) J. Mol. biol . 280:345-353 and New England Biolabs catalog.

[0140] DNA-binding domains from naturally occurring meganucleases, primarily from the LAGLIDADG family, have been used to promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells, and mice, however this approach has been limited to modification of either homologous genes that retain the meganuclease recognition sequence (Monet et al. (1999), Biochem. Biophysics. Res . Common . 255: 88-93), or pre-engineered genomes into which the recognition sequence has been introduced (Route et al . (1994), Mol Cell Biol 14: 8096-106 Chilton et al (2003) Plant Physiology 133: 956-65 Puchta et al (1996) Proc Natl Acad Sci USA 93: 5055-60 ; Rong et al . (2002), Genes Dev . 16:1568-81; Gouble et al . (2006), J. Gene Med . 8(5):616-622). Accordingly, attempts have been made to construct meganucleases exhibiting novel binding specificities at medically or biotechnologically relevant sites (Porteus et al . (2005), Nat. Biotechnol . 23:967-73; Sussman et al. (2004), J. Mol Biol 342: 31-41 Epinat et al (2003) Nucleic Acids Res 31: 2952-62 Chevalier et al (2002) Molec Cell 10:895-905 Epinat et al (2003 ) Nucleic Acids Res. 31:2952-2962; Ashworth et al . (2006) Nature 441:656-659; Paques et al . (2007) Current Gene Therapy 7:49-66; US Pat. No. 20060153826; No. 20060078552; and No. 20040002092). In addition, naturally occurring or engineered DNA-binding domains from meganucleases can be operably linked to a cleavage domain from a heterologous nuclease ( e.g. Fok I) and/or cleavage domains from meganucleases can be operably linked to a heterologous DNA-binding domain ( e.g. , ZFP or TALE).

[0141] In other embodiments, said nuclease is a zinc finger nuclease (ZFN) or a nuclease-hybridized TALE DNA-binding domain (TALEN). ZFN and TALEN contain a DNA-binding domain (zinc finger protein or TALE DNA-binding domain) designed to bind to a target site in a selected gene and a cleavage domain or cleavage half-domain ( e.g. , from a restriction nuclease and/or meganuclease according to described in this document).

[0142] As detailed above, zinc finger binding domains and TALE DNA binding domains can be designed to bind to a selected sequence. See, for example, Beerli et al . (2002) Nature Biotechnol . 20:135-141; Pabo et al . (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol . 19:656-660; Segal et al . (2001) Curr. Opin. Biotechnol . 12:632-637; Choo et al . (2000) Curr. Opin. Struct. biol . 10:411-416. The engineered zinc finger binding domain or TALE protein may have novel binding specificity compared to a naturally occurring protein. Design methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of databases containing triplets (or quadruplets) of nucleotide sequences and individual zinc finger or TALE amino acid sequences, where each triplet or quadruplet nucleotide sequence is associated with one or more zinc finger amino acid sequences or TALE repeat units linking a particular triplet or quadruplet sequence. See, for example, US Pat. Nos. 6,453,242 and 6,534,261, incorporated herein by reference in their entirety.

[0143] The choice of target site, as well as methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art and are described in detail in US Pat.

[0144] In addition, as described in this and other references, zinc finger domains, TALE, and/or multiple zinc finger proteins can be connected to each other using any suitable linker sequences, including, for example, linkers of length 5 or more than amino acids ( e.g. TGEKP (SEQ ID NO: 9), TGGQRP (SEQ ID NO: 10), TGQKP (SEQ ID NO: 11) and/or TGSQKP (SEQ ID NO: 12). See, for example, examples of linker 6 or more amino acids in U.S. Patent Nos. 6,479,626; No. 6,903,185; and No. 7,153,949. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of said protein. See also U.S. Patent No. 8,772,453.

[0145] Accordingly, nucleases such as ZFN, TALEN, and/or meganucleases may contain any DNA-binding domain and any nuclease (cleavage) domain (cleavage domain, cleavage half-domain). As noted above, the cleavage domain may be heterologous to the DNA binding domain, for example, a zinc finger or a TAL effector DNA binding domain and a cleavage domain from a nuclease, or a meganuclease DNA binding domain and a cleavage domain from another nuclease may be used. Heterologous cleavage domains can be derived from any endonuclease or exonuclease. Examples of endonucleases from which the cleavage domain may originate include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, the 2002-2003 catalog. New England Biolabs, Beverly, Massachusetts; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes are known that cleave DNA ( e.g. , nuclease S1; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn et al . (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source of cleavage domains and cleavage half-domains.

[0146] Similarly, the cleavage half-domain may be from any nuclease, or portion thereof, as described above, whose cleavage activity requires dimerization. In general, two fusion proteins are required for cleavage if said fusion proteins contain cleavage half-domains. Alternatively, a single protein containing two cleavage half-domains can be used. The two cleavage half-domains may be derived from the same endonuclease (or functional fragments thereof), or each cleavage half-domain may be derived from a different endonuclease (or functional fragments thereof). In addition, the target sites of the two fusion proteins are preferably distributed relative to each other such that binding of said two fusion proteins to their respective target sites results in a spatial orientation of the cleavage half-domains relative to each other, allowing said cleavage half-domains to form a functional cleavage domain, e.g., by dimerization. Accordingly, in some embodiments, the near edges of the target sites are separated by 5-10 nucleotides or 15-18 nucleotides. However, any integer number of nucleotides or base pairs ( eg , 2 to 50 base pairs or more) can be located between the two target sites. In general, the cleavage site is located between the target sites.

[0147] Restriction endonucleases (restriction enzymes) are present in many species and are capable of sequence-specific binding to DNA (at the recognition site) and cleaving DNA at or near the binding site. Certain restriction enzymes ( such as type IIS) cleave DNA at sites remote from the recognition site and contain shared binding and cleavage domains. For example, the type IIS Fok I enzyme catalyzes double-stranded DNA cleavage, 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. See, for example, US Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; and Li et al . (1992) Proc. Natl. Acad. sci. USA 89:4275-4279; Li et al . (1993) Proc. Natl. Acad. sci. USA 90:2764-2768; Kim et al . (1994a) Proc. Natl. Acad. sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31.978-31.982. Accordingly, in one embodiment, the fusion proteins comprise a cleavage domain (or cleavage half-domain) of at least one type IIS restriction enzyme, and one or more zinc finger binding domains, which may or may not be engineered.

[0148] An example of a type IIS restriction enzyme whose cleavage domain can be separated from the binding domain is Fok I. This particular enzyme is active in the form of a dimer. Bitinaite et al . (1998) Proc. Natl. Acad. sci. USA 95: 10.570-10.575. Accordingly, for the purposes of the present invention, the portion of the Fok I enzyme used in the described fusion proteins is considered to be the cleavage half-domain. Accordingly, two fusion proteins each containing a Fok I cleavage half-domain can be used for targeted double-strand cleavage (two-strand cleavage) and/or targeted replacement of cellular sequences using Fok I zinc finger fusions, with restoration of a catalytically active cleavage domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok I cleavage half-domains can also be used. Parameters for targeted cleavage and targeted sequencing using zinc finger hybridized Fok I are described in various sections of this document.

[0149] A cleavage domain or cleavage half-domain can be any portion of a protein that retains cleavage activity or the ability to multimerize ( eg , dimerize) to form a functional cleavage domain.

[0150] Examples of type IIS restriction enzymes are described in WO 07/014275, which is incorporated herein in its entirety. Additional restriction enzymes also contain shared binding and cleavage domains; these enzymes are provided by the present invention. See, for example, Roberts et al . (2003) Nucleic Acids Res. 31:418-420.

[0151] In some embodiments, the cleavage domain includes the Fok I cleavage domain used to generate the 1FOK.pdb and 2FOK.pdb crystal structures (see Wah et al (1997) Nature 388:97-100), with the sequence given below:

Wild-type Fok I cleavage half-domain (SEQ ID NO: 1)

QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

[0152] FokI -derived cleavage half-domains may contain a mutation at one or more amino acid residues as shown in SEQ ID NO: 1. amino acids) and/or deletions (of one or more amino acid residues). In some embodiments, one or more of residues 414-426, 443-450, 467-488, 501-502 and/or 521-531 (when numbered according to SEQ ID NO: 1 and the sequences shown in Fig. 17) are mutated, since these residues are localized close to the DNA backbone in the ZFN molecular model associated with the target site, described in the source: Miller et al . ((2007) Nat Biotechnol 25:778-784). In some embodiments, one or more residues at positions 416, 422, 447, 448, and/or 525 are mutated. In some embodiments, said mutation involves replacing a wild-type molecule residue with any other residue, e.g., an alanine (A) residue, a cysteine (C) residue, an aspartic acid (D), a glutamic acid (E), a histidine (H) residue. , a phenylalanine residue (F), a glycine residue (G), an asparagine residue (N), a serine residue (S), or a threonine residue (T). In other embodiments, the wild-type molecule residues at one or more of positions 416, 418, 422, 446, 448, 476, 479, 480, 481, and/or 525 are replaced with any other residues, including, but not limited to, R416D, R416E, S418E, S418D, R422H, S446D, K448A, N476D, I479Q, I479T, G480D, Q481A, Q481E, K525S, K525A, N527D, R416E+R422H, R416D+R422H, R416E+K448A, R416D+R422H, K448A+ I479Q, K448A+Q481A, K448A+K525A.

[0153] In some embodiments, the cleavage domain comprises one or more engineered cleavage half-domains (also referred to as dimerization domain mutants) that minimize or prevent homodimerization, as described in, for example, US Pat. Nos. 7,914,796; #8,034,598 and #8,623,618; and US Patent Publication No. 20110201055, the contents of which are hereby incorporated by reference in their entirety. Amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537 and 538 Fok I (when numbered according to SEQ ID NO: 1 and sequences shown in Fig. 17) are targets for influencing the dimerization of Fok I cleavage half-domains. These mutations may include mutations of residues found in naturally occurring restriction enzymes homologous to Fok I. 448 and/or 525 (when numbered according to SEQ ID NO: 1 and the sequences shown in Fig. 17) involves the replacement of a positively charged amino acid with an uncharged or negatively charged amino acid. In another embodiment, the engineered cleavage half-domains contain mutations at amino acid residues 499, 496, and 486 in addition to mutations at one or more amino acid residues 416, 422, 447, 448, or 525; all numbering corresponds to SEQ ID NO: 1 or the sequences shown in FIG. 17.

[0154] In some embodiments, the compositions described herein include engineered Fok I cleavage half-domains that form obligate heterodimers as described in, for example, US Pat. Nos. 7,914,796; No. 8,034,598; #8,961,281 and #8,623,618; and US Patent Publication Nos. 20080131962 and 20120040398. Accordingly, according to one preferred embodiment of the present invention, fusion proteins are provided wherein the engineered cleavage half-domain comprises a polypeptide in which the Gln(Q) residue present at position 486 of the wild-type molecule is replaced with a Glu(E) residue, an Ile(I) residue , present at position 499 of the wild-type molecule, is replaced by a Leu (L) residue; and an Asn (N) residue present at position 496 of the wild-type molecule is changed to an Asp (D) or Glu (E) (“ELD” or “ELE”) residue, in addition to one or more mutations at positions 416, 422, 447, 448 or 525 (with numbering corresponding to SEQ ID NO: 1 and the sequences shown in Fig. 17). In another embodiment, the engineered cleavage half-domains are derived from the wild-type Fok I cleavage half-domain and contain mutations at amino acid residues 490, 538, and 537, numbered according to wild-type Fok I (SEQ ID NO: 1 and sequences shown in FIG. 17) , in addition to one or more mutations at amino acid residues 416, 422, 447, 448, or 525. According to a preferred embodiment of the present invention, a fusion protein is provided wherein the engineered cleavage half-domain comprises a polypeptide wherein the Glu(E) residue present at position 490 of the wild-type molecule type is replaced with a Lys (K) residue, an Ile (I) residue present at position 538 of the wild-type molecule is replaced with a Lys (K) residue, and a His (H) residue present at position 537 of the wild-type molecule is replaced with Lys (K) or an Arg (R) residue ("KKK" or "KKR") (see US8962281, incorporated herein by reference), in addition to one or more mutations in sex 416, 422, 447, 448, or 525. See, for example, US Pat. Nos. 7,914,796; No. 8,034,598 and No. 8,623,618, the contents of which are incorporated by reference in their entirety for any purpose. In other embodiments, the engineered cleavage half-domains contain "Sharkey" and/or "Sharkey" mutations (see Guo et al , (2010) J. Mol. Biol . 400(1):96-107).

[0155] In another embodiment, the engineered cleavage half-domains are derived from the wild-type Fok I cleavage half-domain and contain mutations at amino acid residues 490 and 538, numbered according to wild-type Fok I or a FokI homologue, in addition to one or more mutations at amino acid residues 416, 422 , 447, 448, or 525. According to a preferred embodiment of the present invention, a fusion protein is provided wherein the engineered cleavage half-domain comprises a polypeptide in which the Glu (E) residue present at position 490 of the wild-type molecule is replaced by a Lys (K) residue. , and the Ile (I) residue present at position 538 of the wild-type molecule is changed to a Lys (K) (“KK”) residue, in addition to one or more mutations at positions 416, 422, 447, 448, or 525. According to a preferred embodiment The present invention provides a fusion protein in which the engineered cleavage half-domain contains a polypeptide in which the Gln (Q ) present at position 486 of the wild-type molecule is replaced with a Glu (E) residue, and an Ile (I) residue present at position 499 of the wild-type molecule is replaced with a Leu (L) (“EL”) residue (see US8034598, incorporated herein by reference), in addition to one or more mutations at positions 416, 422, 447, 448, or 525.

[0156] According to one aspect of the present invention, a fusion protein is provided wherein the engineered cleavage half-domain comprises a polypeptide that has a wild-type molecule amino acid residue mutated at one or more of positions 387, 393, 394, 398, 400, 402, 416, 422, 427, 434, 439, 441, 447, 448, 469, 487, 495, 497, 506, 516, 525, 529, 534, 559, 569, 570, 571 in the catalytic domainFoxI. Proposed nuclease domains containing one or more of the mutations presented in any of the attached tables and drawings. In some embodiments, said one or more mutations change the amino acid of the wild-type molecule from a positively charged residue to a neutral residue or a negatively charged residue. According to any of these embodiments, the described mutants can also be introduced into the domainFoxI containing one or more additional mutations. In preferred embodiments, said additional mutations are in the dimerization domain, e.g., at positions 418, 432, 441, 481, 483, 486, 487, 490, 496, 499, 523, 527, 537, 538, and/or 559. Non-limiting examples mutations include mutations (for example,substitutions) wild type molecule residues of any cleavage domain (e.g. FoxI or homologueFoxI) at positions 393, 394, 398, 416, 421, 422, 442, 444, 472, 473, 478, 480, 525, or 530, replaced by any amino acid residue (for example,K393X, K394X, R398X, R416S, D421X, R422X, K444X, S472X, G473X, S472, P478X, G480X, K525X and A530X, where the first residue corresponds to the wild-type molecule and X refers to any amino acid that is substituted for the rest of the wild-type molecule) . In some embodiments, X is E, D, H, A, K, S, T, D, or N. Other examples of mutations include S418E, S418D, S446D, K448A, I479Q, I479T, Q481A, Q481N, Q481E, A530E, and/ or A530K, where the amino acid residue numbering corresponds to the full-length wild-type FokI cleavage domain and its homologues (Fig. 17). In some embodiments, the combinations may include 416 and 422, mutation at position 416 and K448A, K448A and I479Q, K448A and Q481A; and/or K448A and a mutation at position 525. In one embodiment, the residue present at position 416 of the wild-type molecule can be replaced with a Glu (E) (R416E) residue, the residue present at position 422 of the wild-type molecule is replaced with His (H) (R422H) and the residue present at position 525 of the wild-type molecule is replaced by an Ala (A) residue. Cleavage domains as described herein may also include additional mutations including, but not limited to, mutations at positions 432, 441, 483, 486, 487, 490, 496, 499, 527, 537, 538 and/or 559 , such as dimerization domain mutants (for example,ELD, KKR) and/or nickase mutants (catalytic domain mutations). Cleavage half-domains with mutations as described herein form heterodimers as is known in the art.

[0157] Alternatively, nucleases can be assembled in vivo at the target site of the nucleic acid using the so-called "decoupled enzyme" technology (see, for example, US Patent Publication No. 20090068164). The components of such decoupled enzymes may be expressed either on separate expression constructs, or may be linked in a single open reading frame, with the individual components separated by, for example, a 2A self-cleaving peptide or an IRES sequence. The components may be individual zinc finger binding domains or nucleic acid binding domains of a meganuclease.

[0158] Prior to use, nuclease activity ( eg , ZFN and/or TALEN) may be screened for, for example, in the yeast chromosome system as described in US Pat. No. 8,563,314.

[0159] In some embodiments, said nuclease comprises the CRISPR/Cas system. The CRISPR locus (“clustered regularly interspaced short palindromic repeats”, clustered regularly distributed short palindromic repeats), which encodes the RNA components of the system, and the Cas (“CRISPR-associated”) locus, which encodes proteins (Jansen et al. , 2002. Mol Microbiol 43: 1565-1575 Makarova et al 2002 Nucleic Acids Res 30 482-496 Makarova et al 2006 Biol Direct 1: 7 Haft et al 2005 PLoS Comput. Biol . 1: e60) comprise the gene sequences of the CRISPR/Cas nuclease system. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage.

[0160] Type II CRISPR is one of the most well-characterized systems; it performs a targeted DNA double-strand break in four successive steps. First, two non-coding RNAs, an array of pre-cr RNA and a tracr RNA, are transcribed from the CRISPR locus. In the second step, tracr-RNA hybridizes with regions of pre-cr-RNA repeats and mediates the processing of pre-cr-RNA into mature cr-RNAs containing individual spacer sequences. In the third step, the mature cr-RNA:tracr-RNA complex directs Cas9 to the target DNA by Watson-Crick base-pairing between a spacer on the cr-RNA and a proto-spacer on the target DNA next to the proto-spacer adjacent motif (PAM) - an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA to produce a double-strand break within the protospacer. The activity of the CRISPR/Cas system consists of three steps: (i) insertion of foreign DNA sequences into the CRISPR array to prevent future attacks in a process called "adaptation", (ii) expression of relevant proteins, and expression and processing of said array, followed by ( iii) RNA-mediated interference with a foreign nucleic acid. Accordingly, in the bacterial cell, a number of so-called "Cas" proteins are involved in the natural function of the CRISPR/Cas system and play a role in functions such as the insertion of foreign DNA and the like.

[0161] In some embodiments, a CRISPR-Cpf1 system is used. The CRISPR-Cpf1 system identified in Francisella spp is a class 2 CRISPR-Cas system that mediates robust DNA interference in human cells. Although Cpf1 and Cas9 are functionally conserved, they differ in many aspects, including guide RNAs and substrate specificity (see Fagerlund et al , (2015) Genom Bio 16:251). The main difference between Cas9 and Cpf1 proteins is that Cpf1 does not involve tracr-RNA and, accordingly, only cr-RNA is required. FnCpf1 crRNAs are 42-44 nt long (19-nt repeat and 23-25 nt spacer) and contain a single loop-on-stem that allows for sequence changes while retaining secondary structure. In addition, Cpf1 cr-RNAs are significantly shorter than the ~100-bp engineered sRNAs required for Cas9, and the PAM requirements for FnCpfl are 5'-TTN-3' and 5'-CTA-3' on the substituted strand. Although both Cas9 and Cpf1 create double-strand breaks in the target DNA, Cas9 uses RuvC- and HNH-like domains to create blunt-ended cuts within the guide RNA primer sequence, while Cpf1 uses a RuvC-like domain to generate staggered cuts outside the primer sequence. . Because Cpf1 makes staggered cuts outside of the critical seed region, NHEJ does not destroy the target site, so Cpf1 can continue to make cuts at the same site until the required HDR recombination occurs. Accordingly, in the methods and compositions described herein, the term "Cas" is to be understood to include both Cas9 proteins and Cfp1 proteins. Accordingly, as used herein, "CRISPR/Cas system" refers to both CRISPR/Cas and/or CRISPR/Cfp1 systems, including both nuclease and/or transcription factor systems.

[0162] In some embodiments, the Cas protein may be a "functional derivative" of a naturally occurring Cas protein. A "functional derivative" of a naturally occurring polypeptide sequence is a compound that has a characteristic biological property in common with the naturally occurring polypeptide sequence. "Functional derivatives" include, but are not limited to, natural sequence fragments and derivatives of the natural sequence of a polypeptide and fragments thereof, provided that they have a biological activity in common with the corresponding natural sequence of the polypeptide. The biological activity contemplated by the present invention is represented by the ability of the functional derivative to hydrolyze the DNA substrate to form fragments. The term "derivative" encompasses both amino acid sequence variants of a polypeptide, covalent modifications, and fusion products such as derivatives of Cas proteins. Suitable derivatives of a Cas polypeptide or fragment thereof include, but are not limited to, mutants, fusion products, covalent modifications of the Cas protein or fragment thereof. The Cas protein, which includes the Cas protein or fragment thereof, as well as derivatives of the Cas protein or fragment thereof, can be obtained from a cell or chemically synthesized, or obtained using a combination of these two procedures. Said cell may be a cell that naturally produces a Cas protein, or a cell that naturally produces a Cas protein and is genetically engineered to produce an endogenous Cas protein with a higher level of expression, or to produce a Cas protein with an exogenously introduced nucleic acid that encodes Cas identical to or different from the endogenous Cas. In some cases, the cell does not naturally produce the Cas protein and is genetically engineered to produce the Cas protein. In some embodiments, said Cas protein is a small ortholog of Cas9 for delivery by an AAV vector (Ran et al (2015) Nature 510, p. 186).

[0163] Said nuclease or nucleases can make one or more double-stranded and/or single-stranded cuts at the target site. In some embodiments, said nuclease contains a catalytically inactive cleavage domain ( eg, Fok I and/or Cas protein). See, for example, US patent No. 9,200,266; No. 8,703,489 and Guillinger et al . (2014) Nature Biotech. 32(6):577-582. The catalytically inactive cleavage domain can, in combination with the catalytically active domain, act as a niqase to produce a single strand cut. Accordingly, two nikases can be used in combination to obtain a double-stranded cut in a specific region. Additional nickases are also known in the art, eg McCaffery et al . (2016) Nucleic Acids Res. 44(2):e11. doi:10.1093/nar/gkv878. Epub 2015 Oct 19.

Delivery

[0164] Proteins ( e.g. , nucleases), polynucleotides, and/or compositions containing proteins and/or polynucleotides as described herein can be delivered to a target cell by any suitable means, including, for example, by injecting said protein and/or or mRNA components.

[0165] Suitable cells include, but are not limited to, eukaryotic and prokaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines derived from such cells include T cells, COS cells, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g. HEK293-F, HEK293-H, HEK293-T) and perC6, as well as insect cells such as Spodoptera fugiperda (Sf), or cells of fungi such as Saccharomyces, Pichia and Schizosaccharomyces. In some embodiments, said cell line is a CHO-K1, MDCK, or HEK293 cell line. Suitable cells also include stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells (iPSC cells), hematopoietic stem cells, neural stem cells and mesenchymal stem cells.

[0166] Methods for delivering proteins containing DNA binding domains as described herein are described, for example, in US patents No. 6,453,242; No. 6,503,717; #6,534,261; #6,599,692; No. 6,607,882; No. 6,689,558; No. 6,824,978; No. 6,933,113; #6,979,539; No. 7,013,219; and No. 7,163,824; the contents of all listed patents are hereby incorporated by reference in their entirety.

[0167] DNA binding domains and fusion proteins containing said DNA binding domains as described herein can also be delivered using vectors containing sequences encoding one or more DNA binding proteins. In addition, additional nucleic acids ( for example , donor ones) can also be delivered using these vectors. Any vector systems may be used, including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors, adeno-associated virus vectors; etc. See also US Pat. Nos. 6,534,261; No. 6,607,882; No. 6,824,978; No. 6,933,113; #6,979,539; No. 7,013,219; and No. 7,163,824, incorporated herein by reference in their entirety. In addition, it is understood that any of these vectors may optionally contain one or more DNA-binding protein-encoding sequences and/or additional nucleic acids. Accordingly, when one or more of the DNA-binding proteins as described herein, and optionally additional DNA, are introduced into a cell, they may be carried by the same vector or by different vectors. When using multiple vectors, each vector may contain a sequence encoding one or more DNA-binding proteins and, if necessary, additional nucleic acids.

(eds.) (1995); и Yu et al., Gene Therapy 1:13-26 (1994).[0168] Standard viral or non-viral gene transfer techniques can be used to introduce nucleic acids encoding engineered DNA-binding proteins into cells ( e.g., mammalian cells) and target tissues and, if necessary, to co-administer additional nucleotide sequences. Such methods can also be used to introduce nucleic acids ( eg encoding DNA-binding proteins and/or donor NAs) into cells in vitro . In some embodiments, nucleic acids are administered for use in in vivo or ex vivo gene therapy. Non-viral vector delivery systems include DNA plasmids, deproteinized nucleic acids, and complexes of nucleic acids with a delivery backbone such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses that contain either episomal or integrated genomes after delivery into a cell. An overview of gene therapy procedures can be found in: Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 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

(eds.) (1995); and Yu et al ., Gene Therapy 1:13-26 (1994).

[0169] Methods for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, deproteinized DNA, mRNA, artificial virions, and agent-enhanced DNA uptake. Sonoporation can also be used to deliver nucleic acids using, for example, the Sonitron 2000 system (Rich-Mar). In a preferred embodiment, one or more nucleic acids are delivered as mRNA. It is also preferred to use capped mRNAs to increase translation efficiency and/or mRNA stability. In particular, cap analogs without the possibility of switching in reverse orientation (ARCA, "anti-reverse cap analog") or their variants are preferred. See US Pat. Nos. 7,074,596 and 8,153,773, incorporated herein by reference.

[0170] Additional examples of nucleic acid delivery systems include those supplied by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX Molecular Delivery Systems (Holliston, Massachusetts) and Copernicus Therapeutics Inc, (see eg US6008336). Lipofection is described, for example , in US 5049386, US 4946787; and US 4,897,355); commercially available lipofection reagents (e.g. Transfectam™, Lipofectin™ and Lipofectamine™ (Lipofectamine RNAiMAX). Cationic and neutral lipids that are suitable for efficient lipofection with receptor recognition polynucleotides include those described by Felgner (WO 91/17424, WO 91/16024 May be delivered to cells ( ex vivo administration) or to target tissues ( in vivo administration).

[0171] The preparation of nucleic acid lipid complexes, including targeted liposomes such as immunolipid complexes, is well known to those skilled 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 . al ., Gene Therapy 2:710-722 (1995), Ahmad et al., Cancer Res . 52:4817-4820 (1992), US Pat. , #4,774,085, #4,837,028 and #4,946,787).

[0172] Additional delivery methods include packaging the delivered nucleic acids into EnGeneIC delivery backbones (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies; one arm of said antibody is specific for the target tissue and the other is specific for EDV. The specified antibody transports the EDV to the surface of the target cell, after which the EDV enters the inside of the specified cell by endocytosis. After entering the cell, the contents are released (see MacDiarmid et al (2009) Nature Biotechnology 27(7) p. 643).

[0173] The use of RNA or DNA-based viral delivery systems for nucleic acids encoding engineered DNA-binding proteins and/or donors ( e.g. , CAR or ACTR) as needed provides the advantage of improved processes for targeting virus to specific cells in body and transport the viral load to the nucleus. Viral vectors can be administered directly to patients ( in vivo ) or can be used to treat cells in vitro, after which the modified cells are administered to patients ( ex vivo ). Standard virus-based nucleic acid delivery systems include, but are not limited to, retroviral, lentiviral, adenovirus, adeno-associated virus, vaccinia virus, and herpes simplex gene transfer vectors. Integration into the host genome is possible using retroviral, lentivirus, and adeno-associated virus-based gene transfer techniques, often providing long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different types of cells and target tissues.

[0174] Retrovirus tropism can be altered by incorporating foreign envelope proteins, expanding the potential target cell population. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and generally producing high viral titers. The choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packaging capacity of up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient to replicate and package the vectors, which are then used to integrate the therapeutic gene into the target cell for consistent transgene expression. Commonly used retroviral vectors include murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency 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) Sommerfelt 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).

количествах в относительно простой системе. Векторы на основе аденоассоциированного вируса («AAV») также применяют для трансдукции клеток целевыми нуклеиновыми кислотами, например, при получении нуклеиновых кислот и пептидов in vitro и при процедурах генной терапии in vivo и ex vivo (см., например, West et al., Virology 160:38-47 (1987); патент США №4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Конструирование рекомбинантных AAV-векторов описано в ряде публикаций, в том числе в патенте США №5,173,414; у Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS USA 81:6466-6470 (1984); и Samulski et al., J. Virol. 63:03822-3828 (1989).[0175] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. When using such vectors, high titers and high levels of expression were obtained. The specified vector can be obtained in

quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, for example, in in vitro production of nucleic acids and peptides and in in vivo and ex vivo gene therapy procedures (see, for example, West et al ., Virology 160:38-47 (1987), U.S. Patent No. 4,797,368, WO 93/24641, Kotin, Human Gene Therapy 5:793-801 (1994), Muzyczka, J. Clin Invest 94:1351 (1994). Recombinant AAV vectors have been described in a number of publications, including US Patent No. 5,173,414, Tratschin et al . , Mol . Cell. Biol . 5:3251-3260 (1985); 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS USA 81:6466-6470 (1984), and Samulski et al ., J. Virol 63:03822-3828 (1989).

[0176] At least six viral vector-based gene transfer approaches are currently being used in clinical trials, involving approaches that include complementing defective vectors with genes inserted into helper cell lines to produce a transducing agent.

[0177] pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al ., Blood 85:3048-305 (1995); Kohn et al ., Nat. Med. 1:1017-102 ( 1995); Malech et al., PNAS USA 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vector used in gene therapy trials. (Blaese et al ., Science 270:475-480 (1995)). Transduction efficiencies of 50% or higher have been observed for packaged MFG-S vectors. (Ellem et al ., Immunol Immunother . 44(1):10-20 (1997); Dranoff et al ., Hum. Gene Ther . 1:111-2 (1997).

[0178] Recombinant adeno-associated virus (rAAV) vectors are a promising alternative gene delivery system based on the defective non-pathogenic adeno-associated parvovirus type 2. All vectors are derived from a plasmid that retains only the 145 bp AAV inverted terminal repeats flanking the expression cassette carrying the transgene. Efficient gene transfer and stable delivery of transgenes due to integration into the genomes of transduced cells are the key features of this vector system. (Wagner et al ., Lancet 351:9117 1702-3 (1998), Kearns et al ., Gene Ther . 9:748-55 (1996)). Other AAV serotypes, including AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV8.2, AAV9 and AAVrh10 and pseudotyped AAVs such as AAV2/8, AAV2/5 and AAV2/6, may also be used according to the present invention.

[0179] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titers and easily infect a number of different cell types. Most adenoviral vectors are designed such that the transgene replaces the Ad E1a, E1b and/or E3 genes; the replication-deficient vector is then propagated in human 293 cells which provide the function of the deleted gene in a transactive manner. Ad vectors can transduce several tissue types in vivo, including non-dividing, differentiated cells such as those present in liver, kidney, and muscle. Standard Ad-vectors have a significant bearing capacity. An example of the use of an Ad vector in a clinical trial included polynucleotide therapy for antitumor immunization by intramuscular injection (Sterman et al ., Hum. Gene Ther . 7:1083-9 (1998)). Additional examples of the use of adenoviral vectors for gene transfer in clinical trials include those described in: Rosenecker et al ., Infection 24:1 5-10 (1996); Sterman et al ., Hum. Gene Ther . 9:7 1083-1089 (1998); Welsh et al ., Hum. Gene Ther . 2:205-18 (1995); Alvarez et al ., Hum. Gene Ther . 5:597-613 (1997); Topf et al ., Gene Ther . 5:507-513 (1998); Sterman et al ., Hum. Gene Ther . 7:1083-1089 (1998).

[0180] Packaging cells are used to produce viral particles capable of infecting a host cell. Such cells include 293 cells that package adenoviruses and ψ2 cells or PA317 cells that package retroviruses. Viral vectors for use in gene therapy are usually produced in a producer cell line that packages a nucleic acid vector into a viral particle. These vectors typically contain the minimum viral sequences required for packaging and subsequent integration into the host genome (if appropriate); the other viral sequences are replaced by an expression cassette encoding the protein to be expressed. Missing viral functions are replenished by trans activity by the cells of the packaging cell line. For example, AAV vectors used in gene therapy typically contain only inverted terminal repeat (ITR) sequences from the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged in a cell line containing a helper plasmid encoding other AAV genes, namely rep and cap, but lacking the ITR sequence. Said cell line is also infected with adenovirus as a helper virus. The helper virus promotes replication of the AAV vector and expression of the AAV genes from the helper plasmid. The helper plasmid does not pack in significant amounts due to the lack of ITR sequences. Adenovirus infection can be reduced by, for example, heat treatment, to which adenovirus is more sensitive than AAV.

[0181] In many gene therapy applications, delivery of a gene therapy vector with a high degree of specificity to a particular tissue type is desirable. Accordingly, the viral vector can be modified to be cell type specific by expressing the ligand as a protein hybridized to the viral envelope protein on the outer surface of the virus. A ligand is selected that has affinity for a receptor known to be present on the cell type of interest. For example, Han et al ., Proc. Natl. Acad. Sci. USA 92:9747-9751 (1995)) report that Moloney murine leukemia virus can be modified to express human heregulin hybridized to gp70, and that said recombinant virus infects certain human breast cancer cells expressing the human epidermal growth factor receptor. This principle can be extended to other virus/target cell pairs in which the target cell expresses the receptor and the virus expresses a fusion protein containing the receptor ligand on the cell surface. For example, a filamentous phage can be engineered to display antibody fragments ( eg , FAB or Fv) that have a specific binding affinity for virtually any chosen cellular receptor. Although the description above applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be designed to contain specific uptake sequences to promote uptake by specific target cells.

[0182] Delivery methods for CRISPR/Cas systems may include the methods described above. For example, in animal models, in vitro transcribed Cas-encoding mRNA or recombinant Cas protein can be injected directly into genome-edited animal embryos at the single cell stage using glass needles. Typically, in order to express Cas and guide RNAs in cells in vitro , cells are transfected with plasmids that encode them by lipofection or electroporation. A complex of the recombinant Cas protein with an in vitro transcribed guide RNA can also be made, with the Cas ribonucleoprotein and the guide RNA being taken up by the cells of interest (Kim et al (2014) Genome Res 24(6):1012). In therapeutic applications, Cas and guide RNAs can be delivered using a combination of viral and non-viral techniques. For example, mRNA encoding Cas can be delivered by nanoparticles, and guide RNAs and any desired transgene or repair template delivered by AAV (Yin et al (2016) Nat Biotechnol 34(3) p. 328).

[0183] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration ( e.g. , intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application as described below. Alternatively, vectors can be delivered to ex vivo cells, such as cells explanted from an individual patient ( e.g. , lymphocytes, bone marrow aspirate, tissue biopsy), or universal donor hematopoietic stem cells, followed by reimplantation of the cells into the patient, usually after selection of cells that included the specified vector.

[0184] Ex vivo transfection of cells for diagnosis, research, transplantation, or for gene therapy ( eg , via reinfusion of transfected cells into a host) is well known to those skilled in the art. In a preferred embodiment, cells are isolated from a subject, transfected with a nucleic acid of DNA-binding proteins (gene or cDNA), and re-infused into the subject ( eg , patient). Various cell types suitable for ex vivo transfection are well known to those skilled in the art (see, for example, the discussion of methods for isolating and culturing patient-derived cells in Freshney et al. , Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994) and references cited herein).

[0185] In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. An advantage of using stem cells is that they can differentiate into other cell types in vitro, or can be injected into a mammal (eg, a cell donor) where they become established in the bone marrow. Methods are known to differentiate CD34+ cells in vitro into clinically important immune cell types using cytokines such as GM-CSF, IFN-γ, and TNF-α (see Inaba et al ., J. Exp. Med. 176:1693-1702 ( 1992)).

[0186] Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning bone marrow cells with antibodies that bind unwanted cells such as CD4+ and CD8+ (T cells), CD45+ (pan-B cells), GR-1 (granulocytes), and Iad ( differentiated antigen presenting cells) (see Inaba et al ., J. Exp. Med . 176:1693-1702 (1992)).

[0187] In some embodiments, stem cells that have been modified may also be used. For example, neural stem cells that have been rendered resistant to apoptosis and also contain the ZFP TFs of the present invention can be used as therapeutic compositions. Apoptosis resistance can be achieved, for example, by knocking out BAX and/or BAK using BAX- or BAK-specific ZFNs (see U.S. Patent No. 8,597,912) in stem cells, or destroying caspase, again using, for example, caspase -6-specific ZFNs.

[0188] Vectors ( eg , retroviruses, adenoviruses, liposomes, etc.) containing therapeutic DNA-binding proteins (or nucleic acids encoding said proteins) can also be introduced directly into the body for in vivo cell transduction. Alternatively, deproteinized DNA may be introduced. Administration is via any route commonly used to bring the molecule into maximum contact with blood or tissue cells, including, but not limited to, infection, infusion, topical application, and electroporation. Suitable methods for introducing such nucleic acids are available and well known to those skilled in the art; while more than one route may be used to introduce a particular composition, often a particular route may provide a faster and more efficient response than another route.

[0189] Methods for introducing DNA into hematopoietic stem cells are described, for example, in US patent No. 5,928,638. Suitable vectors for introducing transgenes into hematopoietic stem cells, such as CD34+ cells, include adenovirus type 35.

[0190] Vectors suitable for introducing transgenes into immune cells ( eg , T cells) include non-integrable lentiviral vectors. See, for example, Ory et al . (1996) Proc. Natl. Acad. sci. USA 93:11382-11388; Dull et al . (1998) J. Virol . 72:8463-8471; Zuffery et al . (1998) J. Virol . 72:9873-9880; Follenzi et al . (2000) Nature Genetics 25:217-222.

[0191] Pharmaceutically acceptable carriers are determined in part by the particular composition for administration, as well as the particular method used to administer said composition. Accordingly, a wide range of suitable formulations of pharmaceutical compositions are available as described below (see, for example, Remington's Pharmaceutical Sciences, 17th ed., 1989).

[0192] As noted above, the described methods and compositions are suitable for use in any type of cell, including, but not limited to, prokaryotic cells, fungal cells, archaeal cells, plant cells, insect cells, animal cells, vertebrate cells, cells mammalian and human cells, including T-cells and stem cells of any type. Suitable cell lines for protein expression are known to those skilled in the art and include, but are not limited to, COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6, insect cells such as Spodoptera fugiperda (Sf), and cells fungi such as Saccharomyces, Pichia and Schizosaccharomyces. Progeny, variants and derivatives of these cell lines may also be used.

Applications

[0193] The use of engineered nucleases to treat and prevent disease is expected to represent one of the most significant advances in medicine in the coming years. The methods and compositions described herein serve to increase the specificity of these new tools to ensure that the desired target site is the primary cleavage site. To realize the full potential of all applications specified technologyin vitro,in vivo andex vivo minimization or elimination of off-target splitting is required.

[0194] Examples of genetic diseases include, but are not limited to, achondroplasia, achromatopsia, acid maltase deficiency, adenosine deaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, Ecardi syndrome, alpha-1 antitrypsin deficiency, alpha thalassemia, androgen insensitivity syndrome, Apert, arrhythmogenic right ventricular dysplasia, dysplasia, ataxia-telangiectasia, Barth's syndrome, beta thalassemia, blue blister nevus syndrome, Canavan disease, chronic granulomatous diseases (CGD), crying cat syndrome, cystic fibrosis, Derkum's disease, ectodermal dysplasia, Fanconi anemia , progressive fibrodysplasia ossificans, fragile X syndrome, galactosemia, Gaucher disease, generalized gangliosidoses ( e.g. GM1), hemochromatosis, hemoglobin C mutation at codon 6 of beta-globin (HbC), hemophilia, Huntington's disease, Hurler syndrome, hypophosphatasia, syndrome Klinefelter, Krabbe disease, Langer-Gidion syndrome, a deficiency leukocyte adhesion (LAD, OMIM No. 116920), leukodystrophy, long QT syndrome, Marfan syndrome, Mobius syndrome, mucopolysaccharidosis (MPS), patellar nail syndrome, nephrogenic diabetes insipidus, neurofibromatosis, Niemann-Pick disease, osteogenesis imperfecta, phenylketonuria (PKU) , porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Schwachmann syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome , Tay-Sachs disease, thrombocytopenia with absent radius (TAR), Treacher-Collins syndrome, trisomy, tuberous sclerosis, Turner syndrome, urea cycle disorder, von Hippel-Lindau disease, Waardenburg syndrome, Williams syndrome, Wilson's disease, Wiskott syndrome -Aldrich, X-linked lymphoproliferative syndrome (XLP, OMIM No. 308240).

[0195] Additional examples of diseases that can be treated by targeted DNA cleavage and/or homologous recombination include acquired immunodeficiency, lysosomal storage diseases ( e.g. Gaucher disease, GM1 gangliosidosis, Fabry disease and Tay-Sachs disease), mucopolysaccharidosis ( e.g. Hunter, Hurler's disease), hemoglobinopathies ( eg , sickle cell anemia, HbC, α-thalassemia, β-thalassemia), and hemophilia.

[0196] Such methods also allow the treatment of infections (viral or bacterial) in the host ( for example, by blocking the expression of viral or bacterial receptors, thus preventing infection and/or spread in the host) to treat genetic diseases.

[0197] Targeted cleavage of the genomes of the infecting or integrated viruses can be used to treat viral infections in the host. In addition, targeted cleavage of genes encoding viral receptors can be used to block the expression of such receptors, thereby preventing viral infection and/or spread of the virus in the host. Targeted mutagenesis of genes encoding viral receptors ( eg HIV receptors CCR5 and CXCR4) can be used to render said receptors incapable of binding to a virus, thus preventing new infection and blocking the spread of an existing infection. See US Patent Publication No. 2008/015996. Non-limiting examples of viruses or viral receptors that may be targeted include herpes simplex virus (HSV), such as HSV-1 and HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV), HHV6 and HHV7. The hepatitis virus family includes hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV). Other viruses or their receptors may be targeted, including, but not limited to, Picornaviridae ( eg , polioviruses, etc.); Caliciviridae; Togaviridae ( e.g. rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae ( e.g. rabies virus, etc.); Filoviridae; Paramyxoviridae ( e.g. mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae ( e.g. influenza virus types A, B and C, etc.); Bunyaviridae; arenaviridae; Retroviradae; lentiviruses ( eg HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.), HIV-II); simian immunodeficiency virus (SIV), human papillomavirus (HPV), influenza virus and tick-borne encephalitis viruses. See, for example, descriptions of these and other viruses in Virology, 3rd Edition (WK Joklik ed. 1988); Fundamental Virology, 2nd Edition (BN Fields and DM Knipe, eds. 1991). HIV receptors, for example, include CCR-5 and CXCR-4.

[0198] Accordingly, heterodimeric cleavage domain variants as described herein are widely used to increase the specificity of ZFNs used to modify genes. These cleavage domain variants can easily be incorporated into any existing ZFN using either site-directed mutagenesis or in vivo specificity-enhancing subcloning of any ZFN dimers.

[0199] As noted above, the compositions and methods described herein can be used for gene modification, gene editing, and gene disruption. Non-limiting examples of gene modification include targeted integration based on homology-directed repair (HDR); gene correction based on HDR; gene modification based on HDR; HDR-based gene disruption; gene disruption based on NHEJ and/or a combination of HDR, NHEJ, and/or single strand annealing (SSA). Single strand annealing (SSA) refers to the repair of a double strand break between two repeat sequences in the same orientation by excision of LCBs with 5'-3' exonucleases, exposing 2 complementary regions. The single strands encoding the 2 direct repeats are then annulated with each other, and the annulated intermediate can be processed such that the single stranded "tails" (the portion of the single stranded DNA not annelated to any sequence) are cleaved off, the gaps filled in by DNA polymerase, and DNA ends reconnect. This results in the deletion of sequences located between these direct repeats.

[0200] Compositions containing cleavage domains ( eg, ZFN, TALEN, CRISPR/Cas systems) and the methods described herein may also be used in the treatment of various genetic diseases and/or infectious diseases.

[0201] These compositions and methods may also be used in stem cell-based therapies, including but not limited to: correcting somatic cell mutations by short-sequence gene conversion or targeted integration for monogenic gene therapy; to destroy dominant negative alleles; to destroy genes necessary for entry into cells or productive infection of pathogens in cells; improved tissue engineering, for example, by modifying the activity of a gene to promote differentiation or the formation of functional tissues; and/or disrupting the activity of a gene that promotes differentiation or formation of functional tissues; blocking or inducing differentiation, for example, by destroying genes that block differentiation, promoting differentiation of stem cells towards a specific lineage, targeted insertion of a gene or small interfering RNA (siRNA) expression cassette that can stimulate stem cell differentiation, targeted insertion of a gene or expression cassette with siRNAs that can block stem cell differentiation and allow for better propagation and maintenance of pluripotency, and/or for the targeted insertion of a reporter gene into a frame with an endogenous gene, which is a marker of pluripotency or differentiation status, providing a convenient marker for assessing stem cell differentiation status and how environmental changes, cytokines, growth conditions, gene expression, expression of miRNA, shRNA or miRNA molecules, exposure to antibodies to cell surface markers, or drugs alter the specified hundred tus; for transfer of somatic cell nuclei, for example, the patient's own somatic cells can be isolated, the intended target gene is appropriately modified, cell clones are obtained (and quality control is carried out to ensure genome safety), and the nuclei from these cells are isolated and transferred into unfertilized eggs to obtain a patient -specific hESC cells that can be directly injected or differentiated prior to inoculation to a patient, thereby reducing or eliminating tissue rejection; to obtain universal stem cells by knocking out MHC receptors ( for example, to obtain cells with a weakened or completely eliminated immunological identity). Cell types for this procedure include, but are not limited to, T cells, B cells, hematopoietic stem cells, and embryonic stem cells. In addition, induced pluripotent stem cells (iPSCs), which are also obtained from the patient's own somatic cells, can be used. Accordingly, these stem cells or their derivatives (differentiated types of cells or tissues) can potentially be inoculated into any person, regardless of their origin or histocompatibility.

[0202] These compositions and methods can also be used for somatic cell therapy, thus providing a stock of cells that have been modified to enhance their biological properties. Such cells can be infused into various patients regardless of the donor source of these cells and their histocompatibility with the recipient.

[0203] In addition to therapeutic applications, the increased specificity provided by the variants described herein, when used in engineered nucleases, can be used for the design of crops, the construction of cell lines, and the construction of disease models. Obligate heterodimeric cleavage half-domains provide an easy way to improve the properties of nucleases.

[0204] The described engineered cleavage half-domains may also be used in gene modification protocols requiring simultaneous cleavage to multiple targets, either to delete an intermediate region or to modify two specific loci simultaneously. Cleavage at two targets requires cellular expression of four ZFNs or TALENs, potentially resulting in 10 different combinations of active ZFNs or TALENs. In such applications, replacing the wild-type nuclease domain with these new variants eliminates the activity of undesired combinations and reduces the likelihood of off-target cleavage. In cases where cleavage at a particular desired DNA target requires the activity of a pair of nucleases A + B, and simultaneous cleavage at a second desired DNA target requires the activity of a pair of nucleases X + Y, the use of the mutations described herein can prevent the pairing of A with A, A with X, A with Y, and so on. Accordingly, these Fok I mutations reduce non-specific cleavage activity as a result of the formation of "illegal" pairs and provide more effective orthogonal mutant pairs of nucleases (see joint US patent publications No. 20080131962 and No. 20090305346).

EXAMPLES

Example 1: Receive ZFN

[0205] ZFNs targeted at sites in the BCL11A and TCRA genes (targeting the constant region, also known as TRAC) were designed and incorporated into plasmid vectors essentially as described in the references: Urnov et al . (2005) Nature 435(7042):646-651, Perez et al (2008) Nature Biotechnology 26(7): 808-816, as well as PCT Patent Publication WO 2016183298 and PCT Publication WO 2017106528. AAVS1 as described in US Publication No. 20150110762.

Example 2 Mutants at Fok I Residues Targeting Phosphate Interaction

[0206] Using Fok I cleavage domain models (Miller et al (2007) Nat Biotech 25(7):778-85), positively charged arginine or lysine amino acid residues (FIG. 1) were identified that potentially interact with backbone phosphates. DNA.

[0207] These identified positions in the Fok I domain (amino acids 416, 422, 447, 448 and 525) were then specifically changed (mutated) to serine residues to eliminate the interaction between the original positively charged amino acid and negatively charged phosphates on DNA (see FIG. 2A and 2B). If these mutations contain both ZFN partners, a number of different combinations may occur (see illustration in Fig. 2C). These new Fok I mutants were generated in the "KKR" Fok I partner of ELD/KKR heterodimers (see US Pat. No. 8,962,281) and then coupled to a ZFN pair specific for the BCL11A enhancer region, SBS#51857 ELD/SBS#51949 KKR or SBS#51857_KKR/SBS#51949_ELD; the "original" proteins in FIG. 3 are grayed out. Mutations were generated in each partner and then tested for cleavage activity against the original BCL11A target in CD34+ cells in various combinations as shown in FIG. 3. Non-target sites have been previously identified using an unbiased capture assay (PCT Patent Publication WO 2016/183298). These non-target sites are listed in Table 1 below, where each site is identified using a unique randomly generated identifying letter "registration mark", where the "registration mark" PRJIYLFN is the intended BCL11A target sequence. The table below also lists the locus of each site (coordinates are given according to the hg38 assembly from the UC Santa Cruz Human Genome Browser sequence database (Kent et al (2002), Genome Res .12(6):996-1006).

Table 1: Identified cleavage sites for SBS#51857/SBS#51949

"Register sign" Seq ID Sequence (5'→3') Chromosome: hg38 locus Type of PRJIYLFN
(provided target) 13 CTAATCAGAGGCCAAACCCTTCCTGGAGCCTGTGATAAAAGCAACTGTTAGCTTGCACTAGACTAGCTTCAAAGTT Chromosome 2:60495265 Intron NIFMAEVG fourteen CTTCCTGCGGGGTTTTGTGCAAATAACCCATGCTCGGGAGCGAGGCCCTGGGAAGGAGTATCTCGCTTCTTTGGGT Chromosome 8:119856440 Intergene sequence GJZEIYTO fifteen CCTACCCCCACCACCTCACCTTCCCTCAGCCTTGTGTTTCAGCCCCAGTTAGCTGCCCTTAGTTGCTGATGTATTG Chromosome 6:53805960 Intron RFGIYSHZ 16 AGCACGTTTCTCAGTTTCAGCCTGGGGCCTGGCTATAAAGCAAATGCTCAGTCCAGCATTGCGGAATGCAAGGG Chromosome 2:23702834 Intron RFTBCWPJ 17 ACAGCACCGTGCTGCACGGCGTCCTCCGGCCTTGCTGCCTGGCAATGGGTAGCCACCTGGCGTCTGTCTCAGAATA Chromosome 13:75549354 5'UTR XCVJFHOB eighteen TTCCCAGAAGGCGATTGAACCTGAAGCTGCGCCTGGCGCGTGAGCCTGTGGGGGGGACGCGGCTGAGGGGCTTTGA Chromosome 10:132654830 Intron ZMIYSTJN 19 TATCCCTTCCCCAAGTGCAACCAACAGTTGCTCTAAAGCTAGGCTGGTGGAGTTGGGGAAAGGGCCAGCAAGTGAG Chromosome 10:69571178 Intergene sequence PEVYOHIU twenty TCCTCCATGCTCAGAAAGCCTTTCTTGGAGCCAGGCACACAGGAAATGTTAGCTAGTTAGCATTGGCTCTAATACT Chromosome 2:62164811 Intergene sequence QRMXFJNY 21 TAGCTGGGGAGAGATTGCCTCTCTCAGGGCCTAGCCAGTTCCTAGAAATAGCAAGGGCTCAGCTGAGAGCATGCTT Chromosome 14:67422067 Intergene sequence QBFUYVGW 22 TCCCCCGGAAGTGTCCGCACCTCCTAGAGCCCAGCGAGCGAGCGTTTGTGCTTTTGTCCTTTGAACCGGGTGTGGT Chromosome 1:29182166 Intergene sequence ZJCRPAXW 23 GACTCATTCATCCACTCATTCTGAGCACTTGCTGCACACTAGGCCCTGGGCTGGGGCTTCAGCCCAGGAGTTCACT Chromosome 1:54506813 Intergene sequence LXAFJWRI 24 ATGTAGTCTGACGGCCGCGACTGGTTCGTAGCTTTTGAGTGAGGCGGCGGGAAGGGAGCGAGGGAAGAGCGGCAGT Chromosome 2:84459584 Intergene sequence EMQNZDWX 25 TCATGTTATGGAAGTGGCTTCTTTCCTTAAGCCTTATGAATAAGCCTCTGCTAGCTTCAAACTTTGTGTGCAGCTT Chromosome 20:37707468 Intron YJRBCUNZ 26 CATAAAGCACTTACAACAGTGCCTGGCAAATGCCTAGTGCACAAGCAAGTGTTAGCTATTGTTAATGACTATCCATT Chromosome 1:19916857 Intergene sequence HBXGRVYT 27 ATTTTTGCCCCTGTCTTCTCTTTTCCTCCTTTGCTGCATCCCAGGCTCCAGCCTTTCAGCCCTATTTGCAGTACCC Chromosome 1:204975770 intron sequence ZLRCYHDF 28 ACAAGGGGTTCAAGGTTATGAATAACCTGTGCTAATCCCAGAGGCCCCAGGACAGAGTAAGTGGGAACAAACACTG Chromosome 7:131503657 3'UTR OTHMRBJL 29 CGGAAGTTAATATGATCATTGCTAACATTTGCTGTGTTTCAGGCACTGTAAGCATGTATATGGGTCCTTAAAGGGA Chromosome 11:108224087 Intron RSYQNPLG thirty TGAGCCCAGAAACCCCTTACCCTTCCTCCTGCCTCTTGAGAGGCCAGTGTTAGGTGTTAGCCGGGTGCAAAGCTC Chromosome 20:44683649 Intergene sequence SIJYTMVG 31 AACCTGGTGCGAGCAGCCCGGGCTACAGGGTTGCCTGAGGTGTGGGTCCCAGGATGGAGGAGCCCCAGGCCGGCGG Chromosome 19:45406704 coding sequence BCGJKHUV 32 ACTTCGGTGAAGGAAGTCATCAGTGCAGTTGCCGACAAGCTGGGCTCCGGGGAGGGCCTGATCATAGTCAAGATGA Chromosome 2:173018748 coding sequence RDEWOSIT 33 GGGGCCAGGCAGGAAGAACAGCTAACTCTAGCTCACCTGCAAGGCTCAGCACTGGGTTCATTTGAAGTAGTGTCCT Chromosome 3:47439806 Intron YPLXMQCB 34 CTCCTTTGGGTGTGGACGGGACTAACACTTGCTCCATGTCAGGGCTGCAGGACCTCCTGGCTGTTGACAGCAGGCA Chromosome 16:85317248 Intergene sequence TPHSLVEM 35 GATGTTTGAAAAGCGCTGAGCCTGGCCTGGCACCTAAACAGCTCAGCAAGTGTTAGCCAGGATCACTAGCAGTAAT Chromosome 19:45686044 Intergene sequence WOEIMTLA 36 ACCCTGCCGATTTCCTTCCAGTTGCTCGCTGGGGCAACCGGCTAGGCTGGAGGAAGGGCGAGGACGGTGTCACCCCC Chromosome X:134914734 intron TBUYJIVX 37 GAACATGGTGAGGACAAAATGATGTCCAGGAGCCTTTTCTTTGGCCATTGCTAGCCTGAGACGAAAAGTCAGTGGC Chromosome 17:40557500 intron LPRCNGHU 38 AGTCTGTCAGTCTATCGTCCCTACCTGCAGCCCAAAGCATAAGCAACATCTTGCCCAGCTCAGAGGTGACAACCTC Chromosome 1:42891189 Intergene sequence AKRHSGPT 39 CACCGACCCAAGGACCACTCCTTCCAGGAACCTAGCCTAAAGCAAAGGTGCAGACAGCCCGGGGCCACCGCTGACC Chromosome 17:28854518 Intergene sequence IRSUAEVF 40 TTGCAAAAGGAAGTTTGAAGCTAGCAGAGGCTGATTCATGAGGTTCAAGGAAAGAAGTCATCTCTGTAACATAAAA Chromosome X:30280457 Intergene sequence ATWDXHSC 41 CGTCCCCCAGAGTCCTGTTTCCTCTCCTTCAGCCCCCAAATGAGCAAAGGTTAGGCCCCACCCCTGCTGAGTCAGC Chromosome 12:56636086 intron sequence QDMBZRWI 42 GTCAGGGGACAGGGTTTCCTAGCATCCGCGAGCCTTACAGAAAGGCAACTGTGCAGTGCTCCAGCTGGCTTTCTCA Chromosome 10:26734303 intron WBEAFQRU 43 ACCATCAGAGAAGCTAACCTTTCCTGAGGCCTAACTACTTTGCAGGTCCTGTATTATGTCCTCTATAGACATTAGG Chromosome 12:104048818 intron RXDOKCGI 44 GATCATTTCAAAGAAAAACATTCTGGAACAGTTGCCTATGGGCACGGCAGGGTGACGGTGCTGCTTCTGGGTTTGAC Chromosome 11:11842581 intron sequence TXHULGPN 45 GAAGGGCAGAGAACTATAATGACACAGTTGCTTTATTGCTGGACCCAGGACAGTTTATACAGCAGTTCGGAAAGGC Chromosome 11:33894467 Intergene sequence DYNFTSUP 46 ACTTATTGTAAGTGACGCATGTGACCAGTTGCCAATTGTTCAGGCTACAGCTTGGATCTGTTAGCATCCTCATTTA Chromosome 11:102685873 Intergene sequence XFDECBQY 47 CCCAGGGGCTGTGGGACACTCAGAGAGCCTATGCCTGTGCCCTGGGCTCCGGGAGGGGAGAGGATCTGGGGGCCAG Chromosome 16:2122341 intron ZTIFQRKB 48 CCCCAGGGGCTGTGGGACACTCAGAGAGCCTATGCCTGTGCCCTGGGCTCCGGGAGGGGAGAGGATCTGGGGGCCAG Chromosome 16:15141541 Intergene sequence ZFUQTLMTFC 49 AgCTTCCTGcAGCCTcCCTCAAAGCAgaTGTTAGCaCTATTA Chromosome 10:73205435 intron sequence ZCNLWTPH fifty CCTcaCCTaGAaCCTGGGCCCCtGCAACTGTaAcCTGTGGCA Chromosome 19:1198151 Intergene sequence QYYPVRQWMN 51 CCAGAGGaagAACAGTTGCTTGGGTCtAGGCctCAGGAAGGG Chromosome 10:101133375 intron EKYJXRAHKV 52 CCTgTgCTGGgGCCTGGGAGGCAgGCggCTGcTAGCCATCCTG Chromosome 6:89888014 Intergene sequence ADWZQXZI 53 TGACTTAGgaAgCAGTTGCTACCTGCCAGGCcCCAGGctAGG Chromosome 9:128604027 intron sequence

[0208] Shown in FIG. 3 data demonstrated that certain mutations reduced the activity of proteins towards the BCL11A cognate target site, with a commensurate drop in non-targeted cleavage activity (see e.g. % for the original sequences; activity in the non-target NIFMAEVG also dropped to 0.05% versus 9.04% for the original sequences, and activity in the non-target PEVYOHIU dropped to 0.03% from the value of 0.65% for the original sequences ((Fig. 3A)).However, with other mutations, the target cleavage activity remained stable, with substantially reduced activity at both non-target sites assessed. For example, the 51857-ELD/51949-KKR_R416S pair exhibited a target activity against BCL11A substantially similar to that of the parent proteins (80 .63% indels for the mutant pair versus 80.59% for the original pair at a dose of 2 μg (20 μg/ml)), while the activity in the two non-target sites for substantially decreased (0.75% in the non-target NIFMAEVG site for the mutant pair vs. 9.04% for the originals, and 0.08% in the non-target site PEVYOHIU for the mutant pair vs. 0.65% for the originals) (Fig. 3A).

[0209] Mutant proteins were also assembled using the opposite orientation of the Fok I heterodimeric domains, ie. in FIG. 3A shows the results for 51857-ELD/51949-KKR and FIG. 3B shows the results for 51857-KKR/51949-ELD. And in these cases, some pairs also maintained a stable target activity ( for example , 51857-KKR/51949-ELD_K448S), similar to the activity of the original pair (83.02% vs. 88.28%, respectively), but a decrease in activity was observed in non-target locations (1 .00% vs. 9.26% for NIFMAEVG, 0.33% vs. 0.87% for PEVYOHIU (Fig. 3B).

[0210] Experiments were also performed using a pair of TCRA (TRAC)-specific ZFNs: SBS#52742_ELD/SBS#52774_KKR (US Patent Publication No. US-2017-0211075-A1). These experiments were performed in K562 cells that were treated with either 100 or 400 ng of the mRNA encoding each ZFN. These ZFN pairs consisted of a mutated partner from those listed in Table 2 below and one unmutated partner. In these experiments, all of the positively charged amino acids indicated in FIG. 1 were mutated to serine (S). Briefly, 2 x 10e5 cells per transfection were used, with mRNA delivered to the target cell using an Amaxa 96-well shuttle system. Transfected cells were harvested 3 days post-transfection and processed for MiSeq analysis (Illumina) by standard methods. These data are shown in table 2; they demonstrate that some mutations retained persistent target activity, while others ( eg , 52742 ELD_K469S, identified as an active site residue, in Figure 1) knocked out cleavage activity.

Table 2: Target outcomes for TCRA-targeted (TRAC) ZFN with Fok I mutations

Total % indels Total % indels Option 52742 400 ng 100 ng Option 52774 400 ng 100 ng ELD 90.72 71.46 KKR 89.08 70.58 Fok_ELD_K387S 92.31 66.35 Fok_KKR_K387S 89.06 62.70 Fok_ELD_K393S 73.06 23.09 Fok_KKR_K393S 65.89 17.98 Fok_ELD_K394S 71.15 21.86 Fok_KKR_K394S 67.44 25.09 Fok_ELD_R398S 86.23 41.21 Fok_KKR_R398S 84.61 42.28 Fok_ELD_K400S 88.16 63.12 Fok_KKR_K400S 83.15 0.13 Fok_ELD_K402S 84.49 40.64 Fok_KKR_K402S 79.76 0.15 Fok_ELD_R416S 92.91 67.06 Fok_KKR_R416S 87.11 45.48 Fok_ELD_R422S 88.98 47.48 Fok_KKR_R422S 69.98 20.95 Fok_ELD_K427S 75.15 25.24 Fok_KKR_K427S 73.51 25.40 Fok_ELD_K434S 88.65 57.40 Fok_KKR_K434S 88.50 64.84 Fok_ELD_R439S 85.58 56.54 Fok_KKR_R439S 88.38 70.39 Fok_ELD_K441S 88.15 61.13 Fok_KKR_K441S 78.26 72.33 Fok_ELD_R447S 70.26 25.74 Fok_KKR_R447S 36.08 15.06 Fok_ELD_K448S 90.95 68.02 Fok_KKR_K448S 91.39 79.43 Fok_ELD_K469S 0.16 0.12 Fok_KKR_K469S 0.52 0.29 Fok_ELD_R487S 0.45 0.21 Fok_KKR_R487S 0.30 0.18 Fok_ELD_R495S 2.52 0.58 Fok_KKR_R495S 5.71 1.15 Fok_ELD_K497S 87.46 54.80 Fok_KKR_K497S 87.18 59.54 Fok_ELD_K506S 85.54 58.14 Fok_KKR_K506S 86.05 62.42 Fok_ELD_K516S 42.50 12.38 Fok_KKR_K516S 0.16 25.48 Fok_ELD_K525S 91.00 60.36 Fok_KKR_K525S 88.31 46.52 Fok_ELD_K529S 85.01 46.71 Fok_KKR_K529S 88.34 64.13 Fok_ELD_R534S 2.23 0.57 Fok_KKR_R534S 87.46 60.69 Fok_ELD_K559S 86.45 47.69 Fok_KKR_K559S 88.27 58.47 Fok_ELD_R569S 22.45 5.20 Fok_KKR_R569S 64.99 18.87 Fok_ELD_R570S 84.97 46.82 Fok_KKR_R570S 89.58 62.32 Fok_ELD_K571S 84.93 44.61 Fok_KKR_K571S 87.75 70.77

[0211] Off-target analysis was also performed to determine the most frequent off-target cleavage sites as assessed using an unbiased capture assay (PCT Patent Publication WO 2016/183298). The four genomic loci (provided target in TCRA (TRAC) and three non-target loci) identified for this ZFN pair using an unbiased capture assay are shown in Table 3 below.

Table 3: Cleavage sites for TCRA (TRAC)-specific ZFN

"Register sign" Seq ID Sequence (5'→3') Chromosome: hg38 locus Type of JBSCKVMP (provided target) 54 TCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCG Chromosome 14: 22550603 coding sequence XSKWTVWD 55 CCATATGCAGAAAACAGAAACTGTACCCCCTTCCTTACACC Chromosome 18: 30735897 Intergene sequence XVFENVRX 56 TGGCCCTTTGCAGAAAAGGTTGTTGAACCCTACCGTAAAC Chromosome 16:4780553 intron sequence KXWACMTR 57 CCGGAGAGAAGGCTCCGGTTCAGCACTGAGATCAGGACGG Chromosome 1:53220206 Intergene sequence

[0212] Nuclease activity was analyzed at two non-target sites (referred to as OT11, or XVFENVRX and OT16, or XSKWTVWD, presented in Table 3 above) at a dose of 400 ng of mRNA of each ZFN, with one partner in the ZFN pair containing the specified mutation, while in the other retained the unmodified Fok I ELD or KKR domain. The data presented (Table 4) reflects the percentage of indels (activity) observed with 400 ng of each ZFN partner. These data also showed that some mutations retained nearly equivalent target activity along with a reduction in off-target activity. For example, SBS#52774_KKR_K387S had a target activity of 89.06% indels at 400 ng (compared to 89.08 for the original 52774_KKR) and a combined off-target activity in OT11 and OT16 of 6.39% indels (compared to from 15.07% for the original 52774_KKR). The results included Fok I mutations tested in BCL11A-specific ZFNs, where changing the positive charges at positions 416, 422, 447, 448, and 525 reduced off-target activity.

Table 4: Off-target cleavage of TRAC ZFN, mutant onFoxI

% indels % indels 52742 Target OT16 OT11 OT11+OT16 52774 Target OT16 OT11 OT11+OT16 ELD 90.72 7.39 5.82 13.21 KKR 89.08 7.80 7.27 15.07 Fok_ELD_K387S 92.31 13.15 4.32 17.47 Fok_KKR_K387S 89.06 3.12 3.27 6.39 Fok_ELD_K393S 73.06 0.87 0.60 1.47 Fok_KKR_K393S 65.89 1.04 0.71 1.76 Fok_ELD_K394S 71.15 0.79 0.61 1.40 Fok_KKR_K394S 67.44 1.02 0.77 1.79 Fok_ELD_R398S 86.23 2.64 1.86 4.50 Fok_KKR_R398S 84.61 3.10 2.04 5.14 Fok_ELD_K400S 88.16 4.13 2.55 6.68 Fok_KKR_K400S 83.15 2.49 1.60 4.09 Fok_ELD_K402S 84.49 2.10 1.64 3.74 Fok_KKR_K402S 79.76 2.93 1.21 4.13 Fok_ELD_R416S 92.91 6.29 3.08 9.37 Fok_KKR_R416S 87.11 0.74 0.93 1.68 Fok_ELD_R422S 88.98 1.18 1.09 2.27 Fok_KKR_R422S 69.98 0.26 1.07 1.33 Fok_ELD_K427S 75.15 1.18 0.89 2.07 Fok_KKR_K427S 73.51 2.16 1.30 3.46 Fok_ELD_K434S 88.65 5.52 3.50 9.03 Fok_KKR_K434S 88.5 6.76 5.44 12.20 Fok_ELD_R439S 85.58 4.51 2.77 7.29 Fok_KKR_R439S 88.38 7.39 5.62 13.01 Fok_ELD_K441S 88.15 5.96 3.44 9.40 Fok_KKR_K441S 78.26 4.93 3.38 8.30 Fok_ELD_R447S 70.26 0.32 0.14 0.46 Fok_KKR_R447S 36.08 0.11 0.18 0.29 Fok_ELD_K448S 90.95 5.28 1.96 7.24 Fok_KKR_K448S 91.39 2.05 1.12 3.18 Fok_ELD_K469S 0.16 0.18 0.15 0.33 Fok_KKR_K469S 0.52 0.18 0.14 0.32 Fok_ELD_R487S 0.45 0.16 0.12 0.28 Fok_KKR_R487S 0.3 0.17 0.11 0.28 Fok_ELD_R495S 2.52 0.14 0.12 0.26 Fok_KKR_R495S 5.71 0.15 0.13 0.28 Fok_ELD_K497S 87.46 3.56 2.66 6.23 Fok_KKR_K497S 87.18 6.13 6.44 12.57 Fok_ELD_K506S 85.54 4.06 3.05 7.10 Fok_KKR_K506S 86.05 4.22 2.55 6.77 Fok_ELD_K516S 42.5 0.45 0.34 0.79 Fok_KKR_K516S 0.16 0.11 0.15 0.26 Fok_ELD_K525S 91 0.81 2.13 2.94 Fok_KKR_K525S 88.31 0.96 0.79 1.75 Fok_ELD_K529S 85.01 1.99 1.46 3.45 Fok_KKR_K529S 88.34 6.45 4.83 11.27 Fok_ELD_R534S 2.23 0.14 0.15 0.29 Fok_KKR_R534S 87.46 4.44 3.29 7.74 Fok_ELD_K559S 86.45 2.72 1.84 4.56 Fok_KKR_K559S 88.27 4.27 2.61 6.88 Fok_ELD_R569S 22.45 0.25 0.22 0.47 Fok_KKR_R569S 64.99 1.39 1.05 2.45 Fok_ELD_R570S 84.97 2.73 1.96 4.68 Fok_KKR_R570S 89.58 7.98 6.20 14.18 Fok_ELD_K571S 84.93 2.27 1.96 4.23 Fok_KKR_K571S 87.75 8.24 6.11 14.34

от молекулы ДНК (Фиг. 4). Отмечены точки данных, соответствующие парам ZFN, где одна ZFN несет мутацию FokI в положении 416, 422, 447, 448 или 525.[0213] These data were then compared with the calculated distance between the mutated amino acid residue and the DNA molecule to study the effect on both targeted and non-targeted cleavage (FIG. 4). The activity of each ZFN pair is represented by one data point; it was demonstrated that proteins with the most desirable profiles (high target activity and low non-target activity) were represented by proteins with mutations within 10

from the DNA molecule (Fig. 4). Data points are marked corresponding to pairs of ZFNs where one ZFN carries a Fok I mutation at position 416, 422, 447, 448, or 525.

[0214] These results demonstrated that mutations of one or more residues 416, 422, 447, 448, or 525 can increase target activity while decreasing off-target activity.

Example 3: Design of New Engineered Zinc Finger Backbone Mutations

[0215] Previous studies indicate the possibility of some interaction between positively charged amino acid residues in the "backbone" with zinc fingers (regions of the structure not involved in site-specific DNA nucleotide recognition) and phosphates on the DNA molecule (Elrod-Erickson et al , ibid. ), see FIG. 5A. The amino acids at positions -14, -9, and -5 (all positions are relative to the standard alpha-helical region numbering) are often positively charged and can interact with negatively charged phosphates in the DNA backbone (see Fig. 5B). Accordingly, 4867 zinc finger sequences were analyzed for the presence of amino acid residues at each position in the finger sequence (see FIG. 6). At position -5, the neutral amino acids alanine, leucine and glutamine were observed with low but non-zero frequency, thus these amino acids were used in the modifications of the backbone fingers. Positions in ZFP with 6 and 5 fingers were also identified along with potential substitutions (FIGS. 7A and 7B).

[0216] Mutations were carried out in the TCRA (TRAC)-specific ZFN pair SBS#52774/SBS#52742 (see PCT publication WO 2017106528). For these proteins, a total of 21 variants of each partner ((F1, F3, F5, F1+F3, F1+F5, F3+F5, F1+F3+F5) × (R→A, R→Q, R→L )). A representative set of data (Table 5) showed that many couples showed a decrease in off-target activity against the three off-targets analyzed. In this table, ZFN 52774 was combined with ZFN 52742 variants carrying the indicated mutations at position -5 of finger 1 (F1), finger 3 (F3), and finger 5 (F5). The type of mutation is also indicated; all mutants in this dataset are arginine (R) to glutamine (Q) substitution mutants. For example, the protein designated 52742-F1RQ corresponds to a mutant where the arginine at position -5 in finger 1 has been changed to glutamine. The top half of the table shows activity expressed as % indels and the bottom half of the table shows activity expressed as the relative level of the mean activity of the two replicates of the original ZFN 52774/52742 pair. These experiments demonstrated that these mutations can affect off-target cleavage. For example, for mutant 52742-F1RQ; F3RQ; F5RQ while maintaining a stable level of target cleavage (target activity of 69.96% relative to the activity of 62.59% for the original proteins at a dose of 6 μg), the level of non-target cleavage fell (for OT16, a cleavage activity of 19.16% was shown for the original proteins and an activity of 1.43 % for the triple mutant).

[0217] Starting with TCRA (TRAC)-specific parent ZFNs 52742 and 52774, arginine (R) in the first finger of each module was replaced with alanine (A), glutamine (Q), or leucine (L). These constructs were tested in CD34+ cells by treating them with 6 μg of the mRNA encoding each ZFN partner (see Fig. 8A). In the indicated data sets, each column corresponds to the mean of the data for all mutations of each type, and the standard error is indicated by error bars. For example, in FIG. 8A, the black left bar corresponding to the target activity indicates a value representing the average of the target activity for all 6 mutants in a ZFN pair that may contain a single mutation. In the diagram in Fig. 7, these mutations include, respectively, a single mutation in the N-terminal finger of module A 52742 (F1 in the full-length protein), a single mutation in the N-terminal finger of module B 52742 (F3 in the full-length protein), a single mutation in the N-terminal finger of module C 52742 (F5 in the full-length protein) and a similar set of mutations in partner protein 52774. The pooled level of target activity in the second column in black (corresponding to target activity) is the average value for all proteins with 2 mutations (a set of 6 possible options when introducing mutations in one ZFN partner) and in the third column in black the pooled level of target activity is the mean of the 2 possible variants with mutations in all three left or right ZFN modules. For off-target activity data corresponding to gray bars, similar pooled levels were obtained, except that the data for the three off-target sites were combined such that each pooled data set for single or double mutations in the off-target activity data set included 18 data points. , and the pooled data set for the three mutations included 6 data points. These mutations provided a decrease in off-target activity up to 4.8-fold.

[0218] Also conducted experiments in which both partners in a pair of ZFN were mutated in a similar way (right half of Fig. 8B). For example, if the N-terminal finger in module A is changed to alanine, (52742-F1RA), the partner protein is also mutated in the N-terminal finger in module A (52774-F1RA), giving a total of 2 mutations in the ZFN dimer. Alanine-only substitutions were tested simultaneously in both ZFNs in the dimer; these data are shown on the right half of FIG. 8B (designated as 2, 4 or 6). R→A mutations are introduced into only one ZFN in the dimer (designated as 1, 2, or 3) from the left third of FIG. 8A, shown again on the left half of FIG. 8B. Fig. 8C is similar to the right side of FIG. 8A except for using only 2 μg (20 μg/ml) of RNA. These experiments demonstrated that these mutations can provide a 27-fold reduction in off-target activity with a total of six mutations in both ZFNs in the dimer.

[0219] These experiments were also performed with the BCL11A-specific ZFN 51857-ELD/51949-KKR pair described above. Experimental design was similar to that described for the TCRA (TRAC)-specific ZFN pair; the results are shown in FIG. 9A. Data shown reflects results for pairs containing either R→Q (Gln) or R→L (Leu) mutations at 2 μg (20 μg/mL), with only results for one non-target site reported for off-target activity. (NIFMAEVG, see table 1).

[0220] Additional amino acid variants at position -5 were generated by replacing R with E, N, Y, A, or L. In addition to changing the amino acids at position -5, a number of mutations were introduced at positions -9 and -14 in Finger 1 of the BCL11A-specific ZFN pair 51857/51949. These mutations were tested for target and non-target activity as described above, alone and in combination with changes at position -5 in fingers 2-6. The data are shown in Table 6 below. Each protein contained the same DNA-specific helical regions as described for the original proteins above, but received a new SBS identification number reflecting ZFP backbone mutations. Briefly, the full names of the proteins reflect the listed variants of mutations in the finger. Accordingly, the "cR" part of the full name refers to changes made to the C-terminal finger of the two-finger module (see Fig. 7), and "nR" refers to changes made to the N-terminal finger of the two-finger module. The description "rQa" corresponds to the changes made at position -5 in module A, and the finger to which these changes were made is designated either cR or nR. Accordingly, SBS#65461 (full name 51857-NELD-cR-5Qa) is a derivative of SBS#51857 where changes are made to the C-terminal finger at position -5, with Q replacing R in module A. These notations are also used in table, in cells where Q is present in column -5 for F2. When changes are made at position -14, as in the case of SBS#65459 (full name 51857-NELD-nR-14Q-5Qabc), this is indicated in the table. Accordingly, SBS#65459 is a derivative of SBS#51857 where module changes are made to the N-terminal finger, with R replaced by Q at position -14 of Finger 1 and at positions -5 in the N-terminal finger of modules A, B and C. In the case of SBS#65460, at position -14 of Finger 1, R is replaced by S. This is also reflected in the table. The designations "NELD" and "CKKR" indicate the type of Fok I nuclease domains (the "ELD" or "KKR" Fok I domain variant) and other aspects of said vector (see PCT Publication WO 2017/136049). Table 6A shows a selection of pairs of mutations introduced into SBS#51857 derived partners, with either SBS#51949 or SBS#51949 partner containing changes with Q insertions in the N-terminal fingers at positions -5 in modules A, B, and C , where the specified partner additionally contains the R416S mutation in the Fok I domain, while the experiments were performed using 2 μg of mRNA encoding each ZFN. These experiments were also performed using mutations in the protein SBS#51949 (see table 6B), while mutations interacting with phosphate amino acids of the Fok I domain were also tested in combination with backbone mutations. These data indicate that additional changes were able to influence the specificity of the ZFN pairs.

Table 6A: Changes in positions in the ZFN backbone SBS#51857

Finger 1 (F1) F2 F3 F4 F5 F6 AK change Right partner: 51949-CKKR Right partner: 51949-CKKR-nR-5Qabc-R416S SBS# Full title -fourteen -9 -5 -5 -5 -5 -5 -5 Bcl11a OT1 TXHULGPN Bcl11a OT1 TXHULGPN 51857 51857-NELD (AV-A) 0 70.1 3.83 0.11 59.2 0.15 0.08 65461 51857-NELD_cR-5Qa Q one 68.4 2.18 0.11 55.5 0.35 0.07 65462 51857-NELD_cR-5Qb Q one 69.2 1.18 0.14 63.4 0.09 0.04 65463 51857-NELD_cR-5Qc Q one 59.7 0.85 0.08 55.5 0.20 0.07 65464 51857-NELD_cR-5Qab Q Q 2 N/A 1.00 0.04 53.7 0.05 0.07 65465 51857-NELD_cR-5Qac Q Q 2 62.3 0.74 0.03 55.1 0.13 0.06 65466 51857-NELD_cR-5Qbc Q Q 2 62.4 0.38 0.05 57.9 0.15 0.08 65467 51857-NELD_cR-5Qabc Q Q Q 3 59.2 0.27 0.05 53.2 0.08 0.08 63014 51857-NELD_nR-5Qabc (AV-B) Q Q Q 3 55.7 0.73 0.07 55.5 0.15 0.02 65453 51857-NELD_nR-5Eabc E E E 3 41.6 0.20 0.04 44.9 0.04 0.07 65454 51857-NELD_nR-5Nabc N N N 3 7.8 0.13 0.02 48.3 0.08 0.07 65455 51857-NELD_nR-5Yabc Y Y Y 3 42.8 0.50 0.03 42.4 0.09 0.10 65459 51857-NELD_nR-14Q-5Qabc Q Q Q Q four 55.9 0.53 0.05 52.9 0.19 0.07 65460 51857-NELD_nR-14S-5Qabc S Q Q Q four 54.3 0.66 0.06 53.3 0.07 0.07 65469 51857-NELD_nR-5Qabc,cR-5Qa Q Q Q Q four 44.6 0.24 0.06 50.8 0.01 0.08 65470 51857-NELD_nR-5Qabc,cR-5Qb Q Q Q Q four 44.5 0.22 0.07 49.8 0.06 0.02 65471 51857-NELD_nR-5Qabc,cR-5Qc Q Q Q Q four N/A 0.09 0.05 53.9 0.05 0.06 65472 51857-NELD_nR-5Qabc,cR-5Qab Q Q Q Q Q 5 37.3 0.15 0.04 41.2 0.06 0.08 65473 51857-NELD_nR-5Qabc,cR-5Qac Q Q Q Q Q 5 26.9 0.13 0.05 33.2 0.08 0.02 65474 51857-NELD_nR-5Qabc,cR-5Qbc Q Q Q Q Q 5 25.2 0.11 0.04 37.4 0.08 0.09 65475 51857-NELD_nR-5Qabc,cR-5Qabc Q Q Q Q Q Q 6 20.1 0.15 0.06 29.1 0.13 0.09 65477 51857-NELD_nR-5Eabc,cR-5Qabc E Q E Q E Q 6 3.3 0.07 0.05 4.5 0.09 0.08 65478 51857-NELD_nR-5Nabc,cR-5Qabc N Q N Q N Q 6 0.1 0.09 0.05 0.2 0.09 0.05 65479 51857-NELD_nR-5Yabc,cR-5Qabc Y Q Y Q Y Q 6 0.2 0.09 0.05 0.1 0.06 0.03 65481 51857-NELD_nR-9Na-5Qabc,cR-5Qabc N Q Q Q Q Q Q 7 15.1 0.11 0.03 27.3 0.15 0.03 65482 51857-NELD_nR-9Sa-5Qabc,cR-5Qabc S Q Q Q Q Q Q 7 18.8 0.12 0.05 23.7 0.06 0.07 65483 51857-NELD_nR-14Q-5Qabc,cR-5Qabc Q Q Q Q Q Q Q 7 12.4 0.19 0.07 21.0 0.07 0.07 65484 51857-NELD_nR-14S-5Qabc,cR-5Qabc S Q Q Q Q Q Q 7 12.5 0.06 0.06 22.5 0.05 0.04 65468 51857-NELD_nR-14S-5Qabc,cR-5Aabc Q Q A Q A Q A 7 24.0 0.13 0.04 30.2 0.07 0.02 65476 51857-NELD_nR-14S-5Qabc,cR-5Labc Q Q L Q L Q L 7 5.4 0.11 0.04 12.9 0.10 0.04

Table 6B: Changes to other positions in the skeleton and Fok I in ZFN SBS#51949

Finger 1 (F1) F2 F3 F4 F5 AK change Right partner: 51949-CKKR Right Partner: 51949-CKKR-nR-5Qabc-R416S SBS# Full title -fourteen -9 -5 -5 -5 -5 -5 Bcl11a OT1 TXHULGPN Bcl11a OT1 TXHULGPN 51949 51949-CKKR (AV-C) 0 64.3 3.24 0.10 33.1 0.28 0.09 51949-R416S 51949-CKKR-R416S (AV-D) 0 68.8 0.52 0.06 38.7 0.07 0.11 65529 51949-CKKR-R416S_cR-5Qb Q one 69.2 0.17 0.08 44.9 0.13 0.10 65530 51949-CKKR-R416S_cR-5Qc Q one 67.7 0.20 0.04 56.4 0.08 0.08 65528 51949-CKKR-R416S_cR-5Qbc Q Q 2 73.3 0.12 0.06 53.6 0.07 0.06 63022-R416S* 51949-CKKR-R416S_nR-5Qabc (AV-G) Q Q Q 3 71.9 0.18 0.04 50.1 0.15 0.06 65509 51949-CKKR-R416S_nR-5Eabc E E E 3 49.8 0.07 0.04 37.3 0.08 0.04 65510 51949-CKKR-R416S_nR-5Nabc N N N 3 67.6 0.19 0.06 46.7 0.05 0.05 65511 51949-CKKR-R416S_nR-5Yabc Y Y Y 3 64.5 0.17 0.14 41.8 0.10 0.08 65515 51949-CKKR-R416S_nR-14Q-5Qabc Q Q Q Q four 66.4 0.15 0.06 47.2 0.15 0.14 65516 51949-CKKR-R416S_nR-14S-5Qabc S Q Q Q four 65.1 0.12 0.06 43.4 0.12 0.10 65525 51949-CKKR-R416S_nR-5Qabc,cR-5Qb Q Q Q Q four 71.3 0.12 0.07 45.7 0.08 0.06 65526 51949-CKKR-R416S_nR-5Qabc,cR-5Qc Q Q Q Q four 69.7 0.08 0.05 53.0 0.18 0.11 65527 51949-CKKR-R416S_nR-5Qabc,cR-5Qbc Q Q Q Q Q 5 66.7 0.09 0.06 43.2 0.10 0.07 65517 51949-CKKR-R416S_nR-5Eabc,cR-5Qbc E E Q E Q 5 12.9 0.09 0.02 10.6 0.07 0.11 65518 51949-CKKR-R416S_nR-5Nabc,cR-5Qbc N N Q N Q 5 60.6 0.02 0.05 N/A 0.07 0.05 65519 51949-CKKR-R416S_nR-5Yabc,cR-5Qbc Y Y Q Y Q 5 65.5 0.12 0.04 43.8 0.08 0.03 65521 51949-CKKR-R416S_nR-9N-5Qabc,cR-5Qbc N Q Q Q Q Q 6 70.8 0.09 0.05 51.8 0.12 0.07 65523 51949-CKKR-R416S_nR-14Q-5Qabc,cR-5Qbc Q Q Q Q Q Q 6 61.0 0.10 0.07 42.5 0.10 0.04 65524 51949-CKKR-R416S_nR-14S-5Qabc,cR-5Qbc S Q Q Q Q Q 6 51.9 0.08 0.09 38.8 0.07 0.07 65531 51949-CKKR-R416S_nR-14Q-5Qabc,cR-5Abc Q Q Q A Q A 6 50.7 0.06 0.04 38.7 0.05 0.10 65532 51949-CKKR-R416S_nR-14Q-5Qabc,cR-5Lbc Q Q Q L Q L 6 40.5 0.05 0.07 31.0 0.16 0.05 51949-K525S 51949-CKKR-K525S (AV-E) 0 49.0 0.16 0.05 31.1 0.08 0.05 65554 51949-CKKR-K525S_cR-5Qc Q one 35.5 0.10 0.13 37.9 0.10 0.06 65552 51949-CKKR-K525S_cR-5Qbc Q Q 2 55.9 0.10 0.05 37.4 0.58 0.02 63022-K525S** 51949-CKKR-K525S_nR-5Qabc (AV-H) Q Q Q 3 59.5 0.04 0.05 44.9 0.14 0.10 65533 51949-CKKR-K525S_nR-5Eabc E E E 3 0.1 0.09 0.04 0.1 0.11 0.08 65534 51949-CKKR-K525S_nR-5Nabc N N N 3 52.8 0.03 0.04 N/A 0.06 N/A 65535 51949-CKKR-K525S_nR-5Yabc Y Y Y 3 53.1 0.12 0.06 35.6 0.07 0.19 65539 51949-CKKR-K525S_nR-14Q-5Qabc Q Q Q Q four 52.1 0.10 0.10 38.1 0.10 0.05 65540 51949-CKKR-K525S_nR-14S-5Qabc S Q Q Q four 54.2 0.08 0.10 34.1 0.11 0.06 65549 51949-CKKR-K525S_nR-5Qabc,cR-5Qb Q Q Q Q four 56.7 0.03 0.12 37.3 0.10 0.05 65550 51949-CKKR-K525S_nR-5Qabc,cR-5Qc Q Q Q Q four 57.3 0.12 0.09 36.0 0.11 0.00 65551 51949-CKKR-K525S_nR-5Qabc,cR-5Qbc Q Q Q Q Q 5 50.8 0.09 0.05 34.5 0.06 0.00 65541 51949-CKKR-K525S_nR-5Eabc,cR-5Qbc E E Q E Q 5 2.5 0.09 0.08 1.9 0.18 0.05 65542 51949-CKKR-K525S_nR-5Nabc,cR-5Qbc N N Q N Q 5 36.4 0.06 0.02 22.8 0.07 0.03 65543 51949-CKKR-K525S_nR-5Yabc,cR-5Qbc Y Y Q Y Q 5 53.1 0.09 0.08 32.0 0.13 0.15 65545 51949-CKKR-K525S_nR-9N-5Qabc,cR-5Qbc N Q Q Q Q Q 6 51.2 0.15 0.07 33.0 0.08 0.13 65547 51949-CKKR-K525S_nR-14Q-5Qabc,cR-5Qbc Q Q Q Q Q Q 6 36.1 0.09 0.05 22.5 0.08 0.02 65548 51949-CKKR-K525S_nR-14S-5Qabc,cR-5Qbc S Q Q Q Q Q 6 28.7 0.06 0.05 20.1 0.09 0.10 65555 51949-CKKR-K525S_nR-14Q-5Qabc,cR-5Abc Q Q Q A Q A 6 36.3 0.14 0.08 21.8 0.10 0.11 65556 51949-CKKR-K525S_nR-14Q-5Qabc,cR-5Lbc Q Q Q L Q L 6 25.0 0.06 0.05 N/A 0.10 0.04

* 63022-R416S is also known as SBS#65721.

** 63022-K525S is also known as SBS#65722.

[0221] The experiments were repeated on CD34+ cells, with RNA delivered to these cells using the BTX transfection system under conditions optimized according to the manufacturer's instructions. Three concentrations of RNA were used: a final concentration of 60, 20, and 5 μg/ml. The data are shown in Table 6C below and demonstrate that robust target cleavage can be detected even at very low levels of ZFN mRNA, thus non-target cleavage is substantially reduced (>100x). Mutations are designated according to the nomenclature in FIG. 2C. The experiment was repeated using only the original pair and the 3x(R→Q)/3x(R→Q) pair from Table 6C below to determine the robustness of the results. As can be seen from Table 6D, the results were highly reproducible.

Table 6C: Target and non-target effects in titration:

ZFN modification % indels BCL11A NIFMAEVG (OT1) PEVYOHIU (OT3) Left ZFN Right ZFN 60 mcg 20 mcg 5 mcg 60 mcg 20 mcg 5 mcg 60 mcg 20 mcg 5 mcg SBS51857 SBS51949 82.7 87.0 76.5 28.0 5.02 0.34 4.77 0.32 (0.03) 3x (R→Q)
SBS63014 3x (R→Q)*
SBS65721 88.3 86.4 78.0 0.55 0.08 (0.01) 0.04 (0.01) (0.02) 3x (R→Q)
SBS63014 4x (R→Q)*
SBS65526 85.4 86.3 73.4 0.17 (0.03) (0.03) (0.02) (0.00) (0.01) 3x (R→Q)
SBS63014 5x (R→Q)*
SBS65527 83.6 86.5 71.7 (0.03) (0.01) (0.02) (0.02) (0.03) (0.00)

The numbers in brackets correspond to the values at which no signs of nuclease cleavage were detected.

* indicates that the right ZFN additionally contained an additional R416S mutation in the Fok I nuclease domain.

Table 6D: Repeated measures of target activity

ZFN modification % indels % BCL11A indels, replicate Average % indels Left ZFN Right ZFN #1 #2 Number 3 #4 SBS51857 SBS51949 76.8 79.0 82.8 80.1 79.7 3x (R→Q)
SBS63014 3x (R→Q)*
SBS65721 80.5 81.7 81.0 83.4 81.6

* indicates that the right ZFN additionally contained an additional R416S mutation in the Fok I nuclease domain.

Example 4 Titration of ZFN Partners for Optimal Target Activity

[0222] Individual titration of each partner in a ZFN pair can determine the optimal concentration of each ZFN partner, and thus the maximum target modification by said pair, while minimizing off-target modifications. Each individual ZFN half-domain may have its own binding kinetics to the cognate target DNA, thus optimal activity can be achieved by titrating each half-domain separately. Accordingly, the BCL11A-specific pair SBS#51949/SBS#51857 was used to study titration in CD34+ cells using ZFN introduced as mRNA, using high concentrations of ZFN to allow detection of off-target cleavage. During the experiments (table 7, below) it was found that the titration of partner SBS#51857 led to a decrease in off-target cleavage (approximately 8-fold) while maintaining a stable target cleavage. For example, when using 60 μg/ml of 51949 mRNA in combination with 6.6 μg/ml of 51857 mRNA, the level of target modification remained approximately the same as when using 60 μg/ml of each ZFN (76.1% indels when using 60 µg/mL of each ZFN, 78.3% indels when using 60 µg/mL 51949 in combination with 6.6 µg/mL 51857), while the cumulative level of off-target modification dropped from 32.4% indels to 4.0%. Note that reducing the introduction of mRNA of both ZFNs equally led to a gradual decrease in the level of target modification, while the level of non-target modification essentially decreased only when the level of target modification was reduced.

Table 7: Titration of one ZFN

RNA, µg/ml % indels at each locus 51949 51857 BCL11A NIFMAEVG
OT1 GJZEIYTO
OT2 PEVYOHIU
OT3 ZJCRPAXW
OT4 TXHULGPN
OT5 Non-target aggregates 60.0 60.0 76.1 24.6 2.81 3.06 1.12 0.79 32.4 40.0 40.0 76.5 14.6 1.69 1.77 0.80 0.43 19.3 20.0 20.0 71.5 4.9 0.62 0.36 0.35 0.08 6.3 10.0 10.0 62.0 1.3 0.10 0.14 0.26 0.08 1.9 60.0 20.0 78.3 11.8 1.38 1.32 0.58 0.40 15.5 60.0 6.6 78.9 2.8 0.27 0.36 0.40 0.19 4.0 60.0 2.2 73.7 0.9 0.15 0.29 0.28 0.10 1.8

[0223] Western blot analysis was also performed to demonstrate that the expression of each ZFN partner correlated with the amount of ZFN-encoding mRNA delivered (FIG. 10). In this experiment, CD34+ cells were transfected with the indicated ZFNs and expression of ZFN proteins was detected 24 hours later using an anti-Flag antibody (the expression constructs contained the encoded Flag tag). The expression of the two proteins was also analyzed when these ZFNs were co-administered as a single RNA separated by a self-cleaving peptide sequence 2a (see lane 2 in Fig. 10).

[0224] The titration was repeated with variations of both ZFNs independently of each other to detect any effect on the target or non-target cleavage activity. The results (FIG. 11) showed that down-titration of both partners reduced off-target cleavage, but the maximum effect was observed for SBS#51857. On FIG. 11 in the table for the BCL11A target, boxed cells correspond to the retention of cleavage activity against the intended BCL11A target when using 60 μg of SBS#51949 mRNA with either 6.6 μg or 60 μg of SBS#51857 mRNA, with non-target activity at the site NIFMAEVG decreased from 27% to 4% of indels when the dose of SBS#51857 was reduced.

Example 5: Combination of ZFN partner titrations and phosphate-contacting Fok I residue mutations

[0225] Assays were then performed to measure titration activity of ZFN partners in combination with exemplary Fok I mutations. BCL11A-specific ZFNs containing Fok mutations were used in the ratios shown above to maintain target activity while reducing the level of off-target cuts. These experiments were performed in CD34+ cells transfected with mRNA encoding the indicated ZFNs. These data are presented in Table 8 below. "Target/non-target split ratio" means the ratio of target and total combined non-target activity for the specified sample. The Fok I mutant with the combination of 6.6 μg/ml SBS#51857 and 60 μg/ml SBS#51949-R416S provided similar levels of target activity (84.85% vs. at all five non-target sites monitored, resulting in a 32-fold increase in target/non-target cleavage ratio (89.52 vs. 2.76 for 60 μg/ml of each of the original ZFNs).

Table 8: Combination of Fok I mutants and dose reduction titration of ZFN partners:

% NHEJ activity

mRNA1 mcg/ml mRNA2 mcg/ml BCL11A NIFMAEVG
OT1 GJZEIYTO
OT2 PEVYOHIU
OT3 ZJCRPAXW
OT4 TXHULGPN
OT5 Target/non-target cleavage ratio 51857 60 51949 60 78.17 21.96 2.09 2.92 0.73 0.61 2.76 51857 60 51949 R416S 60 84.87 6.11 0.31 1.08 0.65 0.12 10.27 51857 60 51949 K448S 60 81.03 8.90 0.72 4.40 1.29 0.64 5.08 51857 60 51949 K525S 60 80.17 0.89 0.09 0.13 0.20 0.04 59.47 51857 6.6 51949 60 85.21 3.39 0.38 0.49 0.14 0.10 18.98 51857 6.6 51949 R416S 60 84.85 0.68 0.06 0.11 0.08 0.03 89.52 51857 6.6 51949 K448S 60 83.46 1.01 0.09 0.34 0.22 0.06 48.55 51857 6.6 51949 K525S 60 67.92 0.14 0.01 0.02 0.04 0.01 292.27

[0226] All off-target sites listed above in Table 1 were examined for effect on off-target activity by a combination of titration and Fok I mutant approach (see FIG. 12).

[0227] The data demonstrated an approximately 30-fold cumulative reduction in off-target activity.

[0228] The data was also analyzed for the number of captures detected using the unbiased capture assay described above after digestion. The BCL11A-specific pairs described in Table 6D above, both the original pair (SBS51857/SBS51949) and the variant pair (SBS63014/SBS65721) were used and the number of off-target captures was analyzed. In this experiment, ZFN was injected into CD34+ cells either in equal amounts (final concentration 60 μg/ml) or at a final concentration of 6.6 μg and 60 μg for the original pair, and 20 μg and 60 μg for the variant.

[0229] The results are shown in FIG. 13 and demonstrate that although both the parent pair and the variant exhibited consistent cleavage activity at both concentrations (>80% indels), the number of off-target uptakes was significantly reduced, particularly for the variant of the pair when delivered at unequal doses. The combination of Fok I ZFN mutations and unequal concentrations of ZFN partners resulted in a 350-fold increase in cleavage specificity.

Example 6 Combination of ZFP backbone mutations with mutations in phosphate contacting Fok I residues

[0230] We also generated ZFNs containing the zinc finger backbone mutations described in Example 3 with the phosphate-contacting Fok I residue mutations described in Example 2. This combination was tested with a BCL11A-specific ZFN pair in CD34+ cells at two doses: 6 mcg or 2 mcg. The results are presented in Table 9 below and demonstrate that a combination of these two types of approaches can significantly affect the level of off-target activity. In this table, backbone mutations are presented as type of mutation per module (A, B and/or C, see Fig. 7). For example, in the sample designated 51949 LeuABC R416S, the protein contained R→L substitutions in the backbones of fingers 1 (module A), 3 (module B), and 5 (module C), and additionally carried the Fok I R416S mutation. In several examples, ZFN pairs showed no detectable off-target activity while maintaining full target activity. These examples are boxed in Table 9.

Example 7 Combination of partner titrations, ZFP backbone mutations and phosphate contacting Fok I residues

[0231] Activity measurements were also performed by combining optimal titration partners with ZFNs containing ZFP backbone mutations and ZFN Fok I mutations. These ZFNs are tested in CD34+ cells as described above; they demonstrate an increase in the specificity of the ZFN pair at the expense of a decrease in the level of off-target activity.

Example 8 Clinical Scale Specificity of ZFN in CD34+ Cells

[0232] The specificity for the BCL11A variant of the SBS63014/SBS65722 pair was also tested in a large scale procedure suitable for obtaining cellular materials for a clinical trial. Briefly, approximately 95-130 million CD34+ cells per batch were transduced using the Maxcyte device according to the manufacturer's specification. 80 μg/ml SBS63014 mRNA and 20 μg/ml SBS#65722 mRNA were used and off-target cleavage in cells was analyzed two days later using an unbiased capture assay.

[0233] The results showed that PCR analysis of 47 different potential capture loci (see Fig. 14) did not detect significant modifications, with the exception of the target localization, where 79.54% of indels were found. This data demonstrates that the nucleases as described herein are highly specific even when used in a large scale manufacturing procedure.

Example 9 Additional Specificity Studies

[0234] Specificity studies were also performed using AAVS1-targeted ZFNs with various mutations as described above. In particular, for activity assays as described above, ZFN SBS#30035 and SBS#30054 as described in US Publication No. 20150110762 were used to study various mutants, including dimerization domain mutants ( e.g., ELD, KKR and additional mutants), other mutations ( for example, Sharkey), as well as mutants in contact with phosphate residues.

[0235] To obtain the results presented in the tables below, the specified Fok I mutant(s) were introduced into either the Fok I domain of the ELD SBS 30035 (designated as ELD_X, where X is a Fok I mutation), the Fok I domain of the KKR SBS 30054 (designated as KKR_X, where X is a Fok I mutation), or into the Fok I domains of both constructs (designated as ELD_KKR_X, where X is the same Fok I mutation introduced into both Fok I ELD and KKR domains) . The results for the combination of unmodified "original constructs" 30035 and 30054 are marked as "original", "original", "original ZFN", or the like. The lower dose of each parent construct is often labeled as a "half dose". The negative control without nucleases is usually labeled "GFP". The ratio, which is the % indels at the intended locus (often referred to as "AAVS1") divided by the sum of the % indels at all non-target loci as assessed in a particular experiment, is often referred to as "ratio", "target/non-target cleavage ratio", etc. .

[0236] The localization of the AAVS1 target and non-targets is shown below, where "hg38" means genome assembly data according to the UCSC Genome Browser database, hg38 layout variant:

Locus hg38 coordinates AAVS1 Chromosome 19 55115767 OT1 Chromosome 3 184229822 OT2 Chromosome 1 198172185 OT3 Chromosome 3 50189772 OT4 Chromosome 20 35020706 OT5 Chromosome 1 181141477

[0237] Tables 10A-10C present the results of cleavage in 2 different experiments of the target target (AAVS1) and three non-target targets (OT1, OT2, OT3), as well as the ratio of target and non-target cleavage for ELD, KKR and ELD-KKR dimerization mutants with an additional substitution mutation (each amino acid per wild-type AA molecule) at R416 or K525. Also shown are the results for the ELD_S418D, ELD_N476D, ELD_I479T, ELD_Q481E, ELD_N527D, and ELD_Q531R mutants.

Table 10A

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 57.77 9.73 1.31 1.67 1.20 4.15 Initial 55.40 9.06 1.27 1.38 0.98 4.37 Initial 60.63 11.83 1.81 1.87 1.39 3.59 Initial 55.39 13.05 2.15 2.26 2.05 2.84 Half dose 34.13 2.50 0.23 0.31 0.12 10.81 Half dose 36.73 2.67 0.19 0.41 0.23 10.49 Half dose 42.08 3.72 0.32 0.51 0.30 8.67 Half dose 40.22 3.17 0.47 0.57 0.32 8.87 ELD_R416A 59.22 1.32 0.80 0.70 0.22 19.46 ELD_R416C 61.50 2.50 1.31 1.08 0.27 11.93 ELD_R416D 52.20 0.12 0.61 0.16 0.02 57.66 ELD_R416E 76.17 0.49 1.83 0.24 0.05 29.04 ELD_R416F 75.24 3.87 2.09 1.24 0.24 10.12 ELD_R416G 48.30 0.66 0.90 0.71 0.15 19.98 ELD_R416H 82.77 6.04 4.48 1.81 0.31 6.55 ELD_R416I 28.47 3.86 0.68 0.71 0.50 4.94 ELD_R416K 18.07 0.92 0.33 0.37 0.15 10.14 ELD_R416L 51.90 4.62 1.46 1.22 0.51 6.65 ELD_R416M 60.27 2.91 1.09 0.99 0.29 11.43 ELD_R416N 74.77 0.83 1.12 0.59 0.11 28.19 ELD_R416P 2.10 0.08 0.03 0.02 0.06 10.70 ELD_R416Q 76.84 3.23 3.09 1.30 0.25 9.76 ELD_R416S 58.67 0.61 0.85 0.72 0.06 26.28 ELD_R416T 49.52 1.82 0.93 0.81 0.15 13.37 ELD_R416V 47.52 7.94 1.70 1.36 0.94 3.98 ELD_R416W 54.95 2.33 1.09 1.02 0.36 11.44 ELD_R416Y 81.38 20.16 7.28 3.53 1.44 2.51 ELD_K525A 63.30 0.30 0.12 0.14 0.11 94.56 ELD_K525C 74.28 2.89 0.48 0.80 0.51 15.90 ELD_K525D 41.76 0.11 0.04 0.03 0.07 163.80 ELD_K525E 64.31 0.07 0.08 0.10 0.10 188.65 ELD_K525F 23.10 1.13 0.04 0.18 0.35 13.67 ELD_K525G 54.21 0.34 0.25 0.21 0.08 61.86 ELD_K525H 41.07 0.79 0.17 0.24 0.28 27.65 ELD_K525I 68.23 2.29 0.28 0.47 0.61 18.71 ELD_K525L 27.08 0.48 0.09 0.21 0.20 27.89 ELD_K525M 32.75 0.25 0.08 0.13 0.10 58.51 ELD_K525N 41.84 0.56 0.14 0.30 0.21 34.59 ELD_K525P 8.45 0.02 0.03 0.02 0.03 92.43 ELD_K525Q 47.10 0.48 0.31 0.34 0.11 38.14 ELD_K525R 51.08 10.86 1.17 1.35 1.93 3.34 ELD_K525S 65.83 0.53 0.22 0.23 0.15 58.28 ELD_K525T 74.60 2.75 0.48 0.94 0.44 16.19 ELD_K525V 72.57 2.82 0.36 0.58 0.40 17.40 ELD_K525W 18.26 0.88 0.06 0.11 0.34 13.16 ELD_K525Y 37.75 2.21 0.14 0.21 0.82 11.17 ELD_S418D 57.94 0.33 0.11 0.21 0.09 77.35 ELD_N476D 70.96 0.10 0.06 0.04 0.03 304.50 ELD_I479T 60.88 0.10 0.11 0.04 0.07 196.27 ELD_Q481E 68.89 0.16 0.07 0.12 0.08 162.31 ELD_N527D 48.34 0.64 0.13 0.29 0.07 42.42 ELD_Q531R 54.06 0.97 0.12 0.13 0.09 41.65 GFP 0.00 0.01 0.02 0.03 0.03 N/A GFP 0.00 0.04 0.01 0.03 0.02 N/A Initial 1.01 0.89 0.80 0.93 0.85 1.11 Initial 0.97 0.83 0.78 0.77 0.70 1.17 Initial 1.06 1.08 1.11 1.04 0.99 0.96 Initial 0.97 1.20 1.32 1.26 1.46 0.76 Half dose 0.60 0.23 0.14 0.17 0.08 2.89 Half dose 0.64 0.24 0.12 0.23 0.16 2.81 Half dose 0.73 0.34 0.20 0.28 0.21 2.32 Half dose 0.70 0.29 0.29 0.32 0.23 2.37 ELD_R416A 1.03 0.12 0.49 0.39 0.15 5.21 ELD_R416C 1.07 0.23 0.80 0.60 0.19 3.19 ELD_R416D 0.91 0.01 0.37 0.09 0.02 15.43 ELD_R416E 1.33 0.05 1.12 0.13 0.04 7.77 ELD_R416F 1.31 0.35 1.28 0.69 0.17 2.71 ELD_R416G 0.84 0.06 0.55 0.39 0.11 5.35 ELD_R416H 1.44 0.55 2.74 1.01 0.22 1.75 ELD_R416I 0.50 0.35 0.42 0.40 0.36 1.32 ELD_R416K 0.32 0.08 0.20 0.21 0.11 2.71 ELD_R416L 0.91 0.42 0.89 0.68 0.36 1.78 ELD_R416M 1.05 0.27 0.67 0.55 0.20 3.06 ELD_R416N 1.30 0.08 0.69 0.33 0.08 7.54 ELD_R416P 0.04 0.01 0.02 0.01 0.04 2.86 ELD_R416Q 1.34 0.30 1.89 0.73 0.18 2.61 ELD_R416S 1.02 0.06 0.52 0.40 0.04 7.03 ELD_R416T 0.86 0.17 0.57 0.45 0.11 3.58 ELD_R416V 0.83 0.73 1.04 0.76 0.67 1.06 ELD_R416W 0.96 0.21 0.67 0.57 0.25 3.06 ELD_R416Y 1.42 1.85 4.46 1.96 1.02 0.67 ELD_K525A 1.10 0.03 0.08 0.08 0.08 25.30 ELD_K525C 1.30 0.26 0.29 0.45 0.36 4.26 ELD_K525D 0.73 0.01 0.02 0.02 0.05 43.83 ELD_K525E 1.12 0.01 0.05 0.05 0.07 50.48 ELD_K525F 0.40 0.10 0.03 0.10 0.25 3.66 ELD_K525G 0.95 0.03 0.15 0.12 0.05 16.55 ELD_K525H 0.72 0.07 0.11 0.13 0.20 7.40 ELD_K525I 1.19 0.21 0.17 0.26 0.43 5.01 ELD_K525L 0.47 0.04 0.06 0.12 0.14 7.46 ELD_K525M 0.57 0.02 0.05 0.07 0.07 15.66 ELD_K525N 0.73 0.05 0.09 0.17 0.15 9.26 ELD_K525P 0.15 0.00 0.02 0.01 0.02 24.73 ELD_K525Q 0.82 0.04 0.19 0.19 0.08 10.20 ELD_K525R 0.89 0.99 0.72 0.75 1.37 0.89 ELD_K525S 1.15 0.05 0.13 0.13 0.11 15.60 ELD_K525T 1.30 0.25 0.29 0.53 0.31 4.33 ELD_K525V 1.27 0.26 0.22 0.33 0.29 4.66 ELD_K525W 0.32 0.08 0.04 0.06 0.24 3.52 ELD_K525Y 0.66 0.20 0.08 0.11 0.59 2.99 ELD_S418D 1.01 0.03 0.07 0.12 0.06 20.70 ELD_N476D 1.24 0.01 0.04 0.02 0.02 81.48 ELD_I479T 1.06 0.01 0.07 0.02 0.05 52.52 ELD_Q481E 1.20 0.01 0.04 0.06 0.06 43.43 ELD_N527D 0.84 0.06 0.08 0.16 0.05 11.35 ELD_Q531R 0.94 0.09 0.07 0.07 0.06 11.15 GFP 0.00 0.00 0.01 0.02 0.02 N/A GFP 0.00 0.00 0.00 0.02 0.02 N/A

Table 10B

AAVS1 OT1 OT2 OT3 OT4 ratio Initial 57.77 9.73 1.31 1.67 1.20 4.15 Initial 55.40 9.06 1.27 1.38 0.98 4.37 Initial 60.63 11.83 1.81 1.87 1.39 3.59 Initial 55.39 13.05 2.15 2.26 2.05 2.84 Half dose 34.13 2.50 0.23 0.31 0.12 10.81 Half dose 36.73 2.67 0.19 0.41 0.23 10.49 Half dose 42.08 3.72 0.32 0.51 0.30 8.67 Half dose 40.22 3.17 0.47 0.57 0.32 8.87 KKR_R416A 54.27 3.73 0.13 0.25 0.57 11.60 KKR_R416C 54.00 5.09 0.35 0.41 0.94 7.95 KKR_R416D 44.62 0.27 0.05 0.04 0.12 90.35 KKR_R416E 68.27 1.58 0.03 0.16 0.62 28.47 KKR_R416F 63.29 3.94 0.33 0.38 1.09 11.04 KKR_R416G 48.98 2.04 0.12 0.24 0.55 16.60 KKR_R416H 73.76 9.86 0.52 0.91 2.96 5.18 KKR_R416I 23.42 2.91 0.38 0.61 0.44 5.39 KKR_R416K 25.56 1.96 0.31 0.64 0.30 7.98 KKR_R416L 46.72 6.32 0.47 0.78 0.87 5.53 KKR_R416M 53.48 5.09 0.45 0.64 0.93 7.52 KKR_R416N 69.54 2.49 0.07 0.24 0.75 19.57 KKR_R416P 1.75 0.05 0.02 0.02 0.06 11.88 KKR_R416Q 69.01 6.21 0.13 0.44 2.08 7.79 KKR_R416S 61.68 4.36 0.27 0.32 0.94 10.47 KKR_R416T 55.82 6.64 0.31 0.72 1.18 6.31 KKR_R416V 40.09 4.97 0.79 1.04 0.99 5.15 KKR_R416W 41.18 3.67 0.32 0.40 0.49 8.44 KKR_R416Y 72.44 20.12 1.70 2.41 3.83 2.58 KKR_K525A 61.16 0.92 0.09 0.13 0.21 45.07 KKR_K525C 62.86 3.74 0.13 0.31 0.37 13.80 KKR_K525D 36.17 0.14 0.00 0.03 0.04 168.94 KKR_K525E 52.52 0.24 0.02 0.06 0.07 133.17 KKR_K525F 25.30 0.74 0.58 0.34 0.16 13.86 KKR_K525G 52.93 1.09 0.04 0.13 0.34 33.16 KKR_K525H 41.28 2.61 0.29 0.34 0.24 11.88 KKR_K525I 57.29 2.40 0.16 0.28 0.24 18.57 KKR_K525L 30.26 0.79 0.06 0.15 0.07 28.15 KKR_K525M 46.57 1.29 0.15 0.17 0.19 26.01 KKR_K525N 44.68 1.61 0.15 0.25 0.27 19.51 KKR_K525P 2.44 0.04 0.02 0.01 0.03 25.01 KKR_K525Q 52.58 1.55 0.09 0.12 0.26 25.97 KKR_K525R 52.25 9.51 2.04 1.57 1.18 3.65 KKR_K525S 63.23 1.06 0.10 0.12 0.33 39.16 KKR_K525T 66.66 5.06 0.24 0.32 0.45 10.97 KKR_K525V 58.68 2.62 0.18 0.18 0.18 18.57 KKR_K525W 17.81 0.52 0.09 0.21 0.05 20.68 KKR_K525Y 35.10 1.42 0.70 0.41 0.15 13.09 KKR_S418D 73.70 1.47 0.19 0.23 0.34 32.91 KKR_N476D 36.23 0.05 0.03 0.06 0.02 230.91 KKR_I479T 67.02 0.76 0.14 0.64 0.12 40.47 KKR_Q481E 66.20 0.25 0.04 0.04 0.03 183.51 KKR_N527D 54.55 1.95 0.37 0.57 0.26 17.27 KKR_Q531R 54.73 2.17 0.78 0.80 0.24 13.73 GFP 0.00 0.01 0.02 0.03 0.03 N/A GFP 0.00 0.04 0.01 0.03 0.02 N/A Initial 1.01 0.89 0.80 0.93 0.85 1.11 Initial 0.97 0.83 0.78 0.77 0.70 1.17 Initial 1.06 1.08 1.11 1.04 0.99 0.96 Initial 0.97 1.20 1.32 1.26 1.46 0.76 Half dose 0.60 0.23 0.14 0.17 0.08 2.89 Half dose 0.64 0.24 0.12 0.23 0.16 2.81 Half dose 0.73 0.34 0.20 0.28 0.21 2.32 Half dose 0.70 0.29 0.29 0.32 0.23 2.37 KKR_R416A 0.95 0.34 0.08 0.14 0.40 3.10 KKR_R416C 0.94 0.47 0.21 0.23 0.67 2.13 KKR_R416D 0.78 0.03 0.03 0.02 0.09 24.18 KKR_R416E 1.19 0.14 0.02 0.09 0.44 7.62 KKR_R416F 1.10 0.36 0.20 0.21 0.78 2.95 KKR_R416G 0.85 0.19 0.07 0.14 0.39 4.44 KKR_R416H 1.29 0.90 0.32 0.51 2.11 1.38 KKR_R416I 0.41 0.27 0.23 0.34 0.31 1.44 KKR_R416K 0.45 0.18 0.19 0.36 0.21 2.13 KKR_R416L 0.82 0.58 0.29 0.44 0.62 1.48 KKR_R416M 0.93 0.47 0.28 0.36 0.66 2.01 KKR_R416N 1.21 0.23 0.04 0.14 0.54 5.24 KKR_R416P 0.03 0.00 0.01 0.01 0.04 3.18 KKR_R416Q 1.20 0.57 0.08 0.24 1.48 2.08 KKR_R416S 1.08 0.40 0.16 0.18 0.67 2.80 KKR_R416T 0.97 0.61 0.19 0.40 0.84 1.69 KKR_R416V 0.70 0.46 0.48 0.58 0.70 1.38 KKR_R416W 0.72 0.34 0.19 0.22 0.35 2.26 KKR_R416Y 1.26 1.84 1.04 1.34 2.73 0.69 KKR_K525A 1.07 0.08 0.06 0.07 0.15 12.06 KKR_K525C 1.10 0.34 0.08 0.17 0.26 3.69 KKR_K525D 0.63 0.01 0.00 0.02 0.03 45.21 KKR_K525E 0.92 0.02 0.01 0.03 0.05 35.63 KKR_K525F 0.44 0.07 0.36 0.19 0.11 3.71 KKR_K525G 0.92 0.10 0.03 0.07 0.24 8.87 KKR_K525H 0.72 0.24 0.18 0.19 0.17 3.18 KKR_K525I 1.00 0.22 0.10 0.16 0.17 4.97 KKR_K525L 0.53 0.07 0.04 0.08 0.05 7.53 KKR_K525M 0.81 0.12 0.09 0.10 0.13 6.96 KKR_K525N 0.78 0.15 0.09 0.14 0.20 5.22 KKR_K525P 0.04 0.00 0.01 0.01 0.02 6.69 KKR_K525Q 0.92 0.14 0.06 0.07 0.18 6.95 KKR_K525R 0.91 0.87 1.25 0.87 0.84 0.98 KKR_K525S 1.10 0.10 0.06 0.07 0.24 10.48 KKR_K525T 1.16 0.46 0.15 0.18 0.32 2.94 KKR_K525V 1.02 0.24 0.11 0.10 0.13 4.97 KKR_K525W 0.31 0.05 0.05 0.12 0.03 5.53 KKR_K525Y 0.61 0.13 0.43 0.23 0.11 3.50 KKR_S418D 1.29 0.13 0.12 0.13 0.25 8.81 KKR_N476D 0.63 0.00 0.02 0.03 0.01 61.79 KKR_I479T 1.17 0.07 0.09 0.36 0.08 10.83 KKR_Q481E 1.16 0.02 0.02 0.02 0.02 49.10 KKR_N527D 0.95 0.18 0.23 0.32 0.19 4.62 KKR_Q531R 0.96 0.20 0.48 0.44 0.17 3.68 GFP 0.00 0.00 0.01 0.02 0.02 N/A GFP 0.00 0.00 0.00 0.02 0.02 N/A

Table 10C

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 57.77 9.73 1.31 1.67 1.20 4.15 Initial 55.40 9.06 1.27 1.38 0.98 4.37 Initial 60.63 11.83 1.81 1.87 1.39 3.59 Initial 55.39 13.05 2.15 2.26 2.05 2.84 Half dose 34.13 2.50 0.23 0.31 0.12 10.81 Half dose 36.73 2.67 0.19 0.41 0.23 10.49 Half dose 42.08 3.72 0.32 0.51 0.30 8.67 Half dose 40.22 3.17 0.47 0.57 0.32 8.87 ELD_KKR_R416A 67.11 1.14 0.16 0.37 0.12 37.44 ELD_KKR_R416C 68.42 2.75 0.34 0.72 0.29 16.66 ELD_KKR_R416D 70.61 0.04 0.04 0.05 0.03 457.42 ELD_KKR_R416E 91.13 0.41 0.11 0.19 0.07 116.98 ELD_KKR_R416F 87.13 2.62 0.57 0.81 0.38 19.92 ELD_KKR_R416G 47.93 0.47 0.08 0.17 0.12 57.10 ELD_KKR_R416H 94.25 18.51 3.09 3.36 2.33 3.45 ELD_KKR_R416I 13.03 1.02 0.30 0.33 0.23 6.95 ELD_KKR_R416K 11.52 0.91 0.12 0.23 0.11 8.39 ELD_KKR_R416L 44.91 3.13 0.50 0.74 0.48 9.26 ELD_KKR_R416M 69.25 3.99 0.47 0.85 0.47 11.98 ELD_KKR_R416N 89.31 0.55 0.16 0.35 0.12 76.30 ELD_KKR_R416P 0.11 0.01 0.01 0.01 0.03 1.86 ELD_KKR_R416Q 88.16 7.54 1.10 1.66 0.73 7.99 ELD_KKR_R416S 71.87 0.61 0.22 0.28 0.13 58.20 ELD_KKR_R416T 62.11 3.51 0.50 0.99 0.43 11.45 ELD_KKR_R416V 34.90 4.49 1.08 1.14 0.86 4.62 ELD_KKR_R416W 39.15 1.20 0.21 0.36 0.16 20.34 ELD_KKR_R416Y 85.93 62.16 24.08 16.89 17.97 0.71 ELD_KKR_K525A 73.85 0.07 0.01 0.04 0.03 493.98 ELD_KKR_K525C 82.14 0.55 0.12 0.12 0.19 83.07 ELD_KKR_K525D 5.01 0.02 0.01 0.02 0.04 56.71 ELD_KKR_K525E 23.62 0.02 0.01 0.03 0.02 288.19 ELD_KKR_K525F 12.65 0.28 0.07 0.08 0.04 27.09 ELD_KKR_K525G 56.50 0.07 0.04 0.04 0.02 358.56 ELD_kkR_K525H 36.28 0.27 0.04 0.06 0.07 81.26 ELD_KKR_K525I 80.42 0.51 0.04 0.13 0.12 100.52 ELD_KKR_K525L 14.33 0.02 0.01 0.04 0.04 119.70 ELD_KKR_K525M 27.60 0.09 0.02 0.04 0.03 157.04 ELD_KKR_K525N 36.50 0.23 0.04 0.05 0.04 102.77 ELD_KKR_K525P 0.01 0.03 0.03 0.02 0.02 0.11 ELD_KKR_K525Q 41.98 0.10 0.03 0.07 0.04 169.31 ELD_KKR_K525R 53.62 14.69 2.18 1.97 2.52 2.51 ELD_KKR_K525S 77.66 0.11 0.02 0.06 0.05 316.50 ELD_KKR_K525T 84.74 0.68 0.07 0.21 0.21 72.27 ELD_KKR_K525V 83.33 0.48 0.04 0.06 0.09 122.99 ELD_KKR_K525W 8.64 0.10 0.02 0.03 0.05 44.17 ELD_KKR_K525Y 25.83 0.83 0.07 0.11 0.11 23.03 ELD_KKR_S418D 53.46 0.07 0.01 0.01 0.03 442.19 ELD_KKR_N476D 6.29 0.02 0.01 0.02 0.03 77.38 ELD_KKR_I479T 52.47 0.02 0.01 0.02 0.03 730.00 ELD_KKR_Q481E 69.91 0.03 0.02 0.02 0.02 784.97 ELD_KKR_N527D 36.03 0.07 0.02 0.04 0.02 235.36 ELD_KKR_Q531R 63.71 0.91 0.08 0.29 0.08 46.84 GFP 0.00 0.01 0.02 0.03 0.03 N/A GFP 0.00 0.04 0.01 0.03 0.02 N/A Initial 1.01 0.89 0.80 0.93 0.85 1.11 Initial 0.97 0.83 0.78 0.77 0.70 1.17 Initial 1.06 1.08 1.11 1.04 0.99 0.96 Initial 0.97 1.20 1.32 1.26 1.46 0.76 Half dose 0.60 0.23 0.14 0.17 0.08 2.89 Half dose 0.64 0.24 0.12 0.23 0.16 2.81 Half dose 0.73 0.34 0.20 0.28 0.21 2.32 Half dose 0.70 0.29 0.29 0.32 0.23 2.37 ELD_KKR_R416A 1.17 0.10 0.10 0.21 0.08 10.02 ELD_KKR_R416C 1.19 0.25 0.21 0.40 0.21 4.46 ELD_KKR_R416D 1.23 0.00 0.02 0.03 0.02 122.40 ELD_KKR_R416E 1.59 0.04 0.07 0.10 0.05 31.30 ELD_KKR_R416F 1.52 0.24 0.35 0.45 0.27 5.33 ELD_KKR_R416G 0.84 0.04 0.05 0.10 0.08 15.28 ELD_KKR_R416H 1.64 1.70 1.89 1.87 1.66 0.92 ELD_KKR_R416I 0.23 0.09 0.18 0.18 0.16 1.86 ELD_KKR_R416K 0.20 0.08 0.08 0.13 0.08 2.25 ELD_KKR_R416L 0.78 0.29 0.31 0.41 0.34 2.48 ELD_KKR_R416M 1.21 0.37 0.29 0.48 0.34 3.20 ELD_KKR_R416N 1.56 0.05 0.10 0.19 0.08 20.42 ELD_KKR_R416P 0.00 0.00 0.01 0.00 0.02 0.50 ELD_KKR_R416Q 1.54 0.69 0.67 0.93 0.52 2.14 ELD_KKR_R416S 1.25 0.06 0.14 0.16 0.09 15.57 ELD_KKR_R416T 1.08 0.32 0.31 0.55 0.30 3.06 ELD_KKR_R416V 0.61 0.41 0.66 0.63 0.61 1.24 ELD_KKR_R416W 0.68 0.11 0.13 0.20 0.11 5.44 ELD_KKR_R416Y 1.50 5.69 14.74 9.41 12.79 0.19 ELD_KKR_K525A 1.29 0.01 0.00 0.02 0.02 132.18 ELD_KKR_K525C 1.43 0.05 0.08 0.07 0.14 22.23 ELD_KKR_K525D 0.09 0.00 0.01 0.01 0.03 15.18 ELD_KKR_K525E 0.41 0.00 0.00 0.02 0.02 77.12 ELD_KKR_K525F 0.22 0.03 0.04 0.05 0.03 7.25 ELD_KKR_K525G 0.99 0.01 0.02 0.02 0.01 95.95 ELD_kkR_K525H 0.63 0.03 0.03 0.03 0.05 21.74 ELD_KKR_K525I 1.40 0.05 0.03 0.07 0.08 26.90 ELD_KKR_K525L 0.25 0.00 0.01 0.02 0.03 32.03 ELD_KKR_K525M 0.48 0.01 0.01 0.02 0.02 42.02 ELD_KKR_K525N 0.64 0.02 0.02 0.03 0.03 27.50 ELD_KKR_K525P 0.00 0.00 0.02 0.01 0.02 0.03 ELD_KKR_K525Q 0.73 0.01 0.02 0.04 0.03 45.31 ELD_KKR_K525R 0.94 1.35 1.33 1.10 1.80 0.67 ELD_KKR_K525S 1.36 0.01 0.01 0.03 0.04 84.69 ELD_KKR_K525T 1.48 0.06 0.04 0.12 0.15 19.34 ELD_KKR_K525V 1.45 0.04 0.03 0.04 0.07 32.91 ELD_KKR_K525W 0.15 0.01 0.01 0.02 0.03 11.82 ELD_KKR_K525Y 0.45 0.08 0.04 0.06 0.08 6.16 ELD_KKR_S418D 0.93 0.01 0.01 0.01 0.02 118.32 ELD_KKR_N476D 0.11 0.00 0.01 0.01 0.02 20.71 ELD_KKR_I479T 0.92 0.00 0.01 0.01 0.02 195.34 ELD_KKR_Q481E 1.22 0.00 0.01 0.01 0.02 210.05 ELD_KKR_N527D 0.63 0.01 0.01 0.02 0.01 62.98 ELD_KKR_Q531R 1.11 0.08 0.05 0.16 0.06 12.53 GFP 0.00 0.00 0.01 0.02 0.02 N/A GFP 0.00 0.00 0.00 0.02 0.02 N/A

[0238] Tables 11A-11C present the results of cleavage in 2 different experiments of the target target (AAVS1) and three non-target targets (OT1, OT2, OT3), as well as the ratio of target and non-target cleavage for these mutants, including mutants with substitutions in positions 418, 422 and 525 in combination with ELD and/or KKR dimerization domain mutants.

Table 11A

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 63.63 15.40 3.50 3.29 3.11 2.52 Initial 63.10 18.77 3.50 3.63 3.01 2.18 Initial 60.00 11.17 2.50 2.92 2.79 3.10 Initial 56.67 13.75 2.05 2.62 2.28 2.74 Half dose 43.53 6.13 0.58 0.98 0.58 5.26 Half dose 45.07 6.62 0.81 0.77 0.67 5.08 Half dose 36.53 3.47 0.31 0.48 0.30 8.01 Half dose 34.68 3.72 0.28 0.68 0.43 6.80 ELD_S418A 64.85 6.75 4.13 3.56 1.13 4.16 ELD_S418C 48.50 6.15 2.17 3.19 1.45 3.74 ELD_S418D 63.65 1.90 1.07 0.93 0.51 14.47 ELD_S418E 80.24 0.52 0.20 0.31 0.16 67.61 ELD_S418F 18.36 6.25 0.52 0.77 0.25 2.35 ELD_S418G 41.80 17.17 1.22 1.15 4.53 1.74 ELD_S418H 52.02 6.20 3.38 3.05 0.97 3.82 ELD_S418I 18.67 0.11 0.18 0.14 0.03 40.45 ELD_S418K 47.05 2.90 4.22 3.37 1.19 4.03 ELD_S418L 11.43 0.23 0.16 0.42 0.05 13.30 ELD_S418M 24.02 0.64 0.50 0.67 0.13 12.39 ELD_S418N 51.08 6.04 3.49 2.41 0.62 4.07 ELD_S418P 85.88 25.87 11.22 5.63 3.90 1.84 ELD_S418Q 49.25 1.33 1.93 1.38 0.21 10.18 ELD_S418R 43.75 3.65 6.46 3.11 0.68 3.15 ELD_S418T 41.87 0.63 0.58 0.56 0.08 22.56 ELD_S418V 32.95 0.21 0.34 0.27 0.04 38.58 ELD_S418W 19.69 1.28 1.00 0.88 0.15 5.95 ELD_S418Y 21.45 1.68 0.57 0.72 0.34 6.47 ELD_R422A 4.83 0.09 0.07 0.09 0.04 16.58 ELD_R422C 29.95 1.75 0.47 0.84 0.31 8.89 ELD_R422D 40.01 0.84 0.50 0.40 0.07 22.15 ELD_R422E 45.73 0.85 0.32 0.38 0.07 28.14 ELD_R422F 39.31 3.33 1.22 2.37 0.62 5.22 ELD_R422G 41.33 1.35 0.43 0.92 0.18 14.32 ELD_R422H 62.83 5.04 1.65 2.12 0.40 6.83 ELD_R422I 35.81 3.13 0.31 1.37 0.43 6.83 ELD_R422K 72.35 21.16 8.36 4.17 2.86 1.98 ELD_R422L 53.80 10.24 0.63 2.14 1.15 3.80 ELD_R422M 40.21 5.14 0.67 1.71 1.00 4.71 ELD_R422N 39.71 2.03 0.80 1.24 0.20 9.30 ELD_R422P 15.62 0.13 0.12 0.11 0.05 37.48 ELD_R422Q 56.20 8.88 0.84 2.12 0.73 4.47 ELD_R422S 43.70 2.29 0.87 1.55 0.30 8.72 ELD_R422T 50.86 4.10 0.76 1.53 0.31 7.59 ELD_R422V 44.61 4.71 0.43 1.50 0.55 6.21 ELD_R422W 31.71 1.43 1.03 1.61 0.25 7.34 ELD_R422Y 53.71 4.91 2.23 3.44 0.74 4.75 ELD_S418D 63.93 0.40 0.26 0.47 0.15 49.93 ELD_N476D 77.78 0.09 0.06 0.05 0.13 234.74 ELD_I479T 60.14 0.13 0.08 0.02 0.05 219.23 ELD_Q481E 74.06 0.15 0.08 0.22 0.05 149.62 ELD_N527D 50.14 1.36 0.12 0.65 0.11 22.37 ELD_Q531R 64.05 2.11 0.31 0.40 0.18 21.40 ELD_R416D 49.54 0.16 0.42 0.20 0.14 54.16 ELD_R416E 72.49 0.55 1.40 0.29 0.11 30.73 ELD_R416N 72.79 1.18 1.65 1.20 0.16 17.38 ELD_R416S 52.96 1.24 0.96 0.90 0.27 15.67 ELD_K525A 67.68 0.40 0.37 0.32 0.12 55.85 ELD_K525E 55.90 0.08 0.07 0.04 0.14 170.65 ELD_K525G 54.53 0.44 0.21 0.20 0.18 52.62 ELD_K525S 69.63 0.68 0.42 0.44 0.22 39.85 ELD_Q481A 71.88 0.19 0.09 0.13 0.13 132.21 ELD_Q481C 72.36 0.19 0.19 0.43 0.36 61.88 ELD_Q481D 89.61 2.43 0.21 3.17 0.76 13.64 ELD_Q481S 59.95 0.08 0.05 0.16 0.07 168.18 GFP 0.01 0.02 0.00 0.02 0.09 N/A GFP 0.01 0.01 0.01 0.00 0.12 N/A Initial 1.05 1.04 1.21 1.06 1.11 0.96 Initial 1.04 1.27 1.21 1.17 1.08 0.83 Initial 0.99 0.76 0.87 0.94 1.00 1.18 Initial 0.93 0.93 0.71 0.84 0.81 1.04 Half dose 0.72 0.42 0.20 0.32 0.21 2.00 Half dose 0.74 0.45 0.28 0.25 0.24 1.93 Half dose 0.60 0.23 0.11 0.16 0.11 3.04 Half dose 0.57 0.25 0.10 0.22 0.15 2.58 ELD_S418A 1.07 0.46 1.43 1.14 0.41 1.58 ELD_S418C 0.80 0.42 0.75 1.02 0.52 1.42 ELD_S418D 1.05 0.13 0.37 0.30 0.18 5.49 ELD_S418E 1.32 0.03 0.07 0.10 0.06 25.68 ELD_S418F 0.30 0.42 0.18 0.25 0.09 0.89 ELD_S418G 0.69 1.16 0.42 0.37 1.62 0.66 ELD_S418H 0.85 0.42 1.17 0.98 0.35 1.45 ELD_S418I 0.31 0.01 0.06 0.04 0.01 15.36 ELD_S418K 0.77 0.20 1.46 1.08 0.43 1.53 ELD_S418L 0.19 0.02 0.06 0.14 0.02 5.05 ELD_S418M 0.39 0.04 0.17 0.22 0.05 4.71 ELD_S418N 0.84 0.41 1.21 0.77 0.22 1.54 ELD_S418P 1.41 1.75 3.88 1.81 1.39 0.70 ELD_S418Q 0.81 0.09 0.67 0.44 0.07 3.87 ELD_S418R 0.72 0.25 2.24 1.00 0.24 1.19 ELD_S418T 0.69 0.04 0.20 0.18 0.03 8.57 ELD_S418V 0.54 0.01 0.12 0.09 0.01 14.65 ELD_S418W 0.32 0.09 0.35 0.28 0.05 2.26 ELD_S418Y 0.35 0.11 0.20 0.23 0.12 2.46 ELD_R422A 0.08 0.01 0.02 0.03 0.01 6.30 ELD_R422C 0.49 0.12 0.16 0.27 0.11 3.37 ELD_R422D 0.66 0.06 0.17 0.13 0.02 8.41 ELD_R422E 0.75 0.06 0.11 0.12 0.03 10.69 ELD_R422F 0.65 0.23 0.42 0.76 0.22 1.98 ELD_R422G 0.68 0.09 0.15 0.30 0.07 5.44 ELD_R422H 1.03 0.34 0.57 0.68 0.14 2.59 ELD_R422I 0.59 0.21 0.11 0.44 0.15 2.59 ELD_R422K 1.19 1.43 2.89 1.34 1.02 0.75 ELD_R422L 0.88 0.69 0.22 0.69 0.41 1.44 ELD_R422M 0.66 0.35 0.23 0.55 0.36 1.79 ELD_R422N 0.65 0.14 0.28 0.40 0.07 3.53 ELD_R422P 0.26 0.01 0.04 0.04 0.02 14.24 ELD_R422Q 0.92 0.60 0.29 0.68 0.26 1.70 ELD_R422S 0.72 0.16 0.30 0.50 0.11 3.31 ELD_R422T 0.84 0.28 0.26 0.49 0.11 2.88 ELD_R422V 0.73 0.32 0.15 0.48 0.20 2.36 ELD_R422W 0.52 0.10 0.36 0.52 0.09 2.79 ELD_R422Y 0.88 0.33 0.77 1.10 0.26 1.80 ELD_S418D 1.05 0.03 0.09 0.15 0.06 18.96 ELD_N476D 1.28 0.01 0.02 0.02 0.05 89.15 ELD_I479T 0.99 0.01 0.03 0.01 0.02 83.26 ELD_Q481E 1.22 0.01 0.03 0.07 0.02 56.82 ELD_N527D 0.82 0.09 0.04 0.21 0.04 8.50 ELD_Q531R 1.05 0.14 0.11 0.13 0.07 8.13 ELD_R416D 0.81 0.01 0.15 0.06 0.05 20.57 ELD_R416E 1.19 0.04 0.48 0.09 0.04 11.67 ELD_R416N 1.20 0.08 0.57 0.38 0.06 6.60 ELD_R416S 0.87 0.08 0.33 0.29 0.10 5.95 ELD_K525A 1.11 0.03 0.13 0.10 0.04 21.21 ELD_K525E 0.92 0.01 0.02 0.01 0.05 64.81 ELD_K525G 0.90 0.03 0.07 0.06 0.07 19.99 ELD_K525S 1.14 0.05 0.14 0.14 0.08 15.13 ELD_Q481A 1.18 0.01 0.03 0.04 0.05 50.21 ELD_Q481C 1.19 0.01 0.07 0.14 0.13 23.50 ELD_Q481D 1.47 0.16 0.07 1.02 0.27 5.18 ELD_Q481S 0.99 0.01 0.02 0.05 0.02 63.87 GFP 0.00 0.00 0.00 0.01 0.03 N/A GFP 0.00 0.00 0.00 0.00 0.04 N/A

Table 11B

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 63.63 15.40 3.50 3.29 3.11 2.52 Initial 63.10 18.77 3.50 3.63 3.01 2.18 Initial 60.00 11.17 2.50 2.92 2.79 3.10 Initial 56.67 13.75 2.05 2.62 2.28 2.74 Half dose 43.53 6.13 0.58 0.98 0.58 5.26 Half dose 45.07 6.62 0.81 0.77 0.67 5.08 Half dose 36.53 3.47 0.31 0.48 0.30 8.01 Half dose 34.68 3.72 0.28 0.68 0.43 6.80 KKR_S418A 56.84 12.20 0.70 0.94 1.88 3.61 KKR_S418C 42.72 6.97 1.14 1.05 1.26 4.10 KKR_S418D 65.24 1.15 0.16 0.14 0.27 37.87 KKR_S418E 72.45 0.34 0.09 0.16 0.11 104.85 KKR_S418F 14.90 1.08 0.23 0.34 0.42 7.19 KKR_S418G 44.80 8.06 7.82 3.45 1.44 2.16 KKR_S418H 55.57 12.98 1.14 1.42 3.65 2.89 KKR_S418I 29.78 0.28 0.06 0.03 0.13 61.12 KKR_S418K 52.01 17.69 1.58 1.17 3.44 2.18 KKR_S418L 10.83 0.41 0.03 0.05 0.14 16.89 KKR_S418M 30.93 1.55 0.19 0.17 0.46 13.08 KKR_S418N 59.19 19.05 1.08 1.70 4.67 2.23 KKR_S418P 79.20 28.77 3.90 3.93 9.53 1.72 KKR_S418Q 55.07 7.54 0.22 0.56 1.94 5.37 KKR_S418R 45.79 19.09 2.31 1.60 5.19 1.62 KKR_S418T 57.23 6.33 0.27 0.54 1.25 6.83 KKR_S418V 44.43 0.40 0.04 0.10 0.23 57.69 KKR_S418W 18.26 1.60 0.15 0.37 0.61 6.71 KKR_S418Y 21.75 2.48 0.48 0.51 0.82 5.07 KKR_R422A 57.41 2.68 0.24 0.61 1.20 12.14 KKR_R422C 31.57 1.22 0.13 0.31 0.34 15.77 KKR_R422D 49.90 0.25 0.03 0.05 0.28 82.29 KKR_R422E 49.76 0.17 0.02 0.08 0.14 120.70 KKR_R422F 39.99 2.29 0.20 0.32 0.48 12.16 KKR_R422G 40.90 1.64 0.20 0.36 0.57 14.79 KKR_R422H 63.43 4.33 0.37 0.72 1.45 9.23 KKR_R422I 35.55 1.97 0.31 0.41 0.33 11.76 KKR_R422K 71.20 22.19 0.97 3.16 6.13 2.19 KKR_R422L 52.15 3.79 0.94 1.36 0.91 7.44 KKR_R422M 39.52 2.40 0.56 0.65 0.46 9.75 KKR_R422N 46.21 1.84 0.15 0.44 0.81 14.23 KKR_R422P 28.51 0.20 0.02 0.03 0.06 92.81 KKR_R422Q 60.24 4.53 0.56 1.63 1.31 7.50 KKR_R422S 52.09 1.55 0.17 0.63 0.90 16.02 KKR_R422T 54.57 2.99 0.24 0.81 0.91 11.03 KKR_R422V 41.66 1.61 0.24 0.52 0.30 15.58 KKR_R422W 33.25 3.71 0.14 0.47 0.67 6.66 KKR_R422Y 52.98 4.80 0.37 0.74 1.26 7.39 KKR_S418D 70.21 1.58 0.12 0.12 0.20 34.83 KKR_N476D 36.24 0.11 0.04 0.12 0.02 119.52 KKR_I479T 70.33 1.26 0.21 1.21 0.09 25.35 KKR_Q481E 70.45 0.55 0.13 0.05 0.17 78.95 KKR_N527D 57.06 3.84 0.54 1.35 0.71 8.86 KKR_Q531R 58.48 3.90 1.42 1.97 0.82 7.22 KKR_R416D 57.64 0.37 0.04 0.01 0.28 82.22 KKR_R416E 69.37 1.55 0.04 0.14 1.50 21.50 KKR_R416N 70.11 7.61 0.17 0.53 3.59 5.89 KKR_R416S 58.34 5.62 0.24 0.49 1.25 7.68 KKR_K525A 55.21 0.79 0.03 0.14 0.20 47.44 KKR_K525E 49.76 0.25 0.01 0.02 0.11 126.10 KKR_K525G 54.38 1.97 0.08 0.17 0.45 20.33 KKR_K525S 64.03 2.23 0.11 0.12 0.54 21.34 KKR_Q481A 61.41 0.31 0.02 0.03 0.13 123.36 KKR_Q481C 64.51 4.04 0.09 0.02 0.42 14.11 KKR_Q481D 75.10 6.05 0.09 0.13 0.80 10.64 KKR_Q481S 56.88 0.45 0.00 0.00 0.12 98.94 GFP 0.01 0.02 0.00 0.02 0.09 N/A GFP 0.01 0.01 0.01 0.00 0.12 N/A Initial 1.05 1.04 1.21 1.06 1.11 0.96 Initial 1.04 1.27 1.21 1.17 1.08 0.83 Initial 0.99 0.76 0.87 0.94 1.00 1.18 Initial 0.93 0.93 0.71 0.84 0.81 1.04 Half dose 0.72 0.42 0.20 0.32 0.21 2.00 Half dose 0.74 0.45 0.28 0.25 0.24 1.93 Half dose 0.60 0.23 0.11 0.16 0.11 3.04 Half dose 0.57 0.25 0.10 0.22 0.15 2.58 KKR_S418A 0.93 0.83 0.24 0.30 0.67 1.37 KKR_S418C 0.70 0.47 0.39 0.34 0.45 1.56 KKR_S418D 1.07 0.08 0.06 0.05 0.10 14.38 KKR_S418E 1.19 0.02 0.03 0.05 0.04 39.82 KKR_S418F 0.24 0.07 0.08 0.11 0.15 2.73 KKR_S418G 0.74 0.55 2.71 1.11 0.52 0.82 KKR_S418H 0.91 0.88 0.40 0.46 1.31 1.10 KKR_S418I 0.49 0.02 0.02 0.01 0.04 23.21 KKR_S418K 0.85 1.20 0.55 0.38 1.23 0.83 KKR_S418L 0.18 0.03 0.01 0.02 0.05 6.41 KKR_S418M 0.51 0.10 0.07 0.05 0.16 4.97 KKR_S418N 0.97 1.29 0.37 0.55 1.67 0.85 KKR_S418P 1.30 1.95 1.35 1.26 3.41 0.65 KKR_S418Q 0.90 0.51 0.08 0.18 0.69 2.04 KKR_S418R 0.75 1.29 0.80 0.51 1.86 0.62 KKR_S418T 0.94 0.43 0.09 0.17 0.45 2.60 KKR_S418V 0.73 0.03 0.01 0.03 0.08 21.91 KKR_S418W 0.30 0.11 0.05 0.12 0.22 2.55 KKR_S418Y 0.36 0.17 0.17 0.16 0.29 1.92 KKR_R422A 0.94 0.18 0.08 0.20 0.43 4.61 KKR_R422C 0.52 0.08 0.05 0.10 0.12 5.99 KKR_R422D 0.82 0.02 0.01 0.02 0.10 31.25 KKR_R422E 0.82 0.01 0.01 0.02 0.05 45.84 KKR_R422F 0.66 0.15 0.07 0.10 0.17 4.62 KKR_R422G 0.67 0.11 0.07 0.12 0.20 5.62 KKR_R422H 1.04 0.29 0.13 0.23 0.52 3.51 KKR_R422I 0.58 0.13 0.11 0.13 0.12 4.47 KKR_R422K 1.17 1.50 0.33 1.01 2.19 0.83 KKR_R422L 0.86 0.26 0.33 0.44 0.33 2.83 KKR_R422M 0.65 0.16 0.19 0.21 0.16 3.70 KKR_R422N 0.76 0.12 0.05 0.14 0.29 5.40 KKR_R422P 0.47 0.01 0.01 0.01 0.02 35.25 KKR_R422Q 0.99 0.31 0.19 0.52 0.47 2.85 KKR_R422S 0.86 0.10 0.06 0.20 0.32 6.09 KKR_R422T 0.90 0.20 0.08 0.26 0.32 4.19 KKR_R422V 0.68 0.11 0.08 0.17 0.11 5.92 KKR_R422W 0.55 0.25 0.05 0.15 0.24 2.53 KKR_R422Y 0.87 0.33 0.13 0.24 0.45 2.81 KKR_S418D 1.15 0.11 0.04 0.04 0.07 13.23 KKR_N476D 0.60 0.01 0.01 0.04 0.01 45.39 KKR_I479T 1.16 0.09 0.07 0.39 0.03 9.63 KKR_Q481E 1.16 0.04 0.04 0.02 0.06 29.98 KKR_N527D 0.94 0.26 0.19 0.43 0.26 3.36 KKR_Q531R 0.96 0.26 0.49 0.63 0.29 2.74 KKR_R416D 0.95 0.03 0.01 0.00 0.10 31.22 KKR_R416E 1.14 0.11 0.01 0.04 0.54 8.17 KKR_R416N 1.15 0.52 0.06 0.17 1.28 2.24 KKR_R416S 0.96 0.38 0.08 0.16 0.45 2.92 KKR_K525A 0.91 0.05 0.01 0.04 0.07 18.02 KKR_K525E 0.82 0.02 0.00 0.01 0.04 47.89 KKR_K525G 0.89 0.13 0.03 0.06 0.16 7.72 KKR_K525S 1.05 0.15 0.04 0.04 0.19 8.11 KKR_Q481A 1.01 0.02 0.01 0.01 0.05 46.85 KKR_Q481C 1.06 0.27 0.03 0.01 0.15 5.36 KKR_Q481D 1.23 0.41 0.03 0.04 0.28 4.04 KKR_Q481S 0.93 0.03 0.00 0.00 0.04 37.57 GFP 0.00 0.00 0.00 0.01 0.03 N/A GFP 0.00 0.00 0.00 0.00 0.04 N/A

Table 11C

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 63.63 15.40 3.50 3.29 3.11 2.52 Initial 63.10 18.77 3.50 3.63 3.01 2.18 Initial 60.00 11.17 2.50 2.92 2.79 3.10 Initial 56.67 13.75 2.05 2.62 2.28 2.74 Half dose 43.53 6.13 0.58 0.98 0.58 5.26 Half dose 45.07 6.62 0.81 0.77 0.67 5.08 Half dose 36.53 3.47 0.31 0.48 0.30 8.01 Half dose 34.68 3.72 0.28 0.68 0.43 6.80 ELD_KKR_S418A 56.41 3.20 0.67 0.90 0.57 10.56 ELD_KKR_S418C 24.43 0.74 0.21 0.35 0.28 15.51 ELD_KKR_S418D 48.70 0.08 0.03 0.05 0.12 172.57 ELD_KKR_S418E 58.18 0.04 0.01 0.02 0.05 470.11 ELD_KKR_S418F 5.17 0.18 0.06 0.09 0.13 11.37 ELD_KKR_S418G 26.97 9.45 2.45 1.64 1.93 1.74 ELD_KKR_S418H 40.18 3.40 0.55 0.75 0.76 7.36 ELD_KKR_S418I 5.96 0.02 0.02 0.02 0.10 37.01 ELD_KKR_S418K 34.84 3.16 1.33 1.29 0.92 5.20 ELD_KKR_S418L 1.79 0.03 0.01 0.01 0.14 9.91 ELD_KKR_S418M 8.86 0.09 0.05 0.06 0.10 30.11 ELD_KKR_S418N 44.66 8.43 0.90 1.34 0.95 3.84 ELD_KKR_S418P 91.49 35.58 9.89 6.21 8.86 1.51 ELD_KKR_S418Q 39.47 0.85 0.10 0.31 0.25 26.33 ELD_KKR_S418R 29.54 5.45 3.22 1.52 2.05 2.41 ELD_KKR_S418T 34.61 0.24 0.07 0.14 0.09 64.01 ELD_KKR_S418V 18.75 0.03 0.03 0.05 0.11 87.69 ELD_KKR_S418W 6.01 0.12 0.10 0.08 0.13 13.82 ELD_KKR_S418Y 7.26 0.33 0.09 0.06 0.12 12.07 ELD_KKR_R422A 3.43 0.03 0.01 0.05 0.15 14.34 ELD_KKR_R422C 13.04 0.09 0.07 0.03 0.11 43.05 ELD_KKR_R422D 21.98 0.02 0.02 0.01 0.08 166.09 ELD_KKR_R422E 25.12 0.03 0.00 0.03 0.14 124.28 ELD_KKR_R422F 20.43 0.69 0.03 0.30 0.16 17.27 ELD_KKR_R422G 19.82 0.05 0.02 0.03 0.12 89.17 ELD_KKR_R422H 58.50 0.65 0.08 0.39 0.23 43.21 ELD_KKR_R422I 16.94 0.22 0.05 0.14 0.15 30.61 ELD_KKR_R422K 78.47 23.05 2.17 3.34 3.98 2.41 ELD_KKR_R422L 37.68 0.71 0.17 0.28 0.23 27.22 ELD_KKR_R422M 20.81 0.21 0.05 0.20 0.18 32.91 ELD_KKR_R422N 23.17 0.04 0.03 0.06 0.11 93.57 ELD_KKR_R422P 3.63 0.03 0.02 0.01 0.09 23.37 ELD_KKR_R422Q 43.13 0.53 0.10 0.23 0.14 43.28 ELD_KKR_R422S 32.72 0.15 0.04 0.20 0.20 54.65 ELD_KKR_R422T 37.16 0.29 0.04 0.09 0.17 62.72 ELD_KKR_R422V 23.36 0.18 0.02 0.06 0.14 57.49 ELD_KKR_R422W 13.07 0.32 0.03 0.10 0.10 23.58 ELD_KKR_R422Y 37.28 1.31 0.08 0.46 0.31 17.28 ELD_KKR_S418D 44.22 0.09 0.03 0.01 0.07 219.68 ELD_KKR_N476D 4.51 0.03 0.01 0.02 0.07 36.91 ELD_KKR_I479T 41.46 0.03 0.02 0.01 0.09 278.85 ELD_KKR_Q481E 66.40 0.03 0.04 0.01 0.08 413.30 ELD_KKR_N527D 32.76 0.15 0.05 0.11 0.11 78.86 ELD_KKR_Q531R 56.63 0.60 0.02 0.24 0.12 57.58 ELD_KKR_R416D 67.30 0.06 0.03 0.06 0.09 283.84 ELD_KKR_R416E 90.77 0.45 0.06 0.13 0.14 116.83 ELD_KKR_R416N 88.98 0.73 0.14 0.31 0.19 65.36 ELD_KKR_R416S 62.78 1.06 0.11 0.45 0.17 35.01 ELD_KKR_K525A 66.59 0.04 0.02 0.03 0.13 296.70 ELD_KKR_K525E 18.58 0.04 0.02 0.01 0.09 122.53 ELD_KKR_K525G 49.73 0.12 0.01 0.07 0.08 178.32 ELD_KKR_K525S 74.03 0.09 0.03 0.05 0.11 258.39 ELD_KKR_Q481A 92.58 0.03 0.04 0.00 0.09 570.76 ELD_KKR_Q481C 72.98 0.06 0.04 0.03 0.12 298.84 ELD_KKR_Q481D 91.87 0.43 0.02 0.04 0.22 128.41 ELD_KKR_Q481S 58.67 0.03 0.02 0.01 0.08 422.94 GFP 0.01 0.02 0.00 0.02 0.09 N/A GFP 0.01 0.01 0.01 0.00 0.12 N/A Initial 1.05 1.04 1.21 1.06 1.11 0.96 Initial 1.04 1.27 1.21 1.17 1.08 0.83 Initial 0.99 0.76 0.87 0.94 1.00 1.18 Initial 0.93 0.93 0.71 0.84 0.81 1.04 Half dose 0.72 0.42 0.20 0.32 0.21 2.00 Half dose 0.74 0.45 0.28 0.25 0.24 1.93 Half dose 0.60 0.23 0.11 0.16 0.11 3.04 Half dose 0.57 0.25 0.10 0.22 0.15 2.58 ELD_KKR_S418A 0.93 0.22 0.23 0.29 0.21 4.01 ELD_KKR_S418C 0.40 0.05 0.07 0.11 0.10 5.89 ELD_KKR_S418D 0.80 0.01 0.01 0.01 0.04 65.54 ELD_KKR_S418E 0.96 0.00 0.00 0.01 0.02 178.54 ELD_KKR_S418F 0.08 0.01 0.02 0.03 0.05 4.32 ELD_KKR_S418G 0.44 0.64 0.85 0.53 0.69 0.66 ELD_KKR_S418H 0.66 0.23 0.19 0.24 0.27 2.79 ELD_KKR_S418I 0.10 0.00 0.01 0.01 0.04 14.06 ELD_KKR_S418K 0.57 0.21 0.46 0.41 0.33 1.97 ELD_KKR_S418L 0.03 0.00 0.00 0.00 0.05 3.77 ELD_KKR_S418M 0.15 0.01 0.02 0.02 0.03 11.43 ELD_KKR_S418N 0.73 0.57 0.31 0.43 0.34 1.46 ELD_KKR_S418P 1.50 2.41 3.42 1.99 3.17 0.57 ELD_KKR_S418Q 0.65 0.06 0.03 0.10 0.09 10.00 ELD_KKR_S418R 0.49 0.37 1.12 0.49 0.73 0.92 ELD_KKR_S418T 0.57 0.02 0.02 0.05 0.03 24.31 ELD_KKR_S418V 0.31 0.00 0.01 0.02 0.04 33.30 ELD_KKR_S418W 0.10 0.01 0.03 0.03 0.05 5.25 ELD_KKR_S418Y 0.12 0.02 0.03 0.02 0.04 4.58 ELD_KKR_R422A 0.06 0.00 0.00 0.02 0.05 5.45 ELD_KKR_R422C 0.21 0.01 0.03 0.01 0.04 16.35 ELD_KKR_R422D 0.36 0.00 0.01 0.00 0.03 63.08 ELD_KKR_R422E 0.41 0.00 0.00 0.01 0.05 47.20 ELD_KKR_R422F 0.34 0.05 0.01 0.10 0.06 6.56 ELD_KKR_R422G 0.33 0.00 0.01 0.01 0.04 33.87 ELD_KKR_R422H 0.96 0.04 0.03 0.13 0.08 16.41 ELD_KKR_R422I 0.28 0.02 0.02 0.04 0.05 11.63 ELD_KKR_R422K 1.29 1.56 0.75 1.07 1.42 0.92 ELD_KKR_R422L 0.62 0.05 0.06 0.09 0.08 10.34 ELD_KKR_R422M 0.34 0.01 0.02 0.06 0.06 12.50 ELD_KKR_R422N 0.38 0.00 0.01 0.02 0.04 35.54 ELD_KKR_R422P 0.06 0.00 0.01 0.00 0.03 8.87 ELD_KKR_R422Q 0.71 0.04 0.03 0.07 0.05 16.44 ELD_KKR_R422S 0.54 0.01 0.02 0.06 0.07 20.75 ELD_KKR_R422T 0.61 0.02 0.01 0.03 0.06 23.82 ELD_KKR_R422V 0.38 0.01 0.01 0.02 0.05 21.83 ELD_KKR_R422W 0.21 0.02 0.01 0.03 0.04 8.95 ELD_KKR_R422Y 0.61 0.09 0.03 0.15 0.11 6.56 ELD_KKR_S418D 0.73 0.01 0.01 0.00 0.03 83.43 ELD_KKR_N476D 0.07 0.00 0.00 0.01 0.02 14.02 ELD_KKR_I479T 0.68 0.00 0.01 0.00 0.03 105.90 ELD_KKR_Q481E 1.09 0.00 0.01 0.00 0.03 156.97 ELD_KKR_N527D 0.54 0.01 0.02 0.04 0.04 29.95 ELD_KKR_Q531R 0.93 0.04 0.01 0.08 0.04 21.87 ELD_KKR_R416D 1.11 0.00 0.01 0.02 0.03 107.80 ELD_KKR_R416E 1.49 0.03 0.02 0.04 0.05 44.37 ELD_KKR_R416N 1.46 0.05 0.05 0.10 0.07 24.82 ELD_KKR_R416S 1.03 0.07 0.04 0.14 0.06 13.30 ELD_KKR_K525A 1.09 0.00 0.01 0.01 0.05 112.68 ELD_KKR_K525E 0.31 0.00 0.01 0.00 0.03 46.53 ELD_KKR_K525G 0.82 0.01 0.00 0.02 0.03 67.72 ELD_KKR_K525S 1.22 0.01 0.01 0.02 0.04 98.13 ELD_KKR_Q481A 1.52 0.00 0.02 0.00 0.03 216.77 ELD_KKR_Q481C 1.20 0.00 0.01 0.01 0.04 113.49 ELD_KKR_Q481D 1.51 0.03 0.01 0.01 0.08 48.77 ELD_KKR_Q481S 0.96 0.00 0.01 0.00 0.03 160.62 GFP 0.00 0.00 0.00 0.01 0.03 N/A GFP 0.00 0.00 0.00 0.00 0.04 N/A

[0239] Tables 12A-12C present the results of cleavage in 2 different experiments of the target target (AAVS1) and three non-target targets (OT1, OT2, OT3), as well as the ratio of target and non-target cleavage for these mutants.

Table 12A

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 ELD_K448A 59.76 7.05 0.93 2.04 1.40 5.23 ELD_K448C 63.80 9.43 3.22 2.51 2.11 3.69 ELD_K448D 3.14 0.04 0.05 0.05 0.07 15.10 ELD_K448E 75.48 19.58 6.59 3.86 2.19 2.34 ELD_K448F 5.08 0.06 0.20 0.09 0.05 12.65 ELD_K448G 42.70 2.67 0.33 1.26 0.68 8.65 ELD_K448H 6.19 0.19 0.27 0.15 0.09 8.89 ELD_K448I 3.65 0.07 0.10 0.13 0.08 9.54 ELD_K448L 5.82 0.13 0.15 0.24 0.11 9.22 ELD_K448M 69.53 9.69 5.97 3.41 1.87 3.32 ELD_K448N 14.00 0.77 0.44 0.78 0.43 5.79 ELD_K448P 0.10 0.02 0.03 0.03 0.03 0.95 ELD_K448Q 76.82 16.60 4.64 3.11 3.12 2.80 ELD_K448R 73.43 9.83 2.60 3.52 1.87 4.12 ELD_K448S 45.95 6.15 1.53 1.88 1.16 4.29 ELD_K448T 34.16 3.41 2.85 1.96 0.82 3.78 ELD_K448V 13.46 1.47 0.58 0.70 0.53 4.09 ELD_K448W 0.93 0.02 0.04 0.05 0.05 5.74 ELD_K448Y 7.36 0.21 0.52 0.28 0.15 6.37 ELD_I479A 41.45 0.03 0.04 0.04 0.06 248.65 ELD_I479C 32.55 0.02 0.09 0.05 0.10 124.76 ELD_I479D 0.98 0.03 0.01 0.02 0.06 7.69 ELD_I479E 1.46 0.02 0.03 0.05 0.03 11.56 ELD_I479F 22.48 0.02 0.04 0.03 0.11 112.11 ELD_I479G 11.09 0.02 0.04 0.01 0.04 88.84 ELD_I479H 18.97 0.04 0.03 0.02 0.05 142.61 ELD_I479K 0.44 0.02 0.01 0.04 0.07 3.22 ELD_I479L 39.31 0.29 0.13 0.22 0.96 24.66 ELD_I479M 41.44 1.30 0.41 0.41 0.61 15.17 ELD_I479N 20.15 0.03 0.01 0.03 0.06 168.10 ELD_I479P 3.79 0.03 0.01 0.04 0.04 32.04 ELD_I479Q 64.22 0.03 0.05 0.03 0.10 294.99 ELD_I479R 3.21 0.03 0.01 0.03 0.10 18.05 ELD_I479S 36.20 0.02 0.03 0.06 0.09 184.80 ELD_I479T 45.91 0.05 0.07 0.04 0.09 185.53 ELD_I479V 49.92 0.20 0.24 0.11 0.11 75.50 ELD_I479W 3.90 0.00 0.04 0.04 0.04 31.51 ELD_I479Y 23.34 0.02 0.04 0.04 0.03 178.07 ELD_S418D 43.61 0.12 0.14 0.17 0.10 82.58 ELD_N476D 60.68 0.03 0.05 0.05 0.04 342.95 ELD_I479T 41.58 0.05 0.08 0.04 0.07 175.66 ELD_Q481E 54.21 0.05 0.08 0.15 0.07 159.57 ELD_N527D 35.49 0.36 0.11 0.32 0.07 41.19 ELD_Q531R 45.27 0.51 0.11 0.23 0.17 44.55 ELD_R416N 59.39 0.33 0.56 0.42 0.11 41.97 ELD_R416D 30.56 0.12 0.21 0.04 0.03 76.33 ELD_R416E 51.84 0.18 0.74 0.30 0.12 38.76 ELD_R416S 38.94 0.40 0.39 0.46 0.13 28.23 ELD_K525A 45.40 0.11 0.21 0.12 0.11 81.02 ELD_K525E 33.51 0.02 0.01 0.09 0.06 196.02 ELD_K525G 33.61 0.07 0.09 0.09 0.10 95.62 ELD_K525S 50.08 0.23 0.18 0.11 0.11 80.29 ELD_Q481A 46.06 0.02 0.06 0.09 0.04 227.61 ELD_Q481D 73.79 0.48 0.11 0.53 0.20 56.16 ELD_Q481C 47.38 0.06 0.10 0.23 0.17 86.03 ELD_Q481S 36.39 0.01 0.03 0.06 0.02 304.40 ELD_S418E 50.36 0.07 0.09 0.07 0.08 162.21 ELD_R422H 38.80 0.66 0.38 0.58 0.12 22.30 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A Initial pair 1.07 1.35 1.23 1.18 1.25 0.80 Initial pair 0.92 0.73 0.75 0.88 0.69 1.19 Initial pair 1.05 1.13 1.14 1.15 1.22 0.89 Initial pair 0.95 0.80 0.88 0.79 0.85 1.12 Half dose 0.67 0.37 0.26 0.22 0.31 1.99 Half dose 0.68 N/A 0.23 0.30 0.30 N/A Half dose 0.57 0.22 0.17 0.16 0.17 2.80 Half dose 0.57 0.24 0.28 0.25 0.17 2.31 ELD_K448A 1.38 1.44 0.78 1.71 1.38 0.97 ELD_K448C 1.47 1.92 2.71 2.11 2.08 0.68 ELD_K448D 0.07 0.01 0.04 0.04 0.07 2.79 ELD_K448E 1.74 3.99 5.54 3.24 2.16 0.43 ELD_K448F 0.12 0.01 0.17 0.07 0.05 2.33 ELD_K448G 0.98 0.54 0.28 1.06 0.67 1.60 ELD_K448H 0.14 0.04 0.22 0.13 0.09 1.64 ELD_K448I 0.08 0.01 0.08 0.11 0.08 1.76 ELD_K448L 0.13 0.03 0.13 0.20 0.10 1.70 ELD_K448M 1.60 1.97 5.02 2.86 1.84 0.61 ELD_K448N 0.32 0.16 0.37 0.65 0.42 1.07 ELD_K448P 0.00 0.00 0.02 0.03 0.03 0.17 ELD_K448Q 1.77 3.38 3.90 2.61 3.07 0.52 ELD_K448R 1.69 2.00 2.19 2.96 1.84 0.76 ELD_K448S 1.06 1.25 1.29 1.58 1.14 0.79 ELD_K448T 0.79 0.69 2.40 1.64 0.80 0.70 ELD_K448V 0.31 0.30 0.49 0.59 0.53 0.76 ELD_K448W 0.02 0.01 0.04 0.04 0.05 1.06 ELD_K448Y 0.17 0.04 0.44 0.24 0.14 1.18 ELD_I479A 0.96 0.01 0.03 0.03 0.06 45.88 ELD_I479C 0.75 0.00 0.07 0.04 0.10 23.02 ELD_I479D 0.02 0.01 0.01 0.02 0.06 1.42 ELD_I479E 0.03 0.00 0.02 0.04 0.03 2.13 ELD_I479F 0.52 0.00 0.03 0.03 0.11 20.69 ELD_I479G 0.26 0.01 0.04 0.01 0.04 16.39 ELD_I479H 0.44 0.01 0.02 0.02 0.05 26.31 ELD_I479K 0.01 0.00 0.01 0.03 0.07 0.59 ELD_I479L 0.91 0.06 0.11 0.18 0.94 4.55 ELD_I479M 0.96 0.26 0.35 0.34 0.60 2.80 ELD_I479N 0.46 0.01 0.01 0.02 0.06 31.02 ELD_I479P 0.09 0.01 0.01 0.03 0.04 5.91 ELD_I479Q 1.48 0.01 0.04 0.03 0.10 54.43 ELD_I479R 0.07 0.01 0.01 0.03 0.10 3.33 ELD_I479S 0.83 0.00 0.03 0.05 0.09 34.10 ELD_I479T 1.06 0.01 0.06 0.03 0.09 34.23 ELD_I479V 1.15 0.04 0.20 0.09 0.10 13.93 ELD_I479W 0.09 0.00 0.04 0.03 0.04 5.81 ELD_I479Y 0.54 0.00 0.03 0.04 0.03 32.86 ELD_S418D 1.01 0.02 0.12 0.15 0.10 15.24 ELD_N476D 1.40 0.01 0.04 0.05 0.04 63.28 ELD_I479T 0.96 0.01 0.07 0.03 0.07 32.41 ELD_Q481E 1.25 0.01 0.07 0.12 0.06 29.44 ELD_N527D 0.82 0.07 0.09 0.27 0.07 7.60 ELD_Q531R 1.04 0.10 0.09 0.19 0.17 8.22 ELD_R416N 1.37 0.07 0.47 0.35 0.11 7.74 ELD_R416D 0.70 0.03 0.18 0.03 0.03 14.08 ELD_R416E 1.20 0.04 0.62 0.25 0.12 7.15 ELD_R416S 0.90 0.08 0.33 0.38 0.13 5.21 ELD_K525A 1.05 0.02 0.18 0.10 0.11 14.95 ELD_K525E 0.77 0.00 0.01 0.07 0.06 36.17 ELD_K525G 0.77 0.01 0.08 0.08 0.10 17.64 ELD_K525S 1.15 0.05 0.15 0.09 0.11 14.81 ELD_Q481A 1.06 0.00 0.05 0.07 0.04 42.00 ELD_Q481D 1.70 0.10 0.09 0.44 0.19 10.36 ELD_Q481C 1.09 0.01 0.08 0.19 0.16 15.87 ELD_Q481S 0.84 0.00 0.02 0.05 0.02 56.17 ELD_S418E 1.16 0.02 0.08 0.06 0.08 29.93 ELD_R422H 0.89 0.13 0.32 0.49 0.12 4.11 GFP 0.00 0.00 0.05 0.02 0.05 N/A GFP 0.00 0.00 0.04 0.05 0.03 N/A AAVS1 OT1 OT2 OT3 OT4 ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 ELD_K448A 59.76 7.05 0.93 2.04 1.40 5.23 ELD_K448C 63.80 9.43 3.22 2.51 2.11 3.69 ELD_K448D 3.14 0.04 0.05 0.05 0.07 15.10 ELD_K448E 75.48 19.58 6.59 3.86 2.19 2.34 ELD_K448F 5.08 0.06 0.20 0.09 0.05 12.65 ELD_K448G 42.70 2.67 0.33 1.26 0.68 8.65 ELD_K448H 6.19 0.19 0.27 0.15 0.09 8.89 ELD_K448I 3.65 0.07 0.10 0.13 0.08 9.54 ELD_K448L 5.82 0.13 0.15 0.24 0.11 9.22 ELD_K448M 69.53 9.69 5.97 3.41 1.87 3.32 ELD_K448N 14.00 0.77 0.44 0.78 0.43 5.79 ELD_K448P 0.10 0.02 0.03 0.03 0.03 0.95 ELD_K448Q 76.82 16.60 4.64 3.11 3.12 2.80 ELD_K448R 73.43 9.83 2.60 3.52 1.87 4.12 ELD_K448S 45.95 6.15 1.53 1.88 1.16 4.29 ELD_K448T 34.16 3.41 2.85 1.96 0.82 3.78 ELD_K448V 13.46 1.47 0.58 0.70 0.53 4.09 ELD_K448W 0.93 0.02 0.04 0.05 0.05 5.74 ELD_K448Y 7.36 0.21 0.52 0.28 0.15 6.37 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A AAVS1 OT1 OT2 OT3 OT4 ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 ELD_I479A 41.45 0.03 0.04 0.04 0.06 248.65 ELD_I479C 32.55 0.02 0.09 0.05 0.10 124.76 ELD_I479D 0.98 0.03 0.01 0.02 0.06 7.69 ELD_I479E 1.46 0.02 0.03 0.05 0.03 11.56 ELD_I479F 22.48 0.02 0.04 0.03 0.11 112.11 ELD_I479G 11.09 0.02 0.04 0.01 0.04 88.84 ELD_I479H 18.97 0.04 0.03 0.02 0.05 142.61 ELD_I479K 0.44 0.02 0.01 0.04 0.07 3.22 ELD_I479L 39.31 0.29 0.13 0.22 0.96 24.66 ELD_I479M 41.44 1.30 0.41 0.41 0.61 15.17 ELD_I479N 20.15 0.03 0.01 0.03 0.06 168.10 ELD_I479P 3.79 0.03 0.01 0.04 0.04 32.04 ELD_I479Q 64.22 0.03 0.05 0.03 0.10 294.99 ELD_I479R 3.21 0.03 0.01 0.03 0.10 18.05 ELD_I479S 36.20 0.02 0.03 0.06 0.09 184.80 ELD_I479T 45.91 0.05 0.07 0.04 0.09 185.53 ELD_I479V 49.92 0.20 0.24 0.11 0.11 75.50 ELD_I479W 3.90 0.00 0.04 0.04 0.04 31.51 ELD_I479Y 23.34 0.02 0.04 0.04 0.03 178.07 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A

Table 12B

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 KKR_K448A 58.79 4.46 0.58 1.06 0.89 8.42 KKR_K448C 57.00 7.57 1.34 1.41 2.28 4.52 KKR_K448D 2.66 0.04 0.04 0.04 0.02 18.94 KKR_K448E 64.43 6.96 0.21 0.73 1.86 6.60 KKR_K448F 5.15 0.07 0.05 0.02 0.13 18.91 KKR_K448G 13.56 0.05 0.05 0.03 0.06 74.17 KKR_K448H 7.39 0.09 0.02 0.07 0.17 21.44 KKR_K448I 4.04 0.16 0.05 0.06 0.08 11.63 KKR_K448L 5.85 0.08 0.08 0.04 0.08 21.19 KKR_K448M 62.00 6.17 0.51 0.84 2.83 5.99 KKR_K448N 15.19 0.75 0.21 0.20 0.22 10.96 KKR_K448P 0.04 0.02 0.03 0.03 0.04 0.31 KKR_K448Q 65.53 7.52 1.10 1.28 3.51 4.89 KKR_K448R 58.33 7.45 0.71 1.00 1.49 5.48 KKR_K448S 46.58 4.04 0.75 0.90 0.87 7.10 KKR_K448T 34.43 3.18 0.43 0.41 1.45 6.28 KKR_K448V 14.04 1.09 0.31 0.36 0.35 6.68 KKR_K448W 0.64 0.03 0.05 0.06 0.04 3.70 KKR_K448Y 8.97 0.11 0.18 0.09 0.10 18.95 KKR_I479A 39.97 0.06 0.05 0.10 0.07 144.06 KKR_I479C 41.45 0.51 0.17 0.27 0.05 41.20 KKR_I479D 6.45 0.01 0.03 0.04 0.04 54.74 KKR_I479E 13.23 0.02 0.04 0.02 0.07 83.65 KKR_I479F 25.15 0.08 0.11 0.12 0.02 76.19 KKR_I479G 18.48 0.03 0.04 0.02 0.11 95.39 KKR_I479H 19.66 0.04 0.03 0.04 0.07 113.04 KKR_I479K 0.78 0.01 0.04 0.01 0.06 6.35 KKR_I479L 40.10 1.91 2.96 0.90 0.20 6.71 KKR_I479M 43.43 2.12 1.33 1.35 0.19 8.68 KKR_I479N 25.03 0.04 0.05 0.11 0.08 91.41 KKR_I479P 1.28 0.03 0.04 0.06 0.07 6.30 KKR_I479Q 59.57 1.32 0.54 0.77 0.05 22.11 KKR_I479R 3.17 0.03 0.04 0.01 0.04 26.74 KKR_I479S 35.64 0.05 0.06 0.06 0.03 167.22 KKR_I479T 48.64 0.44 0.06 0.50 0.08 45.12 KKR_I479V 49.23 0.48 0.18 0.32 0.25 40.12 KKR_I479W 6.86 0.02 0.01 0.06 0.05 46.92 KKR_I479Y 21.50 0.04 0.04 0.04 0.05 120.50 KKR_S418D 47.81 0.26 0.07 0.10 0.10 90.39 KKR_N476D 19.63 0.06 0.03 0.04 0.04 119.60 KKR_I479T 46.95 0.30 0.11 0.28 0.06 62.71 KKR_Q481E 47.06 0.16 0.09 0.05 0.07 127.97 KKR_N527D 39.17 0.85 0.20 0.40 0.37 21.48 KKR_Q531R 36.99 1.08 0.30 0.63 0.28 16.15 KKR_R416N 54.55 0.92 0.05 0.18 0.49 33.30 KKR_R416D 36.70 0.11 0.03 0.04 0.14 120.60 KKR_R416E 51.32 0.35 0.03 0.06 0.33 66.66 KKR_R416S 39.97 1.41 0.18 0.22 0.62 16.48 KKR_K525A 39.26 0.46 0.05 0.07 0.18 50.61 KKR_K525E 32.52 0.07 0.01 0.11 0.07 128.95 KKR_K525G 34.47 0.43 0.10 0.08 0.24 40.02 KKR_K525S 40.86 0.39 0.02 0.12 0.19 56.36 KKR_Q481A 46.97 0.04 0.02 0.08 0.02 294.46 KKR_Q481D 55.80 1.06 0.05 0.05 0.17 42.03 KKR_Q481C 39.74 0.81 0.08 0.05 0.04 40.79 KKR_Q481S 36.46 0.12 0.05 0.07 0.05 128.50 KKR_S418E 50.83 0.20 0.06 0.06 0.05 137.52 KKR_R422H 40.44 0.77 0.10 0.15 0.40 28.46 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A Initial pair 1.07 1.35 1.23 1.18 1.25 0.80 Initial pair 0.92 0.73 0.75 0.88 0.69 1.19 Initial pair 1.05 1.13 1.14 1.15 1.22 0.89 Initial pair 0.95 0.80 0.88 0.79 0.85 1.12 Half dose 0.67 0.37 0.26 0.22 0.31 1.99 Half dose 0.68 N/A 0.23 0.30 0.30 N/A Half dose 0.57 0.22 0.17 0.16 0.17 2.80 Half dose 0.57 0.24 0.28 0.25 0.17 2.31 KKR_K448A 1.36 0.91 0.49 0.89 0.87 1.55 KKR_K448C 1.31 1.54 1.13 1.18 2.25 0.83 KKR_K448D 0.06 0.01 0.04 0.03 0.02 3.50 KKR_K448E 1.49 1.42 0.18 0.61 1.84 1.22 KKR_K448F 0.12 0.01 0.04 0.02 0.13 3.49 KKR_K448G 0.31 0.01 0.04 0.02 0.06 13.69 KKR_K448H 0.17 0.02 0.02 0.06 0.16 3.96 KKR_K448I 0.09 0.03 0.04 0.05 0.08 2.15 KKR_K448L 0.13 0.02 0.07 0.04 0.08 3.91 KKR_K448M 1.43 1.26 0.43 0.71 2.79 1.10 KKR_K448N 0.35 0.15 0.18 0.17 0.22 2.02 KKR_K448P 0.00 0.00 0.03 0.02 0.04 0.06 KKR_K448Q 1.51 1.53 0.93 1.07 3.45 0.90 KKR_K448R 1.34 1.52 0.60 0.84 1.47 1.01 KKR_K448S 1.07 0.82 0.63 0.75 0.86 1.31 KKR_K448T 0.79 0.65 0.36 0.35 1.43 1.16 KKR_K448V 0.32 0.22 0.26 0.30 0.34 1.23 KKR_K448W 0.01 0.01 0.04 0.05 0.04 0.68 KKR_K448Y 0.21 0.02 0.15 0.07 0.10 3.50 KKR_I479A 0.92 0.01 0.04 0.08 0.07 26.58 KKR_I479C 0.96 0.10 0.14 0.23 0.05 7.60 KKR_I479D 0.15 0.00 0.02 0.04 0.04 10.10 KKR_I479E 0.31 0.00 0.03 0.02 0.07 15.44 KKR_I479F 0.58 0.02 0.09 0.10 0.02 14.06 KKR_I479G 0.43 0.01 0.03 0.02 0.11 17.60 KKR_I479H 0.45 0.01 0.03 0.03 0.07 20.86 KKR_I479K 0.02 0.00 0.03 0.01 0.06 1.17 KKR_I479L 0.92 0.39 2.49 0.76 0.20 1.24 KKR_I479M 1.00 0.43 1.12 1.13 0.19 1.60 KKR_I479N 0.58 0.01 0.04 0.09 0.08 16.87 KKR_I479P 0.03 0.01 0.03 0.05 0.07 1.16 KKR_I479Q 1.37 0.27 0.45 0.65 0.05 4.08 KKR_I479R 0.07 0.01 0.03 0.01 0.04 4.93 KKR_I479S 0.82 0.01 0.05 0.05 0.03 30.85 KKR_I479T 1.12 0.09 0.05 0.42 0.08 8.33 KKR_I479V 1.14 0.10 0.15 0.27 0.24 7.40 KKR_I479W 0.16 0.00 0.01 0.05 0.05 8.66 KKR_I479Y 0.50 0.01 0.03 0.04 0.05 22.23 KKR_S418D 1.10 0.05 0.06 0.09 0.10 16.68 KKR_N476D 0.45 0.01 0.02 0.03 0.04 22.07 KKR_I479T 1.08 0.06 0.09 0.24 0.06 11.57 KKR_Q481E 1.08 0.03 0.07 0.04 0.07 23.61 KKR_N527D 0.90 0.17 0.17 0.33 0.37 3.96 KKR_Q531R 0.85 0.22 0.25 0.53 0.28 2.98 KKR_R416N 1.26 0.19 0.04 0.15 0.48 6.14 KKR_R416D 0.85 0.02 0.02 0.03 0.13 22.25 KKR_R416E 1.18 0.07 0.02 0.05 0.32 12.30 KKR_R416S 0.92 0.29 0.15 0.18 0.61 3.04 KKR_K525A 0.91 0.09 0.05 0.06 0.18 9.34 KKR_K525E 0.75 0.01 0.01 0.09 0.07 23.79 KKR_K525G 0.79 0.09 0.09 0.07 0.24 7.38 KKR_K525S 0.94 0.08 0.02 0.10 0.19 10.40 KKR_Q481A 1.08 0.01 0.02 0.06 0.02 54.33 KKR_Q481D 1.29 0.22 0.04 0.04 0.17 7.75 KKR_Q481C 0.92 0.16 0.07 0.04 0.04 7.53 KKR_Q481S 0.84 0.02 0.04 0.06 0.05 23.71 KKR_S418E 1.17 0.04 0.05 0.05 0.05 25.37 KKR_R422H 0.93 0.16 0.08 0.13 0.39 5.25 GFP 0.00 0.00 0.05 0.02 0.05 N/A GFP 0.00 0.00 0.04 0.05 0.03 N/A AAVS1 OT1 OT2 OT3 OT4 ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 KKR_K448A 58.79 4.46 0.58 1.06 0.89 8.42 KKR_K448C 57.00 7.57 1.34 1.41 2.28 4.52 KKR_K448D 2.66 0.04 0.04 0.04 0.02 18.94 KKR_K448E 64.43 6.96 0.21 0.73 1.86 6.60 KKR_K448F 5.15 0.07 0.05 0.02 0.13 18.91 KKR_K448G 13.56 0.05 0.05 0.03 0.06 74.17 KKR_K448H 7.39 0.09 0.02 0.07 0.17 21.44 KKR_K448I 4.04 0.16 0.05 0.06 0.08 11.63 KKR_K448L 5.85 0.08 0.08 0.04 0.08 21.19 KKR_K448M 62.00 6.17 0.51 0.84 2.83 5.99 KKR_K448N 15.19 0.75 0.21 0.20 0.22 10.96 KKR_K448P 0.04 0.02 0.03 0.03 0.04 0.31 KKR_K448Q 65.53 7.52 1.10 1.28 3.51 4.89 KKR_K448R 58.33 7.45 0.71 1.00 1.49 5.48 KKR_K448S 46.58 4.04 0.75 0.90 0.87 7.10 KKR_K448T 34.43 3.18 0.43 0.41 1.45 6.28 KKR_K448V 14.04 1.09 0.31 0.36 0.35 6.68 KKR_K448W 0.64 0.03 0.05 0.06 0.04 3.70 KKR_K448Y 8.97 0.11 0.18 0.09 0.10 18.95 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A AAVS1 OT1 OT2 OT3 OT4 ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 KKR_I479A 39.97 0.06 0.05 0.10 0.07 144.06 KKR_I479C 41.45 0.51 0.17 0.27 0.05 41.20 KKR_I479D 6.45 0.01 0.03 0.04 0.04 54.74 KKR_I479E 13.23 0.02 0.04 0.02 0.07 83.65 KKR_I479F 25.15 0.08 0.11 0.12 0.02 76.19 KKR_I479G 18.48 0.03 0.04 0.02 0.11 95.39 KKR_I479H 19.66 0.04 0.03 0.04 0.07 113.04 KKR_I479K 0.78 0.01 0.04 0.01 0.06 6.35 KKR_I479L 40.10 1.91 2.96 0.90 0.20 6.71 KKR_I479M 43.43 2.12 1.33 1.35 0.19 8.68 KKR_I479N 25.03 0.04 0.05 0.11 0.08 91.41 KKR_I479P 1.28 0.03 0.04 0.06 0.07 6.30 KKR_I479Q 59.57 1.32 0.54 0.77 0.05 22.11 KKR_I479R 3.17 0.03 0.04 0.01 0.04 26.74 KKR_I479S 35.64 0.05 0.06 0.06 0.03 167.22 KKR_I479T 48.64 0.44 0.06 0.50 0.08 45.12 KKR_I479V 49.23 0.48 0.18 0.32 0.25 40.12 KKR_I479W 6.86 0.02 0.01 0.06 0.05 46.92 KKR_I479Y 21.50 0.04 0.04 0.04 0.05 120.50 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A

Table 12C

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 ELD_KKR_K448A 61.79 1.69 0.26 0.70 0.46 19.89 ELD_KKR_K448C 64.98 6.21 1.34 1.40 1.79 6.05 ELD_KKR_K448D 0.08 0.03 0.02 0.01 0.04 0.86 ELD_KKR_K448E 84.95 18.50 0.87 1.02 2.25 3.75 ELD_KKR_K448F 0.72 0.02 0.02 0.05 0.04 5.26 ELD_KKR_K448G 8.15 0.03 0.05 0.06 0.09 36.59 ELD_KKR_K448H 1.10 0.02 0.02 0.04 0.08 7.22 ELD_KKR_K448I 0.19 0.02 0.01 0.04 0.03 1.94 ELD_KKR_K448L 0.59 0.03 0.05 0.02 0.05 4.00 ELD_KKR_K448M 81.10 9.18 1.14 1.81 2.50 5.55 ELD_KKR_K448N 4.51 0.16 0.19 0.10 0.11 8.02 ELD_KKR_K448P 0.04 0.03 0.03 0.04 0.08 0.24 ELD_KKR_K448Q 90.81 20.83 4.03 3.99 8.91 2.41 ELD_KKR_K448R 82.04 6.56 0.76 1.63 0.95 8.29 ELD_KKR_K448S 40.23 2.31 0.42 0.67 0.41 10.57 ELD_KKR_K448T 19.29 1.14 0.43 0.37 0.37 8.40 ELD_KKR_K448V 3.93 0.34 0.15 0.28 0.19 4.07 ELD_KKR_K448W 0.04 0.04 0.04 0.02 0.10 0.20 ELD_KKR_K448Y 1.79 0.01 0.13 0.02 0.08 7.54 ELD_KKR_I479A 0.79 0.01 0.02 0.03 0.06 7.35 ELD_KKR_I479C 14.11 0.06 0.04 0.03 0.07 70.49 ELD_KKR_I479D 0.01 0.04 0.04 0.01 0.03 0.07 ELD_KKR_I479E 0.04 0.02 0.04 0.06 0.04 0.23 ELD_KKR_I479F 5.13 0.03 0.01 0.03 0.03 50.10 ELD_KKR_I479G 0.08 0.03 0.03 0.03 0.05 0.58 ELD_KKR_I479H 0.09 0.02 0.04 0.04 0.04 0.66 ELD_KKR_I479K 0.04 0.03 0.04 0.01 0.03 0.37 ELD_KKR_I479L 27.72 0.05 0.17 0.12 0.08 66.94 ELD_KKR-I479M 34.75 0.22 0.22 0.10 0.10 53.87 ELD_KKR_I479N 0.12 0.02 0.06 0.04 0.03 0.83 ELD_KKR_I479P 0.04 0.03 0.02 0.04 0.04 0.33 ELD_KKR_I479Q 53.80 0.03 0.04 0.07 0.04 303.42 ELD_KKR_I479R 0.05 0.02 0.07 0.06 0.01 0.27 ELD_KKR_I479S 0.46 0.01 0.05 0.03 0.06 3.16 ELD_KKR_I479T 28.74 0.03 0.04 0.03 0.09 154.73 ELD_KKR_I479V 36.57 0.05 0.04 0.06 0.05 184.04 ELD_KKR_I479W 0.04 0.04 0.01 0.03 0.04 0.28 ELD_KKR_I479Y 1.00 0.01 0.04 0.04 0.03 8.20 ELD_KKR_S418D 22.59 0.02 0.05 0.04 0.08 115.05 ELD_KKR_N476D 2.29 0.03 0.03 0.04 0.08 12.55 ELD_KKR_I479T 20.33 0.03 0.03 0.06 0.06 116.86 ELD_KKR_Q481E 43.53 0.01 0.03 0.04 0.11 234.62 ELD_KKR_N527D 15.86 0.03 0.03 0.06 0.02 112.24 ELD_KKR_Q531R 31.62 0.17 0.06 0.13 0.04 80.56 ELD_KKR_R416N 70.71 0.06 0.06 0.07 0.12 227.36 ELD_KKR_R416D 32.67 0.03 0.02 0.01 0.04 349.76 ELD_KKR_R416E 66.68 0.13 0.06 0.10 0.05 198.02 ELD_KKR_R416S 36.47 0.20 0.05 0.13 0.08 79.49 ELD_KKR_K525A 36.88 0.02 0.05 0.02 0.04 292.90 ELD_KKR_K525E 8.26 0.01 0.01 0.02 0.08 74.88 ELD_KKR_K525G 24.82 0.02 0.03 0.07 0.05 142.86 ELD_KKR_K525S 46.15 0.04 0.02 0.12 0.07 184.82 ELD_KKR_Q481A 82.60 0.04 0.02 0.04 0.04 559.53 ELD_KKR_Q481D 89.32 0.19 0.03 0.04 0.06 279.60 ELD_KKR_Q481C 50.98 0.06 0.01 0.02 0.04 410.14 ELD_KKR_Q481S 36.72 0.02 0.04 0.06 0.05 214.56 ELD_KKR_S418E 38.52 0.04 0.02 0.05 0.02 303.63 ELD_KKR_R422H 37.96 0.14 0.09 0.11 0.08 93.14 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A Initial pair 1.07 1.35 1.23 1.18 1.25 0.80 Initial pair 0.92 0.73 0.75 0.88 0.69 1.19 Initial pair 1.05 1.13 1.14 1.15 1.22 0.89 Initial pair 0.95 0.80 0.88 0.79 0.85 1.12 Half dose 0.67 0.37 0.26 0.22 0.31 1.99 Half dose 0.68 N/A 0.23 0.30 0.30 N/A Half dose 0.57 0.22 0.17 0.16 0.17 2.80 Half dose 0.57 0.24 0.28 0.25 0.17 2.31 ELD_KKR_K448A 1.42 0.34 0.22 0.59 0.45 3.67 ELD_KKR_K448C 1.50 1.26 1.12 1.18 1.76 1.12 ELD_KKR_K448D 0.00 0.01 0.02 0.01 0.03 0.16 ELD_KKR_K448E 1.96 3.77 0.73 0.86 2.21 0.69 ELD_KKR_K448F 0.02 0.00 0.02 0.04 0.04 0.97 ELD_KKR_K448G 0.19 0.01 0.04 0.05 0.09 6.75 ELD_KKR_K448H 0.03 0.00 0.01 0.03 0.07 1.33 ELD_KKR_K448I 0.00 0.00 0.01 0.03 0.03 0.36 ELD_KKR_K448L 0.01 0.01 0.05 0.01 0.05 0.74 ELD_KKR_K448M 1.87 1.87 0.95 1.52 2.46 1.02 ELD_KKR_K448N 0.10 0.03 0.16 0.08 0.11 1.48 ELD_KKR_K448P 0.00 0.01 0.03 0.03 0.08 0.04 ELD_KKR_K448Q 2.09 4.24 3.39 3.35 8.77 0.44 ELD_KKR_K448R 1.89 1.34 0.64 1.37 0.93 1.53 ELD_KKR_K448S 0.93 0.47 0.35 0.57 0.40 1.95 ELD_KKR_K448T 0.44 0.23 0.36 0.31 0.36 1.55 ELD_KKR_K448V 0.09 0.07 0.13 0.24 0.19 0.75 ELD_KKR_K448W 0.00 0.01 0.03 0.02 0.10 0.04 ELD_KKR_K448Y 0.04 0.00 0.11 0.02 0.08 1.39 ELD_KKR_I479A 0.02 0.00 0.02 0.02 0.06 1.36 ELD_KKR_I479C 0.33 0.01 0.03 0.03 0.07 13.01 ELD_KKR_I479D 0.00 0.01 0.03 0.01 0.03 0.01 ELD_KKR_I479E 0.00 0.01 0.03 0.05 0.04 0.04 ELD_KKR_I479F 0.12 0.01 0.01 0.03 0.03 9.24 ELD_KKR_I479G 0.00 0.01 0.03 0.02 0.04 0.11 ELD_KKR_I479H 0.00 0.00 0.04 0.04 0.04 0.12 ELD_KKR_I479K 0.00 0.01 0.03 0.01 0.03 0.07 ELD_KKR_I479L 0.64 0.01 0.14 0.10 0.08 12.35 ELD_KKR-I479M 0.80 0.05 0.19 0.08 0.10 9.94 ELD_KKR_I479N 0.00 0.00 0.05 0.03 0.03 0.15 ELD_KKR_I479P 0.00 0.01 0.02 0.04 0.03 0.06 ELD_KKR_I479Q 1.24 0.01 0.03 0.06 0.04 55.98 ELD_KKR_I479R 0.00 0.00 0.06 0.05 0.01 0.05 ELD_KKR_I479S 0.01 0.00 0.05 0.02 0.05 0.58 ELD_KKR_I479T 0.66 0.01 0.03 0.02 0.09 28.55 ELD_KKR_I479V 0.84 0.01 0.04 0.05 0.05 33.96 ELD_KKR_I479W 0.00 0.01 0.01 0.02 0.04 0.05 ELD_KKR_I479Y 0.02 0.00 0.03 0.04 0.03 1.51 ELD_KKR_S418D 0.52 0.00 0.04 0.03 0.08 21.23 ELD_KKR_N476D 0.05 0.01 0.03 0.03 0.08 2.32 ELD_KKR_I479T 0.47 0.01 0.02 0.05 0.05 21.56 ELD_KKR_Q481E 1.00 0.00 0.02 0.04 0.11 43.29 ELD_KKR_N527D 0.37 0.01 0.03 0.05 0.02 20.71 ELD_KKR_Q531R 0.73 0.03 0.05 0.11 0.04 14.86 ELD_KKR_R416N 1.63 0.01 0.05 0.06 0.11 41.95 ELD_KKR_R416D 0.75 0.01 0.02 0.01 0.04 64.54 ELD_KKR_R416E 1.54 0.03 0.05 0.08 0.05 36.54 ELD_KKR_R416S 0.84 0.04 0.04 0.11 0.08 14.67 ELD_KKR_K525A 0.85 0.00 0.04 0.01 0.04 54.04 ELD_KKR_K525E 0.19 0.00 0.01 0.01 0.07 13.82 ELD_KKR_K525G 0.57 0.00 0.03 0.06 0.05 26.36 ELD_KKR_K525S 1.06 0.01 0.02 0.10 0.07 34.10 ELD_KKR_Q481A 1.90 0.01 0.02 0.03 0.04 103.24 ELD_KKR_Q481D 2.06 0.04 0.03 0.04 0.05 51.59 ELD_KKR_Q481C 1.18 0.01 0.01 0.02 0.04 75.68 ELD_KKR_Q481S 0.85 0.00 0.04 0.05 0.05 39.59 ELD_KKR_S418E 0.89 0.01 0.02 0.04 0.02 56.02 ELD_KKR_R422H 0.88 0.03 0.07 0.09 0.08 17.19 GFP 0.00 0.00 0.05 0.02 0.05 N/A GFP 0.00 0.00 0.04 0.05 0.03 N/A AAVS1 OT1 OT2 OT3 OT4 Ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 ELD_KKR_K448A 61.79 1.69 0.26 0.70 0.46 19.89 ELD_KKR_K448C 64.98 6.21 1.34 1.40 1.79 6.05 ELD_KKR_K448D 0.08 0.03 0.02 0.01 0.04 0.86 ELD_KKR_K448E 84.95 18.50 0.87 1.02 2.25 3.75 ELD_KKR_K448F 0.72 0.02 0.02 0.05 0.04 5.26 ELD_KKR_K448G 8.15 0.03 0.05 0.06 0.09 36.59 ELD_KKR_K448H 1.10 0.02 0.02 0.04 0.08 7.22 ELD_KKR_K448I 0.19 0.02 0.01 0.04 0.03 1.94 ELD_KKR_K448L 0.59 0.03 0.05 0.02 0.05 4.00 ELD_KKR_K448M 81.10 9.18 1.14 1.81 2.50 5.55 ELD_KKR_K448N 4.51 0.16 0.19 0.10 0.11 8.02 ELD_KKR_K448P 0.04 0.03 0.03 0.04 0.08 0.24 ELD_KKR_K448Q 90.81 20.83 4.03 3.99 8.91 2.41 ELD_KKR_K448R 82.04 6.56 0.76 1.63 0.95 8.29 ELD_KKR_K448S 40.23 2.31 0.42 0.67 0.41 10.57 ELD_KKR_K448T 19.29 1.14 0.43 0.37 0.37 8.40 ELD_KKR_K448V 3.93 0.34 0.15 0.28 0.19 4.07 ELD_KKR_K448W 0.04 0.04 0.04 0.02 0.10 0.20 ELD_KKR_K448Y 1.79 0.01 0.13 0.02 0.08 7.54 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A AAVS1 OT1 OT2 OT3 OT4 ratio Initial pair 46.50 6.60 1.46 1.41 1.26 4.33 Initial pair 40.06 3.57 0.90 1.05 0.70 6.44 Initial pair 45.74 5.54 1.36 1.37 1.24 4.81 Initial pair 41.20 3.92 1.04 0.94 0.86 6.09 Half dose 29.20 1.82 0.31 0.26 0.31 10.78 Half dose 29.32 N/A 0.27 0.36 0.31 N/A Half dose 24.58 1.06 0.20 0.19 0.17 15.16 Half dose 24.63 1.16 0.34 0.30 0.17 12.53 ELD_KKR_I479A 0.79 0.01 0.02 0.03 0.06 7.35 ELD_KKR_I479C 14.11 0.06 0.04 0.03 0.07 70.49 ELD_KKR_I479D 0.01 0.04 0.04 0.01 0.03 0.07 ELD_KKR_I479E 0.04 0.02 0.04 0.06 0.04 0.23 ELD_KKR_I479F 5.13 0.03 0.01 0.03 0.03 50.10 ELD_KKR_I479G 0.08 0.03 0.03 0.03 0.05 0.58 ELD_KKR_I479H 0.09 0.02 0.04 0.04 0.04 0.66 ELD_KKR_I479K 0.04 0.03 0.04 0.01 0.03 0.37 ELD_KKR_I479L 27.72 0.05 0.17 0.12 0.08 66.94 ELD_KKR-I479M 34.75 0.22 0.22 0.10 0.10 53.87 ELD_KKR_I479N 0.12 0.02 0.06 0.04 0.03 0.83 ELD_KKR_I479P 0.04 0.03 0.02 0.04 0.04 0.33 ELD_KKR_I479Q 53.80 0.03 0.04 0.07 0.04 303.42 ELD_KKR_I479R 0.05 0.02 0.07 0.06 0.01 0.27 ELD_KKR_I479S 0.46 0.01 0.05 0.03 0.06 3.16 ELD_KKR_I479T 28.74 0.03 0.04 0.03 0.09 154.73 ELD_KKR_I479V 36.57 0.05 0.04 0.06 0.05 184.04 ELD_KKR_I479W 0.04 0.04 0.01 0.03 0.04 0.28 ELD_KKR_I479Y 1.00 0.01 0.04 0.04 0.03 8.20 GFP 0.03 0.02 0.06 0.02 0.05 N/A GFP 0.04 0.01 0.04 0.06 0.03 N/A

[0240] Tables 13A-13C show targeted and non-targeted cleavage using AAVS1-targeted ZFNs with the indicated mutations (ELD, KKR, ELD/KKR and other indicated mutations).

Table 13A

AAVS1 OT1 OT2 OT3 OT4 Ratio original 47.96 4.32 2.12 1.05 0.84 5.76 original 46.45 4.34 1.84 1.11 1.06 5.57 original 45.83 3.15 1.28 0.77 0.92 7.49 original 47.63 4.30 1.67 0.99 1.03 5.97 Half dose 30.79 1.21 0.38 0.19 0.18 15.72 Half dose 31.59 1.19 0.23 0.28 0.18 16.78 Half dose 29.49 0.76 0.35 0.21 0.16 19.93 Half dose 28.04 0.85 0.31 0.20 0.17 18.41 ELD_N527A 20.52 0.76 0.34 0.42 0.28 11.37 ELD_N527C 16.30 0.80 0.25 0.25 0.17 11.05 ELD_N527D 45.05 0.42 0.11 0.21 0.12 53.34 ELD_N527E 18.93 0.06 0.08 0.13 0.09 52.04 ELD_N527F 5.32 0.21 0.16 0.15 0.02 9.79 ELD_N527G 25.25 1.22 0.92 0.60 0.71 7.32 ELD_N527H 20.69 1.65 1.10 0.55 0.13 6.05 ELD_N527I 1.58 0.04 0.02 0.07 0.01 11.16 ELD_N527K 14.65 1.31 0.66 0.49 0.11 5.70 ELD_N527L 3.37 0.22 0.12 0.09 0.06 6.90 ELD_N527M 6.61 0.44 0.18 0.23 0.08 7.06 ELD_N527P 34.71 2.50 0.97 0.72 0.63 7.20 ELD_N527Q 25.72 2.62 1.76 1.13 0.99 3.96 ELD_N527R 17.38 1.70 1.07 0.37 0.28 5.08 ELD_N527S 22.51 0.93 0.67 0.37 0.32 9.83 ELD_N527T 25.14 0.25 0.14 0.12 0.04 45.36 ELD_N527V 4.22 0.14 0.09 0.10 0.05 10.88 ELD_N527W 3.33 0.17 0.05 0.11 0.04 9.07 ELD_N527Y 5.34 0.22 0.18 0.12 0.06 9.29 ELD_Q481A 56.06 0.06 0.10 0.14 0.05 160.54 ELD_Q481C 61.83 0.05 0.28 0.35 0.22 69.22 ELD_Q481D 78.20 0.54 0.21 0.87 0.29 40.90 ELD_Q481E 61.09 0.03 0.05 0.12 0.04 248.97 ELD_Q481F 0.50 0.02 0.05 0.02 0.05 3.54 ELD_Q481G 33.52 0.43 0.32 0.74 0.24 19.41 ELD_Q481H 71.31 7.24 5.48 1.76 0.15 4.87 ELD_Q481I 0.95 0.04 0.04 0.07 0.02 5.29 ELD_Q481K 19.90 0.05 0.06 0.05 0.04 102.54 ELD_Q481L 1.22 0.03 0.05 0.04 0.03 7.88 ELD_Q481M 5.62 0.02 0.05 0.04 0.04 35.82 ELD_Q481N 36.71 1.29 0.50 0.65 0.22 13.81 ELD_Q481P 1.99 0.02 0.01 0.07 0.04 14.21 ELD_Q481R 5.62 0.04 0.02 0.04 0.01 45.70 ELD_Q481S 50.84 0.05 0.07 0.14 0.04 171.76 ELD_Q481T 36.86 0.05 0.07 0.11 0.01 150.05 ELD_Q481V 1.45 0.03 0.06 0.06 0.02 8.28 ELD_Q481W 0.24 0.03 0.02 0.04 0.03 1.95 ELD_Q481Y 1.75 0.04 0.04 0.05 0.02 11.96 ELD_S418D 49.76 0.13 0.14 0.15 0.07 100.32 ELD_N476D 63.24 0.10 0.03 0.09 0.06 222.11 ELD_I479T 49.59 0.05 0.20 0.03 0.09 134.46 ELD_Q481E 65.37 0.07 0.12 0.13 0.07 168.40 ELD_N527D 42.99 0.45 0.15 0.31 0.06 44.51 ELD_Q531R 53.22 0.73 0.21 0.23 0.16 40.16 ELD_R416D 40.79 0.07 0.39 0.05 0.05 73.16 ELD_R416E 57.69 0.13 1.39 0.09 0.11 33.54 ELD_R416N 62.32 0.32 1.90 0.45 0.13 22.24 ELD_R416S 47.72 0.37 1.19 0.82 0.19 18.57 ELD_K525A 55.65 0.07 0.23 0.10 0.15 101.13 ELD_K525E 49.34 0.05 0.08 0.09 0.08 164.69 ELD_K525G 47.83 0.12 0.25 0.06 0.12 86.58 ELD_K525S 60.06 0.28 0.37 0.16 0.16 62.41 GFP 0.01 0.02 0.03 0.02 0.05 N/A GFP 0.01 0.03 0.02 0.05 0.06 N/A original 1.02 1.07 1.23 1.08 0.87 0.93 original 0.99 1.08 1.06 1.13 1.10 0.90 original 0.98 0.78 0.74 0.79 0.96 1.21 original 1.01 1.07 0.97 1.01 1.07 0.96 Half dose 0.66 0.30 0.22 0.19 0.19 2.54 Half dose 0.67 0.30 0.13 0.29 0.19 2.71 Half dose 0.63 0.19 0.20 0.21 0.17 3.22 Half dose 0.60 0.21 0.18 0.20 0.17 2.97 ELD_N527A 0.44 0.19 0.20 0.43 0.29 1.84 ELD_N527C 0.35 0.20 0.15 0.25 0.18 1.78 ELD_N527D 0.96 0.10 0.06 0.21 0.12 8.61 ELD_N527E 0.40 0.02 0.05 0.13 0.09 8.40 ELD_N527F 0.11 0.05 0.09 0.15 0.02 1.58 ELD_N527G 0.54 0.30 0.54 0.61 0.74 1.18 ELD_N527H 0.44 0.41 0.63 0.56 0.13 0.98 ELD_N527I 0.03 0.01 0.01 0.07 0.01 1.80 ELD_N527K 0.31 0.32 0.38 0.50 0.11 0.92 ELD_N527L 0.07 0.05 0.07 0.09 0.06 1.11 ELD_N527M 0.14 0.11 0.11 0.24 0.08 1.14 ELD_N527P 0.74 0.62 0.56 0.73 0.66 1.16 ELD_N527Q 0.55 0.65 1.02 1.15 1.03 0.64 ELD_N527R 0.37 0.42 0.62 0.38 0.29 0.82 ELD_N527S 0.48 0.23 0.39 0.38 0.34 1.59 ELD_N527T 0.54 0.06 0.08 0.12 0.04 7.32 ELD_N527V 0.09 0.03 0.05 0.11 0.06 1.76 ELD_N527W 0.07 0.04 0.03 0.11 0.04 1.46 ELD_N527Y 0.11 0.05 0.11 0.12 0.06 1.50 ELD_Q481A 1.19 0.01 0.06 0.14 0.05 25.92 ELD_Q481C 1.32 0.01 0.16 0.36 0.23 11.17 ELD_Q481D 1.67 0.13 0.12 0.89 0.31 6.60 ELD_Q481E 1.30 0.01 0.03 0.12 0.05 40.19 ELD_Q481F 0.01 0.00 0.03 0.02 0.05 0.57 ELD_Q481G 0.71 0.11 0.19 0.75 0.25 3.13 ELD_Q481H 1.52 1.80 3.18 1.80 0.16 0.79 ELD_Q481I 0.02 0.01 0.02 0.07 0.02 0.85 ELD_Q481K 0.42 0.01 0.04 0.05 0.04 16.55 ELD_Q481L 0.03 0.01 0.03 0.04 0.03 1.27 ELD_Q481M 0.12 0.00 0.03 0.04 0.04 5.78 ELD_Q481N 0.78 0.32 0.29 0.66 0.23 2.23 ELD_Q481P 0.04 0.00 0.01 0.07 0.04 2.29 ELD_Q481R 0.12 0.01 0.01 0.04 0.01 7.38 ELD_Q481S 1.08 0.01 0.04 0.14 0.04 27.73 ELD_Q481T 0.78 0.01 0.04 0.11 0.01 24.22 ELD_Q481V 0.03 0.01 0.03 0.06 0.02 1.34 ELD_Q481W 0.01 0.01 0.01 0.04 0.03 0.32 ELD_Q481Y 0.04 0.01 0.02 0.05 0.02 1.93 ELD_S418D 1.06 0.03 0.08 0.16 0.08 16.19 ELD_N476D 1.35 0.03 0.02 0.10 0.06 35.85 ELD_I479T 1.06 0.01 0.11 0.03 0.09 21.71 ELD_Q481E 1.39 0.02 0.07 0.13 0.07 27.18 ELD_N527D 0.92 0.11 0.09 0.32 0.06 7.18 ELD_Q531R 1.13 0.18 0.12 0.24 0.16 6.48 ELD_R416D 0.87 0.02 0.23 0.05 0.06 11.81 ELD_R416E 1.23 0.03 0.80 0.10 0.12 5.41 ELD_R416N 1.33 0.08 1.10 0.46 0.14 3.59 ELD_R416S 1.02 0.09 0.69 0.84 0.20 3.00 ELD_K525A 1.18 0.02 0.13 0.11 0.15 16.33 ELD_K525E 1.05 0.01 0.05 0.09 0.09 26.58 ELD_K525G 1.02 0.03 0.14 0.06 0.13 13.98 ELD_K525S 1.28 0.07 0.21 0.17 0.16 10.07 GFP 0.00 0.01 0.02 0.02 0.06 N/A GFP 0.00 0.01 0.01 0.05 0.06 N/A

Table 13B

AAVS1 OT1 OT2 OT3 OT4 ratio original 47.96 4.32 2.12 1.05 0.84 5.76 original 46.45 4.34 1.84 1.11 1.06 5.57 original 45.83 3.15 1.28 0.77 0.92 7.49 original 47.63 4.30 1.67 0.99 1.03 5.97 Half dose 30.79 1.21 0.38 0.19 0.18 15.72 Half dose 31.59 1.19 0.23 0.28 0.18 16.78 Half dose 29.49 0.76 0.35 0.21 0.16 19.93 Half dose 28.04 0.85 0.31 0.20 0.17 18.41 KKR_N527A 18.75 0.77 0.48 0.29 0.43 9.48 KKR_N527C 9.00 0.30 0.39 0.17 0.21 8.43 KKR_N527D 46.36 0.77 0.48 0.61 0.21 22.42 KKR_N527E 24.41 0.65 0.66 0.25 0.26 13.36 KKR_N527F 3.53 0.14 0.21 0.06 0.06 7.42 KKR_N527G 26.10 1.25 0.46 0.54 0.45 9.69 KKR_N527H 16.67 0.89 0.99 0.40 0.31 6.42 KKR_N527I 0.98 0.03 0.07 0.03 0.02 6.78 KKR_N527K 18.96 0.77 0.77 0.40 0.34 8.32 KKR_N527L N/A N/A N/A N/A N/A N/A KKR_N527M 5.67 0.22 0.43 0.20 0.18 5.48 KKR_N527P 15.11 0.10 0.10 0.13 0.05 39.87 KKR_N527Q 17.36 1.69 1.19 0.40 0.34 4.79 KKR_N527R N/A N/A N/A N/A N/A N/A KKR_N527S 20.83 0.84 0.44 0.49 0.31 10.04 KKR_N527T N/A N/A N/A N/A N/A N/A KKR_N527V 1.40 0.04 0.05 0.07 0.02 7.59 KKR_N527W N/A N/A N/A N/A N/A N/A KKR_N527Y N/A N/A N/A N/A N/A N/A KKR_Q481A 48.27 0.15 0.05 0.03 0.09 150.70 KKR_Q481C 53.52 1.44 0.15 0.04 0.28 27.84 KKR_Q481D 60.01 1.15 0.07 0.10 0.30 37.18 KKR_Q481E 56.82 0.18 0.04 0.03 0.08 167.29 KKR_Q481F 0.88 0.05 0.04 0.04 0.05 5.12 KKR_Q481G 23.60 2.40 0.55 0.25 0.31 6.72 KKR_Q481H 55.88 2.69 0.15 0.51 2.74 9.18 KKR_Q481I 2.97 0.03 0.05 0.04 0.02 21.98 KKR_Q481K 2.17 0.03 0.03 0.02 0.06 16.91 KKR_Q481L 6.95 0.03 0.01 0.03 0.03 66.91 KKR_Q481M 21.59 0.06 0.04 0.06 0.03 116.95 KKR_Q481N 38.65 0.50 0.09 0.12 0.22 41.46 KKR_Q481P 4.78 0.03 0.07 0.02 0.03 31.55 KKR_Q481R 7.85 0.05 0.04 0.05 0.02 47.90 KKR_Q481S 47.95 0.29 0.07 0.03 0.11 93.90 KKR_Q481T 35.61 0.06 0.06 0.04 0.05 170.00 KKR_Q481V 2.27 0.02 0.03 0.07 0.03 15.14 KKR_Q481W 0.11 0.03 0.02 0.04 0.01 1.10 KKR_Q481Y 12.71 0.22 0.03 0.09 0.11 28.31 KKR_S418D 49.74 0.26 0.10 0.08 0.13 89.43 KKR_N476D 27.43 0.03 0.02 0.07 0.03 166.00 KKR_I479T 51.62 0.29 0.13 0.25 0.12 65.91 KKR_Q481E 52.15 0.07 0.09 0.08 0.09 158.02 KKR_N527D 46.85 1.06 0.60 0.70 0.85 14.62 KKR_Q531R 43.62 0.65 0.79 0.41 0.27 20.59 KKR_R416D 47.19 0.14 0.03 0.11 0.20 97.96 KKR_R416E 56.98 0.55 0.04 0.04 0.78 40.63 KKR_R416N 60.68 1.19 0.18 0.28 1.20 21.34 KKR_R416S 44.42 1.37 0.21 0.18 0.51 19.53 KKR_K525A 49.24 0.19 0.03 0.07 0.14 112.49 KKR_K525E 42.36 0.06 0.06 0.06 0.07 163.51 KKR_K525G 44.05 0.47 0.09 0.07 0.27 48.61 KKR_K525S 50.87 0.44 0.08 0.16 0.29 52.53 GFP 0.01 0.02 0.03 0.02 0.05 N/A GFP 0.01 0.03 0.02 0.05 0.06 N/A original 1.02 1.07 1.23 1.08 0.87 0.93 original 0.99 1.08 1.06 1.13 1.10 0.90 original 0.98 0.78 0.74 0.79 0.96 1.21 original 1.01 1.07 0.97 1.01 1.07 0.96 Half dose 0.66 0.30 0.22 0.19 0.19 2.54 Half dose 0.67 0.30 0.13 0.29 0.19 2.71 Half dose 0.63 0.19 0.20 0.21 0.17 3.22 Half dose 0.60 0.21 0.18 0.20 0.17 2.97 KKR_N527A 0.40 0.19 0.28 0.30 0.45 1.53 KKR_N527C 0.19 0.07 0.23 0.17 0.21 1.36 KKR_N527D 0.99 0.19 0.28 0.62 0.22 3.62 KKR_N527E 0.52 0.16 0.38 0.26 0.27 2.16 KKR_N527F 0.08 0.04 0.12 0.06 0.07 1.20 KKR_N527G 0.56 0.31 0.27 0.55 0.47 1.56 KKR_N527H 0.35 0.22 0.58 0.40 0.33 1.04 KKR_N527I 0.02 0.01 0.04 0.03 0.02 1.09 KKR_N527K 0.40 0.19 0.45 0.41 0.36 1.34 KKR_N527L N/A N/A N/A N/A N/A N/A KKR_N527M 0.12 0.06 0.25 0.20 0.19 0.88 KKR_N527P 0.32 0.03 0.06 0.13 0.05 6.44 KKR_N527Q 0.37 0.42 0.69 0.41 0.36 0.77 KKR_N527R N/A N/A N/A N/A N/A N/A KKR_N527S 0.44 0.21 0.25 0.50 0.32 1.62 KKR_N527T N/A N/A N/A N/A N/A N/A KKR_N527V 0.03 0.01 0.03 0.07 0.02 1.23 KKR_N527W N/A N/A N/A N/A N/A N/A KKR_N527Y N/A N/A N/A N/A N/A N/A KKR_Q481A 1.03 0.04 0.03 0.03 0.09 24.33 KKR_Q481C 1.14 0.36 0.09 0.04 0.30 4.49 KKR_Q481D 1.28 0.29 0.04 0.10 0.31 6.00 KKR_Q481E 1.21 0.05 0.02 0.03 0.08 27.00 KKR_Q481F 0.02 0.01 0.02 0.04 0.05 0.83 KKR_Q481G 0.50 0.60 0.32 0.26 0.33 1.08 KKR_Q481H 1.19 0.67 0.09 0.52 2.85 1.48 KKR_Q481I 0.06 0.01 0.03 0.04 0.02 3.55 KKR_Q481K 0.05 0.01 0.02 0.02 0.06 2.73 KKR_Q481L 0.15 0.01 0.01 0.03 0.03 10.80 KKR_Q481M 0.46 0.01 0.02 0.06 0.03 18.88 KKR_Q481N 0.82 0.12 0.05 0.12 0.23 6.69 KKR_Q481P 0.10 0.01 0.04 0.02 0.03 5.09 KKR_Q481R 0.17 0.01 0.02 0.05 0.03 7.73 KKR_Q481S 1.02 0.07 0.04 0.03 0.11 15.16 KKR_Q481T 0.76 0.01 0.04 0.04 0.05 27.44 KKR_Q481V 0.05 0.01 0.02 0.07 0.03 2.44 KKR_Q481W 0.00 0.01 0.01 0.04 0.01 0.18 KKR_Q481Y 0.27 0.05 0.02 0.09 0.11 4.57 KKR_S418D 1.06 0.06 0.06 0.08 0.13 14.44 KKR_N476D 0.58 0.01 0.01 0.08 0.04 26.80 KKR_I479T 1.10 0.07 0.08 0.25 0.12 10.64 KKR_Q481E 1.11 0.02 0.05 0.08 0.09 25.51 KKR_N527D 1.00 0.26 0.35 0.71 0.88 2.36 KKR_Q531R 0.93 0.16 0.46 0.42 0.28 3.32 KKR_R416D 1.00 0.03 0.02 0.11 0.21 15.81 KKR_R416E 1.21 0.14 0.02 0.04 0.81 6.56 KKR_R416N 1.29 0.29 0.10 0.28 1.25 3.44 KKR_R416S 0.95 0.34 0.12 0.19 0.53 3.15 KKR_K525A 1.05 0.05 0.02 0.07 0.15 18.16 KKR_K525E 0.90 0.02 0.03 0.06 0.08 26.39 KKR_K525G 0.94 0.12 0.05 0.08 0.28 7.85 KKR_K525S 1.08 0.11 0.05 0.16 0.30 8.48 GFP 0.00 0.01 0.02 0.02 0.06 N/A GFP 0.00 0.01 0.01 0.05 0.06 N/A

Table 13C

AAVS1 OT1 OT2 OT3 OT4 ratio original 47.96 4.32 2.12 1.05 0.84 5.76 original 46.45 4.34 1.84 1.11 1.06 5.57 original 45.83 3.15 1.28 0.77 0.92 7.49 original 47.63 4.30 1.67 0.99 1.03 5.97 Half dose 30.79 1.21 0.38 0.19 0.18 15.72 Half dose 31.59 1.19 0.23 0.28 0.18 16.78 Half dose 29.49 0.76 0.35 0.21 0.16 19.93 Half dose 28.04 0.85 0.31 0.20 0.17 18.41 ELD_KKR_N527A 8.62 0.25 0.14 0.13 0.17 12.54 ELD_KKR_N527C 3.27 0.10 0.08 0.04 0.09 10.70 ELD_KKR_N527D 34.90 0.05 0.04 0.07 0.07 155.15 ELD_KKR_N527E 4.64 0.03 0.03 0.05 0.06 26.18 ELD_KKR_N527F 0.68 0.03 0.07 0.05 0.04 3.54 ELD_KKR_N527G 12.78 0.30 0.08 0.40 0.34 11.37 ELD_KKR_N527H 7.94 0.58 0.68 0.31 0.22 4.44 ELD_KKR_N527I 0.02 0.03 0.03 0.01 0.06 0.13 ELD_KKR_N527K 6.75 0.36 0.29 0.29 0.12 6.35 ELD_KKR_N527L 3.18 0.13 0.10 0.15 0.06 7.23 ELD_KKR_N527M 1.31 0.07 0.14 0.09 0.09 3.29 ELD_KKR_N527P 10.03 0.04 0.04 0.06 0.06 48.92 ELD_KKR_N527Q 9.15 0.90 0.82 0.33 0.20 4.07 ELD_KKR_N527R 16.97 1.78 1.21 0.54 0.35 4.37 ELD_KKR_N527S 9.71 0.12 0.14 0.15 0.16 16.85 ELD_KKR_N527T 26.89 0.14 0.08 0.16 0.11 55.91 ELD_KKR_N527V 1.39 0.30 0.03 0.09 0.10 2.67 ELD_KKR_N527W 1.86 0.06 0.05 0.05 0.07 8.21 ELD_KKR_N527Y 2.63 0.09 0.10 0.09 0.09 7.19 ELD_KKR_Q481A 86.30 0.02 0.04 0.04 0.09 462.88 ELD_KKR_Q481C 66.07 0.05 0.03 0.05 0.09 300.25 ELD_KKR_Q481D 90.46 0.12 0.05 0.10 0.16 214.39 ELD_KKR_Q481E 57.02 0.04 0.03 0.04 0.05 343.26 ELD_KKR_Q481F 0.02 0.04 0.02 0.08 0.05 0.09 ELD_KKR_Q481G 18.06 0.34 0.13 0.18 0.12 23.46 ELD_KKR_Q481H 84.29 22.17 0.65 4.71 1.07 2.95 ELD_KKR_Q481I 0.05 0.03 0.01 0.02 0.06 0.47 ELD_KKR_Q481K 3.36 0.03 0.05 0.02 0.10 17.13 ELD_KKR_Q481L 0.12 0.03 0.01 0.03 0.06 0.96 ELD_KKR_Q481M 1.92 0.03 0.03 0.09 0.08 8.29 ELD_KKR_Q481N 29.35 0.20 0.05 0.13 0.09 62.01 ELD_KKR_Q481P 0.45 0.03 0.03 0.08 0.10 1.82 ELD_KKR_Q481R 3.75 0.03 0.04 0.03 0.03 28.89 ELD_KKR_Q481S 54.96 0.02 0.04 0.07 0.05 308.59 ELD_KKR_Q481T 33.31 0.02 0.02 0.00 0.07 290.95 ELD_KKR_Q481V 0.11 0.03 0.01 0.03 0.09 0.67 ELD_KKR_Q481W 0.01 0.02 0.04 0.04 0.07 0.06 ELD_KKR_Q481Y 0.40 0.03 0.01 0.02 0.05 3.52 ELD_KKR_S418D 40.13 0.03 0.03 0.03 0.06 278.01 ELD_KKR_N476D 5.72 0.03 0.04 0.02 0.02 53.02 ELD_KKR_I479T 45.10 0.04 0.04 0.06 0.07 214.05 ELD_KKR_Q481E 68.22 0.02 0.04 0.09 0.07 308.56 ELD_KKR_N527D 29.60 0.05 0.04 0.14 0.08 98.76 ELD_KKR_Q531R 46.16 0.20 0.10 0.09 0.09 95.03 ELD_KKR_R416D 54.14 0.03 0.01 0.04 0.10 298.38 ELD_KKR_R416E 79.47 0.13 0.16 0.12 0.09 157.31 ELD_KKR_R416N 80.25 0.32 0.23 0.43 0.26 64.21 ELD_KKR_R416S 51.99 0.25 0.16 0.20 0.12 72.26 ELD_KKR_K525A 56.83 0.03 0.01 0.04 0.03 508.42 ELD_KKR_K525E 19.36 0.03 0.02 0.05 0.07 114.28 ELD_KKR_K525G 46.19 0.03 0.04 0.09 0.08 186.72 ELD_KKR_K525S 68.60 0.03 0.07 0.05 0.08 301.52 GFP 0.01 0.02 0.03 0.02 0.05 N/A GFP 0.01 0.03 0.02 0.05 0.06 N/A original 1.02 1.07 1.23 1.08 0.87 0.93 original 0.99 1.08 1.06 1.13 1.10 0.90 original 0.98 0.78 0.74 0.79 0.96 1.21 original 1.01 1.07 0.97 1.01 1.07 0.96 Half dose 0.66 0.30 0.22 0.19 0.19 2.54 Half dose 0.67 0.30 0.13 0.29 0.19 2.71 Half dose 0.63 0.19 0.20 0.21 0.17 3.22 Half dose 0.60 0.21 0.18 0.20 0.17 2.97 ELD_KKR_N527A 0.18 0.06 0.08 0.14 0.17 2.02 ELD_KKR_N527C 0.07 0.02 0.04 0.04 0.10 1.73 ELD_KKR_N527D 0.74 0.01 0.02 0.07 0.07 25.05 ELD_KKR_N527E 0.10 0.01 0.02 0.05 0.06 4.23 ELD_KKR_N527F 0.01 0.01 0.04 0.05 0.04 0.57 ELD_KKR_N527G 0.27 0.08 0.05 0.40 0.35 1.84 ELD_KKR_N527H 0.17 0.14 0.40 0.31 0.23 0.72 ELD_KKR_N527I 0.00 0.01 0.02 0.01 0.07 0.02 ELD_KKR_N527K 0.14 0.09 0.17 0.29 0.13 1.03 ELD_KKR_N527L 0.07 0.03 0.06 0.15 0.06 1.17 ELD_KKR_N527M 0.03 0.02 0.08 0.10 0.10 0.53 ELD_KKR_N527P 0.21 0.01 0.03 0.07 0.06 7.90 ELD_KKR_N527Q 0.19 0.22 0.48 0.34 0.21 0.66 ELD_KKR_N527R 0.36 0.44 0.70 0.55 0.36 0.71 ELD_KKR_N527S 0.21 0.03 0.08 0.15 0.17 2.72 ELD_KKR_N527T 0.57 0.03 0.04 0.17 0.11 9.02 ELD_KKR_N527V 0.03 0.07 0.02 0.09 0.10 0.43 ELD_KKR_N527W 0.04 0.02 0.03 0.05 0.07 1.33 ELD_KKR_N527Y 0.06 0.02 0.06 0.09 0.10 1.16 ELD_KKR_Q481A 1.84 0.00 0.02 0.04 0.09 74.72 ELD_KKR_Q481C 1.41 0.01 0.02 0.05 0.10 48.47 ELD_KKR_Q481D 1.93 0.03 0.03 0.10 0.16 34.61 ELD_KKR_Q481E 1.21 0.01 0.02 0.04 0.05 55.41 ELD_KKR_Q481F 0.00 0.01 0.01 0.08 0.05 0.01 ELD_KKR_Q481G 0.38 0.08 0.07 0.18 0.13 3.79 ELD_KKR_Q481H 1.79 5.51 0.38 4.81 1.12 0.48 ELD_KKR_Q481I 0.00 0.01 0.00 0.02 0.06 0.08 ELD_KKR_Q481K 0.07 0.01 0.03 0.02 0.10 2.77 ELD_KKR_Q481L 0.00 0.01 0.01 0.03 0.06 0.16 ELD_KKR_Q481M 0.04 0.01 0.02 0.09 0.09 1.34 ELD_KKR_Q481N 0.62 0.05 0.03 0.13 0.09 10.01 ELD_KKR_Q481P 0.01 0.01 0.02 0.08 0.11 0.29 ELD_KKR_Q481R 0.08 0.01 0.02 0.03 0.03 4.66 ELD_KKR_Q481S 1.17 0.00 0.02 0.07 0.05 49.81 ELD_KKR_Q481T 0.71 0.01 0.01 0.00 0.07 46.97 ELD_KKR_Q481V 0.00 0.01 0.01 0.03 0.09 0.11 ELD_KKR_Q481W 0.00 0.00 0.02 0.04 0.08 0.01 ELD_KKR_Q481Y 0.01 0.01 0.01 0.02 0.05 0.57 ELD_KKR_S418D 0.85 0.01 0.02 0.03 0.06 44.88 ELD_KKR_N476D 0.12 0.01 0.02 0.02 0.02 8.56 ELD_KKR_I479T 0.96 0.01 0.02 0.06 0.07 34.55 ELD_KKR_Q481E 1.45 0.00 0.02 0.09 0.08 49.81 ELD_KKR_N527D 0.63 0.01 0.02 0.14 0.08 15.94 ELD_KKR_Q531R 0.98 0.05 0.06 0.09 0.09 15.34 ELD_KKR_R416D 1.15 0.01 0.01 0.04 0.10 48.17 ELD_KKR_R416E 1.69 0.03 0.09 0.12 0.09 25.39 ELD_KKR_R416N 1.71 0.08 0.13 0.44 0.28 10.37 ELD_KKR_R416S 1.11 0.06 0.09 0.20 0.12 11.66 ELD_KKR_K525A 1.21 0.01 0.01 0.04 0.03 82.07 ELD_KKR_K525E 0.41 0.01 0.01 0.05 0.08 18.45 ELD_KKR_K525G 0.98 0.01 0.03 0.09 0.08 30.14 ELD_KKR_K525S 1.46 0.01 0.04 0.05 0.08 48.67 GFP 0.00 0.01 0.02 0.02 0.06 N/A GFP 0.00 0.01 0.01 0.05 0.06 N/A

[0241] Tables 14A-14C show the results for the indicated mutants.

Table 14A

AAVS1 OT1 OT2 OT3 OT4 Ratio original 42.55 7.68 3.88 2.05 2.94 2.57 original 41.57 6.44 3.50 1.64 2.72 2.91 original 41.51 6.14 2.93 1.36 2.25 3.27 original 38.53 6.24 3.14 1.32 2.35 2.95 Half dose 32.45 2.46 1.00 0.46 0.63 7.14 Half dose 31.98 2.41 1.02 0.53 0.51 7.16 Half dose 27.34 1.29 0.45 0.19 0.34 12.05 Half dose 30.04 2.06 0.91 0.36 0.61 7.63 ELD_N476A 43.61 3.28 1.86 1.50 1.15 5.60 ELD_N476C 46.78 3.74 1.92 1.35 0.97 5.86 ELD_N476D 67.25 0.20 0.09 0.18 0.16 106.44 ELD_N476E 65.43 0.07 0.06 0.08 0.07 228.99 ELD_N476F 18.10 1.22 1.12 0.77 0.39 5.16 ELD_N476G 62.70 3.13 0.60 0.56 0.41 13.33 ELD_N476H 41.57 4.94 3.07 1.81 2.48 3.38 ELD_N476I 30.48 0.21 0.55 0.29 0.25 23.42 ELD_N476K 32.00 3.78 1.00 1.62 0.46 4.67 ELD_N476L 12.47 0.13 0.15 0.14 0.10 23.76 ELD_N476M 27.91 2.27 1.14 0.73 0.81 5.64 ELD_N476P 40.20 6.83 3.21 1.40 2.79 2.82 ELD_N476Q 38.61 2.08 0.77 0.80 0.51 9.28 ELD_N476R 40.17 6.10 4.87 2.55 1.41 2.69 ELD_N476S 54.42 9.71 4.38 2.67 2.74 2.79 ELD_N476T 53.11 5.19 2.35 1.77 1.75 4.80 ELD_N476V 34.78 0.49 0.53 0.36 0.22 21.82 ELD_N476W 14.91 0.49 1.13 0.39 0.22 6.66 ELD_N476Y 26.63 2.53 1.63 0.73 1.41 4.24 ELD_Q531A 25.87 1.96 0.48 0.37 0.70 7.39 ELD_Q531C 12.57 0.16 0.11 0.21 0.09 21.86 ELD_Q531D 4.48 0.05 0.01 0.03 0.05 30.17 ELD_Q531E 29.54 0.06 0.02 0.06 0.05 150.94 ELD_Q531F 4.09 0.02 0.02 0.04 0.09 23.42 ELD_Q531G 7.39 0.56 0.20 0.27 0.45 5.00 ELD_Q531H 47.18 10.36 3.04 2.12 6.48 2.14 ELD_Q531I 25.66 0.05 0.02 0.04 0.06 141.34 ELD_Q531K 42.12 7.43 4.18 1.80 6.55 2.11 ELD_Q531L 12.77 0.03 0.04 0.05 0.04 76.78 ELD_Q531M 30.65 0.35 0.40 0.31 0.19 24.46 ELD_Q531N 12.98 0.69 0.24 0.28 0.46 7.78 ELD_Q531P 5.30 0.05 0.03 0.08 0.05 26.25 ELD_Q531R 48.64 1.11 0.43 0.26 0.46 21.54 ELD_Q531S 14.86 0.39 0.17 0.20 0.33 13.64 ELD_Q531T 37.95 0.40 0.37 0.30 0.33 27.16 ELD_Q531V 33.24 0.11 0.12 0.12 0.02 87.92 ELD_Q531W 2.82 0.05 0.02 0.05 0.11 12.47 ELD_Q531Y 18.15 0.04 0.02 0.04 0.03 133.53 ELD_S418D 42.20 0.10 0.24 0.31 0.14 53.28 ELD_N476D 57.06 0.07 0.09 0.09 0.06 184.93 ELD_I479T 42.10 0.04 0.15 0.02 0.09 137.07 ELD_Q481E 52.80 0.04 0.04 0.19 0.09 145.99 ELD_N527D 35.32 0.60 0.18 0.24 0.08 32.04 ELD_Q531R 43.15 0.74 0.25 0.20 0.29 29.03 ELD_R416N 58.18 0.48 2.31 0.46 0.14 17.21 ELD_R416D 36.27 0.11 0.59 0.08 0.04 44.30 ELD_R416E 55.41 0.17 2.40 0.23 0.09 19.15 ELD_R416S 39.03 0.37 1.26 0.47 0.27 16.49 ELD_K525A 46.87 0.27 0.24 0.17 0.06 63.48 ELD_K525E 40.21 0.05 0.05 0.10 0.04 167.71 ELD_K525G 35.64 0.21 0.15 0.08 0.06 70.09 ELD_K525S 49.97 0.23 0.28 0.31 0.20 48.28 ELD_Q481A 55.82 0.04 0.05 0.16 0.05 191.70 ELD_Q481D 71.61 0.83 0.13 1.15 0.38 28.73 ELD_Q481C 50.24 0.17 0.16 0.23 0.20 66.34 ELD_Q481S 40.58 0.09 0.08 0.07 0.10 118.21 ELD_S418E 53.03 0.09 0.15 0.06 0.16 113.62 ELD_R422H 36.38 0.77 1.01 0.48 0.22 14.68 ELD_I479Q 58.23 0.03 0.03 0.05 0.06 337.38 GFP 0.09 0.03 0.01 N/A 0.04 N/A GFP 0.08 0.02 0.03 0.01 0.02 N/A

Table 14B

AAVS1 OT1 OT2 OT3 OT4 Ratio original 42.55 7.68 3.88 2.05 2.94 2.57 original 41.57 6.44 3.50 1.64 2.72 2.91 original 41.51 6.14 2.93 1.36 2.25 3.27 original 38.53 6.24 3.14 1.32 2.35 2.95 Half dose 32.45 2.46 1.00 0.46 0.63 7.14 Half dose 31.98 2.41 1.02 0.53 0.51 7.16 Half dose 27.34 1.29 0.45 0.19 0.34 12.05 Half dose 30.04 2.06 0.91 0.36 0.61 7.63 KKR_N476A 41.76 3.81 1.60 1.20 1.97 4.87 KKR_N476C 44.42 3.25 1.35 0.98 1.44 6.32 KKR_N476D 31.25 0.10 0.16 0.06 0.04 88.08 KKR_N476E 37.65 0.07 0.04 0.04 0.03 220.42 KKR_N476F 15.09 1.21 0.60 0.29 0.38 6.09 KKR_N476G 45.33 2.34 0.59 1.16 0.85 9.19 KKR_N476H 41.14 7.08 2.71 1.20 2.06 3.15 KKR_N476I 33.90 0.59 0.30 0.16 0.30 25.33 KKR_N476K 41.23 2.04 0.87 1.11 0.88 8.40 KKR_N476L 11.80 0.23 0.13 0.09 0.12 20.83 KKR_N476M 30.41 2.34 0.79 0.75 0.77 6.53 KKR_N476P 33.04 3.11 1.21 1.38 2.72 3.92 KKR_N476Q 38.61 1.03 0.42 0.50 0.58 15.21 KKR_N476R 44.34 6.29 3.71 1.38 3.07 3.07 KKR_N476S 49.87 9.00 3.00 2.07 4.76 2.65 KKR_N476T 48.39 4.98 2.03 1.55 2.71 4.29 KKR_N476V 39.89 0.55 0.23 0.22 0.46 27.20 KKR_N476W 13.62 1.05 1.64 0.36 0.38 3.97 KKR_N476Y 26.14 2.86 1.46 1.06 0.50 4.45 KKR_Q531A 33.77 3.29 2.47 0.89 0.94 4.45 KKR_Q531C 13.20 0.33 0.21 0.28 0.12 14.06 KKR_Q531D 6.11 0.05 0.02 0.04 0.08 31.32 KKR_Q531E 18.16 0.32 0.30 0.57 0.13 13.71 KKR_Q531F 4.27 0.04 0.04 0.04 0.06 24.16 KKR_Q531G 7.06 0.63 1.39 0.35 0.25 2.70 KKR_Q531H 38.76 6.91 1.57 0.91 2.35 3.30 KKR_Q531I 32.37 0.15 0.03 0.08 0.07 97.56 KKR_Q531K 38.59 9.56 6.05 1.62 1.05 2.11 KKR_Q531L 30.94 0.06 0.02 0.05 0.04 181.72 KKR_Q531M 39.71 1.07 0.57 0.46 0.37 16.08 KKR_Q531N 33.22 5.31 1.85 0.84 1.27 3.59 KKR_Q531P 3.89 0.08 0.09 0.06 0.05 13.73 KKR_Q531R 41.38 1.89 1.96 1.07 0.70 7.37 KKR_Q531S 20.10 1.59 0.79 0.52 0.35 6.18 KKR_Q531T 38.72 2.20 0.31 0.37 0.43 11.69 KKR_Q531V 33.72 0.81 0.05 0.07 0.18 30.51 KKR_Q531W 3.69 0.13 0.06 0.05 0.06 12.37 KKR_Q531Y 6.03 0.23 0.06 0.08 0.06 14.16 KKR_S418D 45.54 0.37 0.11 0.11 0.22 56.33 KKR_N476D 25.28 0.07 0.05 0.04 0.06 118.80 KKR_I479T 45.08 0.54 0.17 0.34 0.05 41.10 KKR_Q481E 46.92 0.28 0.03 0.06 0.10 97.21 KKR_N527D 34.74 1.03 0.51 0.56 0.40 13.88 KKR_Q531R 37.42 1.37 1.65 0.72 0.50 8.84 KKR_R416N 52.89 2.05 0.08 0.16 1.09 15.63 KKR_R416D 45.13 0.28 0.04 0.07 0.20 75.06 KKR_R416E 51.18 0.51 0.03 0.08 0.78 36.52 KKR_R416S 42.07 2.94 0.35 0.20 0.98 9.42 KKR_K525A 43.73 0.59 0.08 0.09 0.16 47.64 KKR_K525E 40.94 0.11 0.03 0.03 0.10 150.39 KKR_K525G 37.87 0.70 0.06 0.11 0.29 32.75 KKR_K525S 40.53 0.64 0.07 0.05 0.26 40.01 KKR_Q481A 48.58 0.07 0.03 0.04 0.08 212.03 KKR_Q481D 51.77 2.24 0.04 0.09 0.40 18.71 KKR_Q481C 40.58 1.50 0.10 0.07 0.31 20.44 KKR_Q481S 41.92 0.40 0.04 0.08 0.07 71.54 KKR_S418E 51.17 0.19 0.06 0.07 0.07 129.17 KKR_R422H 41.25 1.53 0.24 0.29 0.75 14.62 KKR_I479Q 47.54 1.66 0.95 0.53 0.09 14.68 GFP 0.09 0.03 0.01 N/A 0.04 N/A GFP 0.08 0.02 0.03 0.01 0.02 N/A

Table 14C

AAVS1 OT1 OT2 OT3 OT4 ratio original 42.55 7.68 3.88 2.05 2.94 2.57 original 41.57 6.44 3.50 1.64 2.72 2.91 original 41.51 6.14 2.93 1.36 2.25 3.27 original 38.53 6.24 3.14 1.32 2.35 2.95 Half dose 32.45 2.46 1.00 0.46 0.63 7.14 Half dose 31.98 2.41 1.02 0.53 0.51 7.16 Half dose 27.34 1.29 0.45 0.19 0.34 12.05 Half dose 30.04 2.06 0.91 0.36 0.61 7.63 ELD_KKR_N476A 38.74 0.88 0.22 0.75 0.33 17.82 ELD_KKR_N476C 46.45 0.54 0.56 0.43 0.24 26.33 ELD_KKR_N476D 8.21 0.02 0.02 0.04 0.07 56.96 ELD_KKR_N476E 1.99 0.04 0.01 0.03 0.05 15.67 ELD_KKR_N476F 6.61 0.21 0.16 0.05 0.11 12.56 ELD_KKR_N476G 58.20 0.10 0.07 0.08 0.07 185.26 ELD_KKR_N476H 36.62 2.95 1.48 1.06 1.09 5.57 ELD_KKR_N476I 13.54 0.03 0.03 0.02 0.02 141.62 ELD_KKR_N476K 25.76 0.31 0.10 0.60 0.10 23.29 ELD_kkR_N476L 1.67 0.04 0.01 0.06 0.04 11.86 ELD_kkR_N476M 16.01 0.29 0.12 0.17 0.12 23.13 ELD_KKR_N476P 28.84 1.82 0.61 0.74 1.21 6.59 ELD_KKR_N476Q 29.68 0.06 0.06 0.09 0.12 90.88 ELD_KKR_N476R 36.35 3.33 3.04 1.67 0.76 4.13 ELD_KKR_N476S 55.29 8.64 2.32 2.13 3.28 3.38 ELD_KKR_N476T 55.05 1.63 0.98 0.97 1.04 11.91 ELD_KKR_N476V 24.38 0.06 0.05 0.06 0.07 101.77 ELD_KKR_N476W 4.04 0.09 0.00 0.08 0.03 19.35 ELD_KKR_N476Y 13.95 0.80 0.30 0.38 0.17 8.47 ELD_KKR_Q531A 17.74 0.53 0.20 0.18 0.22 15.68 ELD_KKR_Q531C 3.11 0.03 0.02 0.02 0.08 20.54 ELD_KKR_Q531D 0.05 0.02 0.01 0.02 0.03 0.62 ELD_KKR_Q531E 5.44 0.04 0.03 0.03 0.04 38.30 ELD_KKR_Q531F 0.19 0.03 0.00 0.04 0.05 1.77 ELD_KKR_Q531G 1.37 0.16 0.11 0.06 0.07 3.36 ELD_KKR_Q531H 42.66 7.88 1.12 1.19 3.73 3.07 ELD_KKR_Q531I 2.61 0.03 0.01 0.04 0.05 20.98 ELD_KKR_Q531K 38.46 8.02 5.20 1.54 2.03 2.29 ELD_KKR_Q531L 0.34 0.03 0.01 0.03 0.09 2.13 ELD_KKR_Q531M 17.43 0.04 0.04 0.01 0.08 100.86 ELD_KKR_Q531N 6.41 0.40 0.22 0.08 0.09 8.17 ELD_KKR_Q531P 0.15 0.03 0.02 0.03 0.04 1.18 ELD_KKR_Q531R 43.88 0.46 0.15 0.18 0.10 48.73 ELD_KKR_Q531S 6.70 0.07 0.06 0.10 0.06 22.49 ELD_KKR_Q531T 29.41 0.06 0.03 0.07 0.05 144.03 ELD_KKR_Q531V 19.78 0.05 0.01 0.01 0.09 125.77 ELD_KKR_Q531W 0.19 0.05 0.02 0.02 0.06 1.27 ELD_KKR_Q531Y 1.50 0.06 0.02 0.02 0.06 9.52 ELD_KKR_S418D 31.26 0.05 0.02 0.04 0.03 226.26 ELD_KKR_N476D 4.18 0.03 0.01 0.02 0.03 44.63 ELD_KKR_I479T 27.06 0.05 0.03 0.06 0.05 139.49 ELD_KKR_Q481E 43.46 0.04 0.02 0.04 0.03 324.28 ELD_KKR_N527D 21.32 0.03 0.02 0.08 0.02 142.33 ELD_KKR_Q531R 35.54 0.16 0.07 0.08 0.06 94.23 ELD_KKR_R416N 68.24 0.22 0.11 0.16 0.14 106.90 ELD_KKR_R416D 38.22 0.05 0.04 0.03 0.03 259.98 ELD_KKR_R416E 69.86 0.05 0.09 0.08 0.12 207.67 ELD_KKR_R416S 37.02 0.33 0.11 0.12 0.11 54.52 ELD_KKR_K525A 44.65 0.04 0.02 0.02 0.10 262.08 ELD_KKR_K525E 15.13 0.02 0.03 0.03 0.05 108.50 ELD_KKR_K525G 31.56 0.04 0.03 0.02 0.07 199.27 ELD_KKR_K525S 46.57 0.02 0.02 0.04 0.04 405.87 ELD_KKR_Q481A 80.87 0.04 0.00 0.06 0.04 567.11 ELD_KKR_Q481D 85.06 0.25 0.03 0.10 0.14 162.56 ELD_KKR_Q481C 50.45 0.02 0.04 0.06 0.03 333.81 ELD_KKR_Q481S 39.61 0.04 0.00 0.03 0.05 342.28 ELD_KKR_S418E 46.25 0.04 0.03 0.01 0.09 273.38 ELD_KKR_R422H 37.06 0.27 0.06 0.07 0.12 72.00 ELD_KKR_I479Q 55.27 0.04 0.00 0.04 0.08 343.98 GFP 0.09 0.03 0.01 N/A 0.04 N/A GFP 0.08 0.02 0.03 0.01 0.02 N/A

[0242] Tables 15A-15C show the results of duplicate experiments with the indicated mutants.

Table 15A

AAVS1 OT1 OT2 OT3 OT4 ratio original 66.27 11.83 3.46 4.41 2.67 2.96 original 66.73 11.97 3.83 4.11 3.12 2.90 original 62.81 10.97 3.22 3.14 2.45 3.18 Half dose 48.04 3.58 0.80 0.88 0.39 8.51 Half dose 43.89 3.26 0.75 0.78 0.66 8.05 Half dose 45.19 2.87 0.64 0.97 N/A 10.07 ELD_T419D 78.79 11.23 3.69 4.89 3.59 3.37 ELD_Q420E 32.41 3.86 1.44 1.99 1.16 3.84 ELD_Q420R 29.10 2.19 1.14 1.88 0.59 5.01 ELD_E425D 0.23 0.04 0.04 0.03 0.02 1.85 ELD_S446A 73.08 21.06 7.32 5.85 3.84 1.92 ELD_S446D 75.31 0.66 0.39 0.23 0.05 56.91 ELD_S446R 72.76 2.75 8.84 5.34 0.22 4.24 ELD_S446T 33.26 2.23 1.63 1.07 0.36 6.29 ELD_R447A 19.26 0.20 0.12 0.18 0.06 34.22 ELD_R447E 9.96 0.06 0.03 0.02 0.03 69.17 ELD_R447Q 21.02 0.38 0.51 0.37 0.06 15.96 ELD_A470D 0.06 0.09 0.01 0.04 0.03 0.34 ELD_A470G 14.66 0.21 0.14 0.42 0.06 17.59 ELD_Y471E 0.34 0.02 0.01 0.03 0.03 3.65 ELD_Y471F 47.88 0.10 0.17 0.45 0.03 63.86 ELD_S472A 74.79 14.00 7.01 6.05 3.59 2.44 ELD_S472D 71.65 0.26 1.22 0.62 0.12 32.28 ELD_S472G 51.69 5.45 4.09 3.94 1.83 3.38 ELD_Y475E 0.15 0.05 0.03 0.03 0.04 1.04 ELD_Y475F 39.94 7.76 1.80 2.48 2.23 2.80 ELD_P478D 86.44 0.39 0.02 0.12 0.13 133.89 ELD_G480A 83.61 23.32 6.80 6.63 12.01 1.71 ELD_G480D 86.31 2.13 0.10 1.26 1.48 17.34 ELD_G480K 83.45 21.94 6.63 4.99 14.57 1.73 ELD_G480N 72.90 20.19 1.90 5.23 4.81 2.27 ELD_G480R 70.85 21.55 6.56 3.58 10.49 1.68 ELD_N492D 0.67 0.06 0.03 0.04 0.06 3.66 ELD_Q493E 15.78 2.79 1.01 1.47 0.97 2.53 ELD_N500D 2.15 0.06 0.04 0.02 0.02 15.13 ELD_N502D 6.77 0.87 0.19 0.63 0.27 3.46 ELD_S521A 78.63 13.46 4.39 6.26 3.30 2.87 ELD_S521D 0.52 0.12 0.03 0.03 0.05 2.34 ELD_H523E 82.23 4.72 6.32 4.82 1.28 4.80 ELD_H523K 69.86 9.95 3.49 4.30 1.94 3.55 ELD_H523S 80.18 17.34 8.96 6.98 4.84 2.10 ELD_H523V 41.49 3.56 2.12 2.32 1.08 4.58 ELD_H523Y 71.52 24.22 6.87 4.50 7.88 1.65 ELD_G526D 9.28 0.05 0.05 0.09 0.04 40.47 ELD_G526N 10.88 0.20 0.08 0.22 0.09 18.55 ELD_G526S 10.79 0.22 0.11 0.30 0.17 13.35 ELD_A530D 57.08 5.87 1.80 2.75 1.01 4.99 ELD_Q531E 48.43 0.08 0.07 0.03 0.06 207.58 ELD_N536D 4.93 0.39 0.07 0.22 0.13 6.10 ELD_N540D 17.44 2.97 1.59 1.72 1.17 2.34 ELD_N542D 82.04 26.01 8.78 6.82 5.77 1.73 ELD_N573D 36.59 6.49 2.30 2.30 1.81 2.84 ELD_N574D 7.62 0.98 0.23 0.53 0.33 3.66 ELD_N578D 23.28 2.69 1.33 1.78 0.96 3.45 ELD_S418E 77.93 0.13 0.16 0.20 0.11 130.43 ELD_S418D 69.74 0.48 0.17 0.63 0.22 46.38 ELD_N476D 74.70 0.01 0.04 0.07 0.09 352.05 ELD_I479T 60.80 0.12 0.12 0.01 0.06 201.14 ELD_Q481E 71.27 0.04 0.10 0.15 0.07 202.50 ELD_N527D 50.11 0.59 0.17 0.34 0.06 42.99 ELD_Q531R 63.19 0.91 0.14 0.28 0.20 41.47 ELD_Q481A 75.33 0.05 0.11 0.08 0.06 254.98 ELD_R416E 76.92 0.28 4.23 0.57 0.10 14.86 ELD_K525A 72.24 0.41 0.42 0.56 0.19 45.87 ELD_K525S 73.75 0.91 0.64 0.69 0.29 29.11 ELD_I479Q 77.13 0.06 0.04 0.05 0.06 373.93 GFP 0.09 0.04 0.04 0.02 0.05 N/A GFP 0.01 0.03 0.02 0.01 0.03 N/A

Table 15B

AAVS1 OT1 OT2 OT3 OT4 Ratio original 66.27 11.83 3.46 4.41 2.67 2.96 original 66.73 11.97 3.83 4.11 3.12 2.90 original 62.81 10.97 3.22 3.14 2.45 3.18 Half dose 48.04 3.58 0.80 0.88 0.39 8.51 Half dose 43.89 3.26 0.75 0.78 0.66 8.05 Half dose 45.19 2.87 0.64 0.97 N/A 10.07 KKR_T419D 75.79 14.88 7.12 6.14 4.34 2.33 KKR_Q420E 37.28 4.82 1.99 2.17 1.48 3.56 KKR_Q420R 27.41 4.09 1.16 1.21 1.28 3.54 KKR_E425D 0.23 0.04 0.04 0.04 0.02 1.59 KKR_S446A 69.60 17.74 5.75 5.60 9.07 1.82 KKR_S446D 71.48 0.11 0.03 0.04 0.05 311.16 KKR_S446R 69.10 3.80 0.35 0.33 2.39 10.05 KKR_S446T 68.15 6.99 3.88 2.40 4.04 3.94 KKR_R447A 32.43 0.01 0.02 0.03 0.06 249.14 KKR_R447E 10.88 0.03 0.01 0.03 0.03 112.41 KKR_R447Q 41.86 0.02 0.02 0.02 0.09 269.85 KKR_A470D 2.09 0.06 0.01 0.04 0.05 12.73 KKR_A470G 14.81 0.43 0.11 0.24 0.22 14.80 KKR_Y471E 0.29 0.01 0.00 0.01 0.01 10.77 KKR_Y471F 59.99 1.03 0.08 0.09 0.44 36.50 KKR_S472A 72.04 18.47 4.06 4.86 5.36 2.20 KKR_S472D 73.49 0.51 0.08 0.09 0.59 57.76 KKR_S472G 58.36 9.39 2.26 2.74 3.37 3.29 KKR_Y475E 0.08 0.01 0.02 0.02 0.01 1.43 KKR_Y475F 40.95 7.01 4.44 3.44 1.91 2.44 KKR_P478D 68.06 0.22 0.18 0.13 0.11 107.13 KKR_G480A 74.73 11.68 13.46 6.92 4.14 2.06 KKR_G480D N/A N/A N/A N/A N/A N/A KKR_G480K 74.95 14.58 4.77 5.59 9.04 2.21 KKR_G480N 72.63 13.59 11.47 6.96 1.87 2.14 KKR_G480R 72.60 13.28 9.20 5.33 6.12 2.14 KKR_N492D 0.49 0.03 0.01 0.03 0.04 4.61 KKR_Q493E 19.03 2.00 1.24 1.14 0.85 3.64 KKR_N500D 2.44 0.02 0.01 0.03 0.03 25.48 KKR_N502D 10.57 0.43 0.18 0.51 0.24 7.77 KKR_S521A 73.75 14.27 4.57 4.50 3.63 2.73 KKR_S521D 0.02 0.04 0.03 0.03 0.03 0.14 KKR_H523E 77.04 12.72 1.14 1.86 4.57 3.80 KKR_H523K 68.39 9.40 2.92 3.75 2.51 3.68 KKR_H523S 73.90 18.71 3.05 5.29 6.00 2.24 KKR_H523V 51.85 7.60 2.23 2.16 2.33 3.62 KKR_H523Y 65.36 9.66 7.27 5.25 3.16 2.58 KKR_G526D 9.91 0.10 0.03 0.11 0.05 34.03 KKR_G526N 9.13 0.48 0.08 0.20 0.36 8.18 KKR_G526S 9.33 0.23 0.04 0.26 0.12 14.26 KKR_A530D 67.48 13.32 5.25 6.01 4.12 2.35 KKR_Q531E 26.70 0.49 0.20 1.08 0.05 14.69 KKR_N536D 4.55 0.26 0.11 0.25 0.05 6.80 KKR_N540D 38.98 4.18 1.89 2.31 1.31 4.03 KKR_N542D 70.17 15.67 5.58 4.86 4.57 2.29 KKR_N573D 43.19 7.22 2.96 2.81 1.90 2.90 KKR_N574D 9.05 1.05 0.58 0.55 0.45 3.43 KKR_N578D 8.46 0.81 0.27 0.47 0.28 4.63 KKR_S418E 69.91 0.22 0.06 0.08 0.04 176.22 KKR_S418D 63.18 0.73 0.12 0.08 0.23 54.44 KKR_N476D 31.97 0.09 0.07 0.09 0.11 89.68 KKR_I479T 67.39 0.56 0.11 0.96 0.08 39.39 KKR_Q481E 67.03 0.16 0.03 0.04 0.08 213.25 KKR_N527D 49.72 1.12 0.19 0.75 N/A 24.10 KKR_Q531R 53.34 1.41 0.71 1.19 0.38 14.45 KKR_Q481A 66.84 0.14 0.04 0.05 0.08 217.35 KKR_R416E 74.69 1.79 0.06 0.11 2.17 18.11 KKR_K525A 65.23 0.90 0.07 0.13 0.37 44.44 KKR_K525S 67.51 2.22 0.20 0.15 0.64 20.95 KKR_I479Q 73.07 1.99 1.11 1.34 0.11 16.06 GFP 0.09 0.04 0.04 0.02 0.05 N/A GFP 0.01 0.03 0.02 0.01 0.03 N/A

Table 15C

AAVS1 OT1 OT2 OT3 OT4 Ratio original 66.27 11.83 3.46 4.41 2.67 2.96 original 66.73 11.97 3.83 4.11 3.12 2.90 original 62.81 10.97 3.22 3.14 2.45 3.18 Half dose 48.04 3.58 0.80 0.88 0.39 8.51 Half dose 43.89 3.26 0.75 0.78 0.66 8.05 Half dose 45.19 2.87 0.64 0.97 N/A 10.07 ELD_KKR_T419D 85.90 11.12 5.15 6.07 4.42 3.21 ELD_KKR_Q420E 13.44 0.53 0.29 0.56 0.27 8.13 ELD_KKR_Q420R 11.95 0.85 0.21 0.41 0.22 7.07 ELD_KKR_E425D 0.08 0.02 0.02 0.04 0.06 0.57 ELD_KKR_S446A 74.69 23.06 7.98 6.61 8.35 1.62 ELD_KKR_S446D N/A N/A N/A N/A N/A N/A ELD_KKR_S446R 65.40 3.05 1.55 1.67 0.40 9.79 ELD_KKR_S446T 60.85 2.91 6.14 1.43 1.80 4.96 ELD_KKR_R447A 3.05 0.01 0.01 0.02 0.01 54.51 ELD_KKR_R447E 0.11 0.01 0.01 0.03 0.04 1.15 ELD_KKR_R447Q 5.65 0.02 0.00 0.02 0.03 80.80 ELD_KKR_A470D 0.06 0.03 0.02 0.02 0.04 0.49 ELD_KKR_A470G 3.42 0.05 0.03 0.03 0.02 26.36 ELD_KKR_Y471E 0.08 0.00 0.01 0.01 0.02 2.19 ELD_KKR_Y471F 40.26 0.02 0.00 0.04 0.02 476.01 ELD_KKR_S472A 80.75 19.68 5.51 6.09 4.95 2.23 ELD_KKR_S472D 75.69 0.11 0.02 0.06 0.09 275.20 ELD_KKR_S472G 42.97 3.47 1.96 2.50 1.45 4.58 ELD_KKR_Y475E 0.04 0.00 0.01 0.02 0.04 0.59 ELD_KKR_Y475F 22.50 3.79 1.65 1.86 1.18 2.66 ELD_KKR_P478D 59.04 0.05 0.01 0.01 0.04 550.41 ELD_KKR_G480A 87.20 17.66 13.65 7.82 9.44 1.80 ELD_KKR_G480D 91.15 0.06 0.06 0.05 0.08 349.99 ELD_KKR_G480K 88.46 5.67 0.26 1.23 11.32 4.79 ELD_KKR_G480N 71.97 16.43 3.72 6.72 2.05 2.49 ELD_KKR_G480R 38.18 9.36 6.54 1.15 6.83 1.60 ELD_KKR_N492D 0.03 0.02 0.01 0.01 0.04 0.41 ELD_KKR_Q493E 5.52 0.58 0.23 0.37 0.23 3.92 ELD_KKR_N500D 0.07 0.02 0.03 0.03 0.04 0.58 ELD_KKR_N502D 1.10 0.07 0.04 0.10 0.04 4.30 ELD_KKR_S521A 81.32 12.31 3.39 6.02 2.84 3.31 ELD_KKR_S521D N/A 0.06 0.01 0.03 0.03 N/A ELD_KKR_H523E 83.69 3.33 1.05 1.37 1.10 12.21 ELD_KKR_H523K 71.56 5.67 1.65 3.19 1.01 6.21 ELD_KKR_H523S 86.20 23.40 6.54 7.59 7.24 1.93 ELD_KKR_H523V 32.38 2.32 1.17 1.35 0.48 6.09 ELD_KKR_H523Y 68.44 20.77 8.90 4.64 6.21 1.69 ELD_KKR_G526D 0.45 0.05 0.01 0.05 0.02 3.23 ELD_KKR_G526N 1.43 0.02 0.03 0.03 0.01 14.61 ELD_KKR_G526S 1.56 0.05 0.02 0.05 0.03 10.66 ELD_KKR_A530D 57.66 4.57 1.75 4.02 1.33 4.94

ELD_K448S 42.71 4.50 1.29 1.49 1.45 0.55 9.29 4.60 ELD_K469S 0.10 0.28 0.25 0.14 0.42 0.13 1.21 0.08 ELD_R487S 0.19 0.23 0.23 0.12 0.41 0.18 1.17 0.16 ELD_R495S 1.37 0.21 0.21 0.12 0.58 0.18 1.30 1.06 ELD_K497S 22.50 2.89 0.65 0.95 0.81 0.47 5.77 3.90 ELD_K506S 20.06 2.87 0.88 0.77 1.15 0.41 6.07 3.30 ELD_K516S 2.67 0.30 0.25 0.31 0.53 0.18 1.56 1.71 ELD_K525S 53.18 0.42 0.31 0.26 0.42 0.26 1.67 31.81 ELD_K529S 16.10 1.48 0.53 0.51 0.69 0.42 3.64 4.43 ELD_R534S 12.81 0.28 0.26 0.30 0.43 0.17 1.44 8.91 ELD_K559S 17.55 2.05 0.54 0.65 0.73 0.44 4.42 3.97 ELD_R569S 51.39 8.56 2.26 2.03 2.08 0.99 15.92 3.23 ELD_R570S 15.32 2.10 0.68 0.59 0.86 0.35 4.57 3.35 ELD_K571S 17.23 2.78 0.98 0.82 1.20 0.50 6.28 2.74 KKR_K387S 53.39 8.19 1.69 1.36 2.75 1.07 15.05 3.55 KKR_K393S 8.26 0.43 0.30 0.35 0.60 0.31 2.00 4.14 KKR_K394S 9.13 0.60 0.50 0.44 0.60 0.28 2.42 3.77 KKR_R398S 17.87 1.43 0.53 0.59 0.64 0.28 3.47 5.16 KKR_K400S 15.10 1.69 0.42 0.64 0.69 0.44 3.88 3.89 KKR_K402S 15.77 1.46 0.70 0.74 0.89 0.41 4.20 3.75 KKR_R416S 52.03 2.77 0.33 0.52 1.32 0.51 5.45 9.56 KKR_R422S 45.06 0.86 0.33 0.32 0.93 0.51 2.95 15.28 KKR_K427S 8.58 0.68 0.42 0.44 0.51 0.29 2.33 3.68 KKR_K434S 29.41 3.49 1.17 1.29 1.23 0.57 7.75 3.79 KKR_R439S 27.32 3.05 0.86 0.82 1.04 0.51 6.27 4.35 KKR_K441S 11.00 0.90 0.48 0.28 0.50 0.27 2.43 4.53 KKR_R447S 24.50 0.15 0.20 0.19 0.35 0.23 1.11 22.06 KKR_K448S 45.19 3.53 0.77 0.80 1.24 0.79 7.13 6.34 KKR_K469S 0.15 0.18 0.24 0.17 0.46 0.26 1.30 0.11 KKR_R487S 0.16 0.24 0.18 0.13 0.37 0.10 1.02 0.15 KKR_R495S 1.78 0.21 0.14 0.16 0.46 0.21 1.19 1.50 KKR_K497S 31.87 4.05 1.24 1.26 1.35 0.83 8.72 3.65 KKR_K506S 30.39 4.27 1.44 1.15 1.39 0.72 8.97 3.39 KKR_K516S 5.60 0.62 0.39 0.42 0.57 0.24 2.25 2.49 KKR_K525S 51.12 1.18 0.37 0.18 0.70 0.21 2.64 19.36 KKR_K529S 17.49 1.78 0.49 0.73 0.77 0.42 4.19 4.18 KKR_R534S 18.27 1.29 0.81 0.66 0.77 0.33 3.85 4.74 KKR_K559S 23.28 2.32 0.71 0.62 0.81 0.46 4.92 4.73 KKR_R569S 5.40 0.20 0.35 0.31 0.44 0.21 1.51 3.57 KKR_R570S 23.22 3.52 1.13 0.90 1.07 0.75 7.36 3.15 KKR_K571S 8.62 1.03 0.53 0.52 0.61 0.34 3.04 2.83 EGFP 0.12 0.16 0.23 0.14 0.40 0.15 0.21 0.57

Table 16B

Sample AAVS1 OT1 OT2 OT3 OT4 Total Ratio Sources 47.56 9.72 2.63 2.84 2.46 17.65 2.70 Sources 49.08 11.10 3.09 3.71 3.06 20.97 2.34 ELD_I414L 37.57 4.36 2.12 2.24 1.18 9.91 3.79 ELD_N417D 67.23 17.94 5.20 5.03 3.71 31.89 2.11 ELD_N417S 29.91 7.81 1.11 1.56 2.17 12.65 2.37 ELD_S418D 46.39 0.29 0.14 0.34 0.16 0.93 49.69 ELD_S418G 32.76 11.10 0.81 1.11 4.23 17.25 1.90 ELD_S418P 67.64 19.29 11.36 5.53 3.85 40.03 1.69 ELD_T419K 54.75 12.34 3.49 3.64 2.83 22.29 2.46 ELD_T419S 56.92 11.83 3.66 3.51 2.26 21.27 2.68 ELD_T419Y 39.15 6.93 1.83 2.27 1.74 12.76 3.07 ELD_Q420A 37.22 9.61 1.07 1.81 2.61 15.10 2.46 ELD_D421N 39.03 13.33 1.77 2.41 5.30 22.80 1.71 ELD_D421S 50.44 16.11 1.73 2.46 6.35 26.65 1.89 ELD_I423D 14.05 1.84 0.39 0.75 0.57 3.55 3.96 ELD_I423L 57.80 15.54 2.48 3.29 3.51 24.82 2.33 ELD_L424F 67.38 20.09 10.08 6.00 4.01 40.18 1.68 ELD_E425Q 8.67 0.07 0.10 0.05 0.07 0.29 30.34 ELD_M426I 61.70 16.65 2.74 4.59 3.84 27.82 2.22 ELD_K441E 32.05 9.23 2.59 2.29 2.31 16.41 1.95 ELD_K441D 5.87 0.94 0.35 0.38 0.24 1.91 3.06 ELD_K441L 13.37 2.77 0.74 0.80 0.95 5.26 2.54 ELD_H442R 18.30 0.45 0.63 0.47 0.14 1.70 10.79 ELD_G445E 2.63 0.15 0.21 0.20 0.06 0.62 4.21 ELD_S446G 35.42 3.07 1.52 1.75 1.09 7.42 4.77 ELD_T468N 0.19 0.06 0.05 0.14 0.07 0.31 0.59 ELD_S472K 44.07 2.77 1.36 1.45 0.54 6.13 7.19 ELD_G473D 29.64 6.26 1.15 1.52 2.12 11.04 2.68 ELD_G473K 25.08 5.70 0.92 1.42 0.70 8.74 2.87 ELD_N476S 55.45 10.94 2.55 3.65 2.18 19.31 2.87 ELD_N476D 59.33 0.13 0.06 0.17 0.10 0.46 128.70 ELD_P478S 63.62 14.02 0.62 2.98 4.36 21.98 2.89 ELD_I479T 41.42 0.09 0.15 0.06 0.11 0.42 99.43 ELD_G480S 61.29 14.12 2.99 4.03 6.03 27.17 2.26 ELD_Q481H 67.73 14.54 6.96 4.37 0.32 26.19 2.59 ELD_Q481E 53.31 0.11 0.11 0.21 0.05 0.48 110.94 ELD_Q481N 35.13 3.37 0.59 1.56 0.35 5.88 5.97 ELD_E484Q 37.48 11.64 1.93 1.39 4.08 19.04 1.97 ELD_P501S 20.02 4.32 1.60 1.47 1.17 8.56 2.34 ELD_G522S 60.02 12.86 2.23 3.29 3.27 21.65 2.77 ELD_H523F 42.63 14.68 2.12 2.27 4.62 23.68 1.80 ELD_N527D 41.63 1.13 0.22 0.68 0.11 2.13 19.59 ELD_N527G 23.28 2.93 0.84 1.27 1.50 6.55 3.56 ELD_N527K 13.62 2.56 0.78 0.89 0.23 4.46 3.06 ELD_Y528F 52.40 12.72 3.52 3.03 2.79 22.05 2.38 ELD_K529E 22.98 2.77 0.58 1.51 0.54 5.40 4.25 ELD_A530E 41.94 3.37 0.57 1.18 0.85 5.97 7.03 ELD_A530K 46.55 6.55 1.31 1.71 1.50 11.07 4.21 ELD_Q531N 16.28 1.30 0.26 0.53 0.37 2.46 6.62 ELD_Q531R 50.98 1.64 0.30 0.48 0.39 2.81 18.14 KKR_I414L 51.17 9.11 1.42 2.80 2.95 16.28 3.14 KKR_N417D 73.10 16.32 2.74 3.50 5.59 28.14 2.60 KKR_N417S 33.80 4.26 2.65 2.30 1.26 10.47 3.23 KKR_S418D 58.61 0.57 0.20 0.15 0.16 1.08 54.37 KKR_S418G 41.59 4.18 5.48 2.79 1.37 13.82 3.01 KKR_S418P 74.02 17.76 3.16 3.61 8.51 33.05 2.24 KKR_T419K 63.65 16.14 3.82 4.06 4.22 28.25 2.25 KKR_T419S 66.12 13.56 2.63 3.54 3.41 23.14 2.86 KKR_T419Y 42.35 4.81 1.45 1.68 1.20 9.14 4.63 KKR_Q420A 44.62 4.92 2.87 2.81 1.17 11.78 3.79 KKR_D421N 39.41 8.14 6.27 2.59 1.84 18.84 2.09 KKR_D421S 51.41 10.28 7.67 3.19 2.50 23.64 2.17 KKR_I423D 29.67 2.84 0.93 1.08 0.66 5.50 5.39 KKR_I423L 66.91 13.71 4.37 4.47 3.16 25.72 2.60 KKR_L424F 73.65 23.87 5.78 6.31 12.31 48.26 1.53 KKR_E425Q 33.35 0.09 0.05 0.08 0.08 0.30 109.99 KKR_M426I 63.92 9.92 2.71 3.45 2.36 18.44 3.47 KKR_K441E 45.72 8.60 5.21 3.29 2.64 19.74 2.32 KKR_K441D 10.66 0.80 0.84 0.58 0.40 2.63 4.05 KKR_K441L 23.57 2.99 1.55 1.40 0.91 6.84 3.44 KKR_H442R 32.85 2.18 0.14 0.51 0.62 3.45 9.51 KKR_G445E 5.57 0.31 0.16 0.25 0.24 0.96 5.80 KKR_S446G 48.86 4.31 1.13 1.13 1.59 8.16 5.99 KKR_T468N 0.94 0.06 0.02 0.05 0.07 0.20 4.79 KKR_S472K 54.49 6.10 0.99 1.93 1.66 10.68 5.10 KKR_G473D 41.00 7.64 3.40 2.26 1.52 14.83 2.76 KKR_G473K 32.02 2.67 2.10 2.03 1.01 7.80 4.11 KKR_N476S 59.29 8.93 1.51 2.82 2.96 16.22 3.66 KKR_N476D 28.98 0.12 0.04 0.08 0.05 0.30 97.89 KKR_P478S 64.26 5.11 1.52 2.45 1.40 10.49 6.13 KKR_I479T 59.75 0.55 0.22 0.87 0.10 1.73 34.49 KKR_G480S 65.65 10.79 4.90 3.14 2.04 20.87 3.15 KKR_Q481H 69.52 6.10 0.12 1.41 6.52 14.16 4.91 KKR_Q481E 65.72 0.28 0.07 0.06 0.11 0.52 125.97 KKR_Q481N 35.21 2.64 0.10 0.83 1.15 4.72 7.46 KKR_E484Q 51.22 10.35 8.22 2.39 2.70 23.66 2.17 KKR_P501S 30.29 3.83 0.79 1.57 1.02 7.21 4.20 KKR_G522S 63.97 12.37 3.37 3.47 2.21 21.42 2.99 KKR_H523F 53.03 9.32 5.28 3.38 2.74 20.72 2.56 KKR_N527D 52.34 1.75 0.56 1.24 0.59 4.13 12.66 KKR_N527G 28.13 2.49 0.55 1.59 0.78 5.41 5.20 KKR_N527K 20.69 1.81 0.72 1.02 0.63 4.19 4.94 KKR_Y528F 57.87 8.88 2.17 2.83 2.86 16.73 3.46 KKR_K529E 30.72 3.64 1.05 1.62 1.22 7.53 4.08 KKR_A530E 58.94 9.65 3.18 4.82 2.87 20.52 2.87 KKR_A530K 60.15 3.86 1.26 1.87 0.86 7.84 7.67 KKR_Q531N 17.46 2.00 1.25 1.49 0.46 5.20 3.36 KKR_Q531R 56.70 1.99 1.28 2.44 0.62 6.33 8.96

[0243] Tables 17A-17C show the results using the indicated examples of mutants, including double mutants.

Table 17A

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 29.64 3.29 1.51 0.82 0.98 4.49 Initial 30.09 3.79 1.91 0.97 1.09 3.88 Initial 29.29 3.92 1.53 0.73 0.75 4.23 Half dose 20.23 1.53 0.49 0.24 0.27 7.96 Half dose 19.34 1.28 0.41 0.21 0.19 9.25 Half dose 21.01 1.37 0.32 0.16 0.34 9.62 ELD_R416E_S418E 20.74 0.03 0.09 0.04 0.06 94.13 ELD_R416E_N476D 40.36 0.06 0.04 0.03 0.07 213.54 ELD_R416E_I479T 21.55 0.06 0.25 0.01 0.03 61.74 ELD_R416E_Q481A 2.83 0.05 0.02 0.01 0.06 19.59 ELD_R416E_Q481E 41.39 0.07 0.03 0.04 0.04 225.41 ELD_R416E_K525S 34.38 0.03 0.07 0.03 0.08 166.24 ELD_S418E_N476D 31.37 0.04 0.03 0.01 0.06 221.16 ELD_S418E_I479T 11.14 0.08 0.02 0.03 0.04 62.37 ELD_S418E_Q481A 3.20 0.03 0.02 0.03 0.11 16.08 ELD_S418E_Q481E 38.73 0.01 0.03 0.01 0.05 400.31 ELD_S418E_K525S 24.79 0.07 0.02 0.01 0.06 160.35 ELD_N476D_I479T 20.03 0.02 0.04 0.01 0.03 180.94 ELD_N476D_Q481A 12.76 0.03 0.04 0.02 0.05 88.57 ELD_N476D_Q481E 50.65 0.07 0.03 0.03 0.08 242.53 ELD_N476D_K525S 42.41 0.05 0.04 0.01 0.06 270.45 ELD_I479T_Q481A 8.92 0.07 0.02 0.01 0.02 72.43 ELD_I479T_Q481E 35.78 0.05 0.06 0.01 0.06 196.60 ELD_I479T_K525S 25.90 0.06 0.04 0.02 0.07 139.72 ELD_R416S_K525S 32.61 0.06 0.07 0.05 0.07 127.78 ELD_R422S_K525S 26.37 0.10 0.05 0.01 0.08 108.57 ELD_K448S_K525S 38.31 0.32 0.31 0.29 0.16 35.35 ELD_Q481A_K525S 15.18 0.09 0.05 0.01 0.07 67.80 ELD_Q481E_K525S 48.15 0.06 0.07 0.01 0.04 274.23 ELD_K525S_Q531R 46.91 0.07 0.06 0.06 0.10 168.33 ELD_R416D_K448A 33.83 0.09 0.41 0.07 0.05 54.89 ELD_R416D_I479Q 4.00 0.11 0.01 0.01 0.03 24.24 ELD_R416D_Q481A 0.98 0.07 0.02 0.04 0.03 6.23 ELD_R416D_K525A 5.58 0.07 0.07 0.02 0.04 26.88 ELD_R416E_R422H 36.93 0.10 0.41 0.10 0.04 56.30 ELD_R416E_K448A 46.84 0.24 1.54 0.27 0.08 22.04 ELD_R416E_I479Q 16.28 0.05 0.01 0.02 0.05 129.05 ELD_R416E_K525A 25.31 0.04 0.04 0.02 0.05 171.87 ELD_R416E_N527D 29.19 0.06 0.15 0.03 0.05 103.79 ELD_S418E_K448A 45.64 0.11 0.08 0.05 0.13 125.00 ELD_S418E_I479Q 11.10 0.05 0.03 0.03 0.07 60.83 ELD_S418E_K525A 18.22 0.05 0.03 0.00 0.06 132.51 ELD_R422H_I479Q 31.90 0.04 0.03 0.02 0.04 232.64 ELD_R422H_Q481A 15.51 0.07 0.07 0.01 0.04 84.46 ELD_R422H_K525A 31.01 0.07 0.05 0.03 0.05 152.49 ELD_K448A_I479Q 51.54 0.04 0.01 0.06 0.01 421.61 ELD_K448A_Q481A 35.37 0.08 0.01 0.06 0.05 173.76 ELD_K448A_K525A 43.95 0.17 0.15 0.22 0.13 65.65 ELD_K448A_N527D 32.41 0.58 0.13 0.21 0.08 32.34 ELD_I479Q_Q481A 4.15 0.04 0.02 0.01 0.08 28.92 ELD_I479Q_K525A 27.87 0.05 0.02 0.00 0.05 252.87 ELD_Q481A_K525A 8.65 0.04 0.03 0.01 0.05 65.76 ELD_Q481A_N527D 15.47 0.07 0.06 0.01 0.07 72.82 ELD_K525A_N527D 23.46 0.07 0.04 0.02 0.07 116.95 ELD_S446D 40.19 0.53 0.44 0.11 0.10 34.11 ELD_G480D 54.97 1.33 0.11 0.38 0.69 21.84 ELD_S418D 32.88 0.10 0.18 0.12 0.16 58.14 ELD_N476D 44.56 0.10 0.05 0.07 0.10 137.79 ELD_I479T 30.54 0.13 0.16 0.02 0.04 88.36 ELD_Q481E 40.57 0.06 0.05 0.15 0.04 139.98 ELD_N527D 26.68 0.41 0.13 0.14 0.07 35.71 ELD_Q531R 32.95 0.63 0.17 0.12 0.19 29.60 ELD_I479Q 50.31 0.06 0.03 0.01 0.05 313.15 ELD_R416E 46.99 0.37 2.36 0.24 0.17 15.03 ELD_K525A 41.41 0.31 0.38 0.14 0.13 42.92 ELD_K525S 39.15 0.18 0.42 0.15 0.12 44.95 ELD_Q481A 41.21 0.05 0.13 0.04 0.09 133.38 GFP 0.04 0.05 0.04 0.00 0.08 N/A GFP 0.05 0.06 0.02 0.02 0.05 N/A GFP 0.02 0.05 0.03 0.01 0.05 N/A

Table 17B

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 29.64 3.29 1.51 0.82 0.98 4.49 Initial 30.09 3.79 1.91 0.97 1.09 3.88 Initial 29.29 3.92 1.53 0.73 0.75 4.23 Half dose 20.23 1.53 0.49 0.24 0.27 7.96 Half dose 19.34 1.28 0.41 0.21 0.19 9.25 Half dose 21.01 1.37 0.32 0.16 0.34 9.62 KKR_R416E_S418E 20.09 0.02 0.01 0.02 0.08 151.13 KKR_R416E_N476D 20.88 0.03 0.02 0.02 0.10 121.14 KKR_R416E_I479T 42.32 0.11 0.03 0.03 0.03 220.35 KKR_R416E_Q481A 0.05 0.08 0.03 0.03 0.05 0.28 KKR_R416E_Q481E 31.75 0.08 0.02 0.01 0.10 163.74 KKR_R416E_K525S 30.56 0.08 0.03 0.04 0.06 146.79 KKR_S418E_N476D 2.68 0.06 0.03 0.02 0.07 15.27 KKR_S418E_I479T 35.49 0.04 0.02 0.02 0.08 234.26 KKR_S418E_Q481A 1.55 0.08 0.01 0.02 0.05 9.60 KKR_S418E_Q481E 17.90 0.05 0.04 0.01 0.04 135.16 KKR_S418E_K525S 10.28 0.08 0.02 0.03 0.05 55.61 KKR_N476D_I479T 5.09 0.05 0.02 0.01 0.10 26.81 KKR_N476D_Q481A 4.44 0.08 0.05 0.01 0.06 22.35 KKR_N476D_Q481E 3.08 0.09 0.04 0.02 0.03 17.28 KKR_N476D_K525S 8.06 0.02 0.02 0.00 0.11 52.10 KKR_I479T_Q481A 25.89 0.05 0.01 0.01 0.05 204.61 KKR_I479T_Q481E 35.07 0.05 0.00 0.02 0.08 218.88 KKR_I479T_K525S 37.19 0.09 0.08 0.02 0.06 151.36 KKR_R416S_K525S 30.89 0.12 0.02 0.04 0.07 125.26 KKR_R422S_K525S 28.13 0.07 0.04 0.01 0.06 160.39 KKR_K448S_K525S 30.31 0.42 0.04 0.04 0.08 52.74 KKR_Q481A_K525S 4.99 0.03 0.02 0.04 0.04 36.01 KKR_Q481E_K525S 24.26 0.03 0.01 0.01 0.12 142.63 KKR_K525S_Q531R 31.33 0.20 0.05 0.01 0.12 83.63 KKR_R416D_K448A 34.82 0.06 0.07 0.05 0.04 160.49 KKR_R416D_I479Q 37.53 0.07 0.01 0.01 0.07 229.85 KKR_R416D_Q481A 1.51 0.05 0.01 0.03 0.05 10.67 KKR_R416D_K525A 8.83 0.09 0.02 0.01 0.11 40.27 KKR_R416E_R422H 39.63 0.19 0.02 0.03 0.08 122.95 KKR_R416E_K448A 43.43 0.13 0.05 0.04 0.19 106.38 KKR_R416E_I479Q 40.55 0.12 0.04 0.07 0.05 140.11 KKR_R416E_K525A 23.98 0.04 0.05 0.03 0.09 112.67 KKR_R416E_N527D 40.12 0.10 0.01 0.04 0.13 141.70 KKR_S418E_K448A 36.67 0.10 0.08 0.02 0.03 158.02 KKR_S418E_I479Q 36.46 0.07 0.09 0.03 0.02 173.06 KKR_S418E_K525A 6.28 0.08 0.01 0.04 0.04 36.37 KKR_R422H_I479Q 35.05 0.29 0.14 0.06 0.03 66.28 KKR_R422H_Q481A 12.81 0.06 0.03 0.01 0.10 64.84 KKR_R422H_K525A 31.03 0.11 0.04 0.05 0.05 126.26 KKR_K448A_I479Q 35.23 0.17 0.19 0.05 0.06 74.29 KKR_K448A_Q481A 19.81 0.11 0.01 0.03 0.03 116.04 KKR_K448A_K525A 36.25 0.08 0.04 0.01 0.07 183.47 KKR_K448A_N527D 29.74 0.30 0.12 0.01 0.10 55.29 KKR_I479Q_Q481A 31.65 0.09 0.07 0.01 0.06 128.89 KKR_I479Q_K525A 37.06 0.06 0.02 0.03 0.08 196.46 KKR_Q481A_K525A 3.09 0.06 0.01 0.03 0.05 21.23 KKR_Q481A_N527D N/A N/A N/A N/A N/A N/A KKR_K525A_N527D N/A N/A N/A N/A N/A N/A KKR_S446D 38.46 0.08 0.03 0.03 0.08 175.19 KKR_G480D 40.11 0.60 0.98 0.14 0.14 21.66 KKR_S418D 35.59 0.35 0.13 0.06 0.08 56.27 KKR_N476D 20.04 0.12 0.05 0.05 0.04 78.85 KKR_I479T 36.62 0.40 0.09 0.24 0.07 45.83 KKR_Q481E 37.26 0.15 0.03 0.01 0.08 137.34 KKR_N527D 29.30 0.98 0.37 0.24 0.30 15.48 KKR_Q531R 27.77 0.61 0.69 0.38 0.19 14.82 KKR_I479Q 40.88 1.28 0.74 0.35 0.09 16.60 KKR_R416E 41.76 0.49 0.06 0.05 0.55 35.99 KKR_K525A 33.81 0.46 0.04 0.07 0.19 44.54 KKR_K525S 35.00 1.05 0.15 0.14 0.24 22.10 KKR_Q481A 36.52 0.12 0.09 0.01 0.10 113.98 GFP 0.04 0.05 0.04 0.00 0.08 N/A GFP 0.05 0.06 0.02 0.02 0.05 N/A GFP 0.02 0.05 0.03 0.01 0.05 N/A

Table 17C

AAVS1 OT1 OT2 OT3 OT4 Ratio Initial 29.64 3.29 1.51 0.82 0.98 4.49 Initial 30.09 3.79 1.91 0.97 1.09 3.88 Initial 29.29 3.92 1.53 0.73 0.75 4.23 Half dose 20.23 1.53 0.49 0.24 0.27 7.96 Half dose 19.34 1.28 0.41 0.21 0.19 9.25 Half dose 21.01 1.37 0.32 0.16 0.34 9.62 ELD_KKR_R416E_S418E 1.12 0.06 0.01 0.04 0.08 5.75 ELD_KKR_R416E_N476D 0.08 0.02 0.01 0.02 0.05 0.90 ELD_KKR_R416E_I479T 9.58 0.01 0.01 0.01 0.07 93.64 ELD_KKR_R416E_Q481A 0.04 0.03 0.03 0.02 0.06 0.34 ELD_KKR_R416E_Q481E 15.79 0.01 0.01 0.02 0.07 146.87 ELD_KKR_R416E_K525S 18.64 0.07 0.03 0.01 0.06 110.61 ELD_KKR_S418E_N476D 0.03 0.05 0.03 0.01 0.06 0.19 ELD_KKR_S418E_I479T 0.07 0.06 0.05 0.02 0.05 0.39 ELD_KKR_S418E_Q481A 0.06 0.06 0.02 0.01 0.07 0.39 ELD_KKR_S418E_Q481E 0.25 0.06 0.02 0.01 0.06 1.60 ELD_KKR_S418E_K525S 0.04 0.07 0.01 0.01 0.06 0.28 ELD_KKR_N476D_I479T 0.01 0.08 0.03 0.01 0.07 0.06 ELD_KKR_N476D_Q481A 0.04 0.02 0.01 0.02 0.02 0.60 ELD_KKR_N476D_Q481E 0.04 0.07 0.04 0.01 0.05 0.22 ELD_KKR_N476D_K525S 0.03 0.05 0.05 0.00 0.04 0.19 ELD_KKR_I479T_Q481A 1.92 0.02 0.03 0.01 0.07 14.25 ELD_KKR_I479T_Q481E 1.71 0.06 0.04 0.02 0.04 11.29 ELD_KKR_I479T_K525S 0.87 0.03 0.02 0.02 0.07 6.00 ELD_KKR_R416S_K525S 16.65 0.08 0.02 0.04 0.08 77.13 ELD_KKR_R422S_K525S 8.09 0.10 0.02 0.01 0.07 39.66 ELD_KKR_K448S_K525S 34.04 0.06 0.03 0.02 0.06 203.03 ELD_KKR_Q481A_K525S 2.27 0.05 0.04 0.01 0.08 12.73 ELD_KKR_Q481E_K525S 1.58 0.04 0.04 0.02 0.05 10.24 ELD_KKR_K525S_Q531R 31.89 0.06 0.02 0.02 0.05 213.09 ELD_KKR_R416D_K448A 34.69 0.02 0.05 0.04 0.09 171.10 ELD_KKR_R416D_I479Q 0.64 0.09 0.04 0.01 0.08 2.97 ELD_KKR_R416D_Q481A 0.72 0.07 0.03 0.00 0.09 3.74 ELD_KKR_R416D_K525A 0.44 0.03 0.01 0.03 0.03 4.38 ELD_KKR_R416E_R422H 51.26 0.06 0.04 0.04 0.08 241.20 ELD_KKR_R416E_K448A 65.65 0.04 0.05 0.03 0.11 296.99 ELD_KKR_R416E_I479Q 9.45 0.03 0.01 0.03 0.06 69.73 ELD_KKR_R416E_K525A 8.87 0.06 0.02 0.03 0.07 50.16 ELD_KKR_R416E_N527D 8.77 0.06 0.02 0.00 0.05 65.60 ELD_KKR_S418E_K448A 12.39 0.07 0.01 0.02 0.02 105.55 ELD_KKR_S418E_I479Q 0.17 0.08 0.04 0.06 0.07 0.68 ELD_KKR_S418E_K525A 0.07 0.05 0.02 0.00 0.04 0.57 ELD_KKR_R422H_I479Q 6.81 0.04 0.01 0.01 0.10 39.89 ELD_KKR_R422H_Q481A 17.21 0.05 0.04 0.04 0.05 97.54 ELD_KKR_R422H_K525A 9.57 0.02 0.04 0.04 0.04 68.82 ELD_KKR_K448A_I479Q 33.17 0.06 0.01 0.01 0.07 209.50 ELD_KKR_K448A_Q481A 52.80 0.05 0.03 0.02 0.05 351.52 ELD_KKR_K448A_K525A 31.94 0.06 0.03 0.02 0.08 164.22 ELD_KKR_K448A_N527D 12.12 0.08 0.03 0.01 0.04 70.27 ELD_KKR_I479Q_Q481A 1.71 0.01 0.00 0.06 0.10 10.33 ELD_KKR_I479Q_K525A 1.27 0.09 0.05 0.02 0.06 5.70 ELD_KKR_Q481A_K525A 0.52 0.01 0.02 0.01 0.05 5.01 ELD_KKR_Q481A_N527D N/A N/A N/A N/A N/A N/A ELD_KKR_K525A_N527D N/A N/A N/A N/A N/A N/A ELD_KKR_S446D 15.36 0.04 0.02 0.04 0.08 83.81 ELD_KKR_G480D 64.90 0.09 0.02 0.06 0.15 200.43 ELD_KKR_S418D 26.63 0.05 0.03 0.00 0.08 167.71 ELD_KKR_N476D 4.22 0.02 0.05 0.02 0.03 36.30 ELD_KKR_I479T 26.01 0.05 0.05 0.04 0.03 153.83 ELD_KKR_Q481E 38.76 0.08 0.02 0.05 0.03 221.20 ELD_KKR_N527D 21.40 0.10 0.03 0.03 0.07 92.64 ELD_KKR_Q531R 30.69 0.17 0.05 0.10 0.03 89.70 ELD_KKR_I479Q 52.76 0.03 0.03 0.02 0.07 350.46 ELD_KKR_R416E 61.25 0.16 0.10 0.09 0.10 138.11 ELD_KKR_K525A 38.71 0.15 0.01 0.03 0.07 152.55 ELD_KKR_K525S 42.82 0.02 0.03 0.02 0.07 299.99 ELD_KKR_Q481A 70.60 0.07 0.02 0.04 0.06 383.30 GFP 0.04 0.05 0.04 0.00 0.08 N/A GFP 0.05 0.06 0.02 0.02 0.05 N/A GFP 0.02 0.05 0.03 0.01 0.05 N/A

[0244] Tables 18A-18C show the results of applying the indicated mutant cleavage domains.

Table 18A

AAVS1 OT1 Ratio Initial 60.28 12.69 4.75 Initial 57.85 9.71 5.96 Initial 54.74 9.42 5.81 Half dose 51.05 5.51 9.26 Half dose 42.72 3.69 11.58 Half dose 50.26 7.75 6.49 ELD_G480A 71.44 10.67 6.70 ELD_G480C 62.08 8.04 7.72 ELD_G480D 64.70 0.79 82.32 ELD_G480E 76.47 0.57 134.21 ELD_G480F 40.12 1.20 33.37 ELD_G480H N/A 15.72 N/A ELD_G480I 41.29 4.05 10.19 ELD_G480K 68.06 14.64 4.65 ELD_G480L 41.04 2.35 17.49 ELD_G480M 39.95 3.93 10.16 ELD_G480N 57.82 11.58 4.99 ELD_G480P 45.73 1.29 35.32 ELD_G480Q 25.33 0.30 85.73 ELD_G480R 56.94 13.22 4.31 ELD_G480S 68.85 11.40 6.04 ELD_G480T 68.02 14.68 4.63 ELD_G480V 43.43 2.22 19.53 ELD_G480W 64.39 0.83 77.94 ELD_G480Y 45.27 2.45 18.51 ELD_R416E 66.43 0.40 164.15 ELD_I479Q 70.95 0.21 345.03 ELD_G480D 72.46 0.65 110.75 ELD_Q481A 58.01 0.34 173.11 ELD_K525A 66.31 0.60 111.25 ELD_R416E_R422H 48.29 0.07 679.24 ELD_R416E_K448A 70.50 0.50 142.30 ELD_K448A_I479Q 68.99 0.11 637.89 ELD_K448A_Q481A 41.36 0.12 333.89 ELD_K448A_K525A 70.69 0.24 300.02 GFP 0.11 0.03 N/A GFP 0.20 0.00 N/A

Table 18B

AAVS1 OT1 Ratio Initial 60.28 12.69 4.75 Initial 57.85 9.71 5.96 Initial 54.74 9.42 5.81 Half dose 51.05 5.51 9.26 Half dose 42.72 3.69 11.58 Half dose 50.26 7.75 6.49 KKR_G480A 65.59 6.54 10.03 KKR_G480C 56.27 5.14 10.94 KKR_G480D 56.11 1.00 56.19 KKR_G480E 56.49 0.73 77.63 KKR_G480F 34.91 0.78 44.55 KKR_G480H 60.07 8.52 7.05 KKR_G480I 38.68 1.60 24.22 KKR_G480K 65.79 11.26 5.84 KKR_G480L 41.92 1.13 36.98 KKR_G480M 55.69 5.66 9.84 KKR_G480N 61.56 9.62 6.40 KKR_G480P 54.84 1.32 41.45 KKR_G480Q 62.68 7.05 8.89 KKR_G480R 60.84 10.34 5.88 KKR_G480S 66.61 12.23 5.45 KKR_G480T 71.73 14.73 4.87 KKR_G480V 41.05 1.63 25.24 KKR_G480W 57.21 0.56 102.21 KKR_G480Y 46.86 3.28 14.27 KKR_R416E 70.79 1.45 48.82 KKR_I479Q 65.46 2.18 29.99 KKR_G480D 56.96 1.09 52.26 KKR_Q481A 62.91 0.17 366.11 KKR_K525A 66.35 1.35 49.21 KKR_R416E_R422H 69.06 0.08 847.00 KKR_R416E_K448A N/A 0.25 N/A KKR_K448A_I479Q 64.17 0.37 175.75 KKR_K448A_Q481A 35.73 0.17 214.80 KKR_K448A_K525A 64.77 0.34 192.29 GFP 0.11 0.03 N/A GFP 0.20 0.00 N/A

Table 18C

AAVS1 OT1 Ratio Initial 60.28 12.69 4.75 Initial 57.85 9.71 5.96 Initial 54.74 9.42 5.81 Half dose 51.05 5.51 9.26 Half dose 42.72 3.69 11.58 Half dose 50.26 7.75 6.49 ELD_KKR_G480A 83.25 15.29 5.44 ELD_KKR_G480C 62.30 6.18 10.09 ELD_KKR_G480D 68.39 0.17 400.26 ELD_KKR_G480E 64.32 0.16 390.88 ELD_KKR_G480F 50.56 2.10 24.03 ELD_KKR_G480H 70.45 19.84 3.55 ELD_KKR_G480I 30.54 1.41 21.66 ELD_KKR_G480K 75.70 4.49 16.88 ELD_KKR_G480L 29.27 0.35 83.87 ELD_KKR_G480M 38.29 2.25 17.05 ELD_KKR_G480N 62.58 10.50 5.96 ELD_KKR_G480P 46.49 0.10 446.75 ELD_KKR_G480Q 21.45 0.45 48.14 ELD_KKR_G480R 34.10 4.91 6.94 ELD_KKR_G480S 70.28 15.69 4.48 ELD_KKR_G480T 74.83 16.53 4.53 ELD_KKR_G480V 30.55 0.92 33.16 ELD_KKR_G480W 40.77 0.11 378.20 ELD_KKR_G480Y 38.97 1.69 23.09 ELD_KKR_R416E 75.31 0.36 207.10 ELD_KKR_I479Q 63.75 0.08 804.35 ELD_KKR_G480D 77.51 0.13 612.83 ELD_KKR_Q481A 86.20 0.12 731.56 ELD_KKR_K525A 56.37 0.06 969.83 ELD_KKR_R416E_R422H 58.35 0.06 996.94 ELD_KKR_R416E_K448A 84.57 0.07 1241.44 ELD_KKR_K448A_I479Q 43.78 0.03 1296.88 ELD_KKR_K448A_Q481A 63.32 0.08 816.13 ELD_KKR_K448A_K525A 48.43 0.13 384.71 GFP 0.11 0.03 N/A GFP 0.20 0.00 N/A

[0245] In FIG. 15 and 16 also provide an overview of the selected results with the indicated mutants.

[0246] These results demonstrate highly specific cleavage using the mutants described herein.

[0247] All patents, patent applications, and publications referred to herein are incorporated herein by reference in their entirety.

While the present invention has been described in sufficient detail with reference to illustrative material and examples for clarity of understanding, it will be clear to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the above descriptions and examples are not to be construed as limiting.

--->

SEQUENCE LIST

<110> SANGAMO THERAPEUTICS, INC./SANGAMO THERAPEUTICS, INC.

<120> ENGINEERED TARGET SPECIFIC NUCLEASES/DESIGNED

TARGET-SPECIFIC NUCLEASE

<130> 8325-0156.40

<140> PCT/US2017/048409

<141> 2017-08-24

<150> 62/443981

<151> 2017-01-09

<150> 62/378978

<151> 2016-08-24

<160> 72

<170> PatentIn, Rev. 3.5

<210> 1

<211> 196

<212> PRT/PROTEIN

<213> Unknown

<220>

<223> Description of the unknown sequence:

Wild-type FokI cleavage half-domain

<400> 1

Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His

1 5 10 15

Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala

20 25 30

Arg Asn Ser Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe

35 40 45

Phe Met Lys Val Tyr Gly Tyr Arg Gly Lys His Leu Gly Gly Ser Arg

50 55 60

Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly

65 70 75 80

Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile

85 90 95

Gly Gln Ala Asp Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg

100 105 110

Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser

115 120 125

Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn

130 135 140

Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly

145 150 155 160

Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys

165 170 175

Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly

180 185 190

Glu Ile Asn Phe

195

<210> 2

<211> 588

<212> DNA

<213> Unknown

<220>

<223> Description of the unknown sequence:

Wild-type FokI cleavage half-domain

<400> 2

cagctggtga agagcgagct ggaggagaag aagtccgagc tgcggcacaa gctgaagtac 60

gtgccccacg agtacatcga gctgatcgag atcgccagga acagcaccca ggaccgcatc 120

ctggagatga aggtgatgga gttcttcatg aaggtgtacg gctacagggg aaagcacctg 180

ggcggaagca gaaagcctga cggcgccatc tatacagtgg gcagccccat cgattacggc 240

gtgatcgtgg acacaaaggc ctacagcggc ggctacaatc tgcctatcgg ccaggccgac 300

gagatgcaga gatacgtgga ggagaaccag acccggaata agcacatcaa ccccaacgag 360

tggtggaagg tgtaccctag cagcgtgacc gagttcaagt tcctgttcgt gagcggccac 420

ttcaagggca actacaaggc ccagctgacc aggctgaacc acatcaccaa ctgcaatggc 480

gccgtgctga gcgtggagga gctgctgatc ggcggcgaga tgatcaaagc cggcaccctg 540

acactggagg aggtgcggcg caagttcaac aacggcgaga tcaacttc 588

<210> 3

<211> 28

<212> PRT/PROTEIN

<213> Unknown

<220>

<223> Description of the unknown sequence:

Protein with zinc fingers

<400> 3

Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp

1 5 10 15

His Leu Thr Thr His Ile Arg Thr His Thr Gly Glu

20 25

<210> 4

<211> 23

<212> PRT/PROTEIN

<213> Unknown

<220>

<223> Description of the unknown sequence:

Protein with zinc fingers

<400> 4

Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp His Leu

1 5 10 15

Thr Thr His Ile Arg Thr His

twenty

<210> 5

<211> 23

<212> PRT/PROTEIN

<213> Unknown

<220>

<223> Description of the unknown sequence:

Protein with zinc fingers

<400> 5

Phe Ala Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg

1 5 10 15

Lys Arg His Thr Lys Ile His

twenty

<210> 6

<211> 23

<212> PRT/PROTEIN

<213> Unknown

<220>

<223> Description of the unknown sequence:

Protein with zinc fingers

<400> 6

Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp His Leu

1 5 10 15

Ser Lys His Ile Lys Thr His

twenty

<210> 7

<211> 179

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<220>

<221> MOD_RES/MODIFIED REMAINS

<222>(8)..(8)

<223> Q, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (13)..(13)

<223> E, N, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (17)..(17)

<223> A, Q, L, E, N, Y or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (21)..(27)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (45)..(45)

<223> A, Q, L, or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (49)..(55)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (70)..(70)

<223> E, N, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (74)..(74)

<223> A, Q, L, E, N, Y or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (78)..(84)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (102)..(102)

<223> A, Q, L, or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (106)..(112)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (127)..(127)

<223> E, N, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (131)..(131)

<223> A, Q, L, E, N, Y or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (135)..(141)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (159)..(159)

<223> A, Q, L, or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (163)..(169)

<223> Any amino acid

<400> 7

Val Pro Ala Ala Met Ala Glu Xaa Pro Phe Gln Cys Xaa Ile Cys Met

1 5 10 15

Xaa Asn Phe Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Ile Arg Thr His

20 25 30

Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Xaa Lys Phe Ala

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Thr Lys Ile His Thr Gly Ser Gln

50 55 60

Lys Pro Phe Gln Cys Xaa Ile Cys Met Xaa Asn Phe Ser Xaa Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Xaa His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala

85 90 95

Cys Asp Ile Cys Gly Xaa Lys Phe Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa

100 105 110

His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe Gln Cys Xaa Ile

115 120 125

Cys Met Xaa Asn Phe Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Ile Arg

130 135 140

Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile Cys Gly Xaa Lys

145 150 155 160

Phe Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr His Thr Lys Ile His Leu

165 170 175

Arg Gly Ser

<210> 8

<211> 151

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<220>

<221> MOD_RES/MODIFIED REMAINS

<222>(8)..(8)

<223> Q, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (13)..(13)

<223> E, N, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (17)..(17)

<223> A, Q, L, E, N, Y or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (21)..(27)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (42)..(42)

<223> E, N, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (46)..(46)

<223> A, Q, L, E, N, Y or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (50)..(56)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (74)..(74)

<223> A, Q, L, or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (78)..(84)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (99)..(99)

<223> E, N, S or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (103)..(103)

<223> A, Q, L, E, N, Y or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (107)..(113)

<223> Any amino acid

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (131)..(131)

<223> A, Q, L, or R

<220>

<221> MOD_RES/MODIFIED REMAINS

<222> (135)..(141)

<223> Any amino acid

<400> 8

Val Pro Ala Ala Met Ala Glu Xaa Pro Phe Gln Cys Xaa Ile Cys Met

1 5 10 15

Xaa Lys Phe Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Thr Lys Ile His

20 25 30

Thr Gly Ser Gln Lys Pro Phe Gln Cys Xaa Ile Cys Met Xaa Asn Phe

35 40 45

Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Ile Arg Thr His Thr Gly Glu

50 55 60

Lys Pro Phe Ala Cys Asp Ile Cys Gly Xaa Lys Phe Ala Xaa Xaa Xaa

65 70 75 80

Xaa Xaa Xaa Xaa His Thr Lys Ile His Thr Gly Ser Gln Lys Pro Phe

85 90 95

Gln Cys Xaa Ile Cys Met Xaa Asn Phe Ser Xaa Xaa Xaa Xaa Xaa Xaa

100 105 110

Xaa His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala Cys Asp Ile

115 120 125

Cys Gly Xaa Lys Phe Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr His Thr

130 135 140

Lys Ile His Leu Arg Gly Ser

145 150

<210> 9

<211> 5

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 9

Thr Gly Glu Lys Pro

fifteen

<210> 10

<211> 6

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 10

Thr Gly Gly Glyn Arg Pro

fifteen

<210> 11

<211> 5

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 11

Thr Gly Gln Lys Pro

fifteen

<210> 12

<211> 6

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 12

Thr Gly Ser Gln Lys Pro

fifteen

<210> 13

<211> 76

<212> DNA

<213> Homo sapiens

<400> 13

ctaatcagag gccaaaccct tcctggagcc tgtgataaaa gcaactgtta gcttgcacta 60

gactagcttc aaagtt 76

<210> 14

<211> 76

<212> DNA

<213> Homo sapiens

<400> 14

cttcctgcgg ggttttgtgc aaataaccca tgctcgggag cgaggccctg ggaaggagta 60

tctcgcttct ttgggt 76

<210> 15

<211> 76

<212> DNA

<213> Homo sapiens

<400> 15

cctaccccca ccacctcacc ttccctcagc cttgtgtttc agccccagtt agctgccctt 60

agttgctgat gtattg 76

<210> 16

<211> 76

<212> DNA

<213> Homo sapiens

<400> 16

agcacgtttc tccagtttcc agcctggggc ctggctataa agcaaatgct cagtccagca 60

ttgcggaatg caaggg 76

<210> 17

<211> 76

<212> DNA

<213> Homo sapiens

<400> 17

acagcaccgt gctgcacggc gtcctccggc cttgctgcct ggcaatgggt agccacctgg 60

cgtctgtctc agaata 76

<210> 18

<211> 76

<212> DNA

<213> Homo sapiens

<400> 18

ttcccagaag gcgattgaac ctgaagctgc gcctggcgcg tgagcctgtg ggggggacgc 60

ggctgagggg ctttga 76

<210> 19

<211> 76

<212> DNA

<213> Homo sapiens

<400> 19

tatcccttcc ccaagtgcaa ccaacagttg ctctaaagct aggctggtgg agttggggaa 60

agggccagca agtgag 76

<210> 20

<211> 76

<212> DNA

<213> Homo sapiens

<400> 20

tcctccatgc tcagaaagcc ttttcttggag ccaggcacac aggaaatgtt agctagttag 60

cattggctct aatact 76

<210> 21

<211> 76

<212> DNA

<213> Homo sapiens

<400> 21

tagctgggga gagattgcct ctctcaggggc ctagccagtt cctagaaata gcaagggctc 60

agctgagagc atgctt 76

<210> 22

<211> 76

<212> DNA

<213> Homo sapiens

<400> 22

tccccgggaa gtgtccgcac ctcctagagc ccagcgagcg agcgtttgtg cttttgtcct 60

ttgaaccggg tgtggt 76

<210> 23

<211> 76

<212> DNA

<213> Homo sapiens

<400> 23

gactcattca tccactcatt ctgagcactt gctgcacact aggccctggg ctggggcttc 60

agcccaggag ttcact 76

<210> 24

<211> 76

<212> DNA

<213> Homo sapiens

<400> 24

atgtagtctg acggccgcga ctggttcgta gcttttgagt gaggcggcgg gaagggagcg 60

agggaagagc ggcagt 76

<210> 25

<211> 76

<212> DNA

<213> Homo sapiens

<400> 25

tcatgttatg gaagtggctt ctttccttaa gccttatgaa taagcctctg ctagcttcaa 60

actttgtgtg cagctt 76

<210> 26

<211> 76

<212> DNA

<213> Homo sapiens

<400> 26

cataaagcac ttacaacagt gcctggcaaa tgcctagtgc acagcaagtg ttagctattg 60

ttaatgacta tccatt 76

<210> 27

<211> 76

<212> DNA

<213> Homo sapiens

<400> 27

atttttgccc ctgtcttctc ttttcctcct ttgctgcatc ccaggctcca gcctttcagc 60

cctatttgca gtaccc 76

<210> 28

<211> 76

<212> DNA

<213> Homo sapiens

<400> 28

acaaggggtt caaggttatg aataacctgt gctaatccca gaggccccag gacagagtaa 60

gtgggaacaa acactg 76

<210> 29

<211> 76

<212> DNA

<213> Homo sapiens

<400> 29

cggaagttaa tatgatcatt gctaacattt gctgtgtttc aggcactgta agcatgtata 60

tgggtcctta aaggga 76

<210> 30

<211> 76

<212> DNA

<213> Homo sapiens

<400> 30

tgagcccaga aaccccttac ccttcctcct gcctcttgag aggccagtgt taggtgttag 60

ccggggtgca aagctc 76

<210> 31

<211> 76

<212> DNA

<213> Homo sapiens

<400> 31

aacctggtgc gagcagcccg ggctacaggg ttgcctgagg tgtgggtccc aggatgggagg 60

agccccaggc cggcgg 76

<210> 32

<211> 76

<212> DNA

<213> Homo sapiens

<400> 32

acttcggtga aggaagtcat cagtgcagtt gccgacaagc tgggctccgg ggagggcctg 60

atcatagtca agatga 76

<210> 33

<211> 76

<212> DNA

<213> Homo sapiens

<400> 33

ggggccaggc aggaagaaca gctaactcta gctcacctgc aaggctcagc actgggttca 60

tttgaagtag tgtcct 76

<210> 34

<211> 76

<212> DNA

<213> Homo sapiens

<400> 34

ctcctttggg tgtggacggg actaacactt gctccatgtc agggctgcag gacctcctgg 60

ctgttgacag caggca 76

<210> 35

<211> 76

<212> DNA

<213> Homo sapiens

<400> 35

gatgtttgaa aagcgctgag cctggcctgg cacctaaaca gctcagcaag tgttagccag 60

gatcactagc agtaat 76

<210> 36

<211> 76

<212> DNA

<213> Homo sapiens

<400> 36

accctgccga ttttccttcca gttgctcgct ggggcaaccg gctaggctgg aggaagggcg 60

aggacggtgt cacccc 76

<210> 37

<211> 76

<212> DNA

<213> Homo sapiens

<400> 37

gaacatggtg aggacaaaat gatgtccagg agccttttct ttggccattg ctagcctgag 60

acgaaaagtc agtggc 76

<210> 38

<211> 76

<212> DNA

<213> Homo sapiens

<400> 38

agtctgtcag tctatcgtcc ctacctgcag cccaaagcat aagcaacatc ttgcccagct 60

cagaggtgac aacctc 76

<210> 39

<211> 76

<212> DNA

<213> Homo sapiens

<400> 39

caccgaccca aggaccactc cttccaggaa cctagcctaa agcaaaggtg cagacagccc 60

ggggccaccg ctgacc 76

<210> 40

<211> 76

<212> DNA

<213> Homo sapiens

<400> 40

ttgcaaaagg aagtttgaag ctagcagagg ctgattcatg aggttcaagg aaagaagtca 60

tctctgtaac ataaaa 76

<210> 41

<211> 76

<212> DNA

<213> Homo sapiens

<400> 41

60

cccctgctga gtcagc 76

<210> 42

<211> 76

<212> DNA

<213> Homo sapiens

<400> 42

gtcaggggac agggtttcct agcatccgcg agccttacag aaaggcaact gtgcagtgct 60

ccagctggct ttctca 76

<210> 43

<211> 76

<212> DNA

<213> Homo sapiens

<400> 43

accatcagag aagctaacct ttcctgaggc ctaactactt tgcaggtcct gtattatgtc 60

ctctatagac attagg 76

<210> 44

<211> 76

<212> DNA

<213> Homo sapiens

<400> 44

gatcatttca aagaaaacat tctggaacag ttgcctatgg gcacggcagg gtgacggtgc 60

tgcttctggg tttgac 76

<210> 45

<211> 76

<212> DNA

<213> Homo sapiens

<400> 45

gaagggcaga gaactataat gacacagttg ctttattgct ggacccagga cagtttatac 60

agcagttcgg aaaggc 76

<210> 46

<211> 76

<212> DNA

<213> Homo sapiens

<400> 46

acttattgta agtgacgcat gtgaccagtt gccaattgtt caggctacag cttggatctg 60

ttagcatcct cattta 76

<210> 47

<211> 76

<212> DNA

<213> Homo sapiens

<400> 47

cccaggggct gtgggacact cagagagcct atgcctgtgc cctgggctcc gggaggggag 60

aggatctggg ggccag 76

<210> 48

<211> 77

<212> DNA

<213> Homo sapiens

<400> 48

ccccaggggc tgtgggacac tcagagagcc tatgcctgtg ccctgggctc cgggagggga 60

gaggatctgg gggccag 77

<210> 49

<211> 42

<212> DNA

<213> Homo sapiens

<400> 49

agcttcctgc agcctccctc aaagcagatg ttagcactat ta 42

<210> 50

<211> 42

<212> DNA

<213> Homo sapiens

<400> 50

cctcacctag aacctgggcc cctgcaactg taacctgtgg ca 42

<210> 51

<211> 42

<212> DNA

<213> Homo sapiens

<400> 51

ccagaggaag aacagttgct tgggtctaggg cctcaggaag gg 42

<210> 52

<211> 43

<212> DNA

<213> Homo sapiens

<400> 52

cctgtgctgg ggcctgggag gcaggcggct gctagccatc ctg 43

<210> 53

<211> 42

<212> DNA

<213> Homo sapiens

<400> 53

tgacttagga agcagttgct acctgccagg ccccaggcta gg 42

<210> 54

<211> 40

<212> DNA

<213> Homo sapiens

<400> 54

tcagtgattg ggttccgaat ccctcctcctg aaagtggccg 40

<210> 55

<211> 40

<212> DNA

<213> Homo sapiens

<400> 55

ccatatgcag aaaacagaaa ctgtacccct tccttacacc 40

<210> 56

<211> 40

<212> DNA

<213> Homo sapiens

<400> 56

tggccctttg cagaaaaggt tgttgaaccc taccgtaaac 40

<210> 57

<211> 40

<212> DNA

<213> Homo sapiens

<400> 57

ccggagagaa ggctccggtt cagcactgag atcaggacgg 40

<210> 58

<211> 195

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 58

Leu Val Lys Gly Glu Met Glu Lys Lys Lys Ser Asp Leu Arg His Lys

1 5 10 15

Leu Lys His Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Gln

20 25 30

Asp Ser Lys Gln Asn Arg Leu Phe Glu Phe Lys Val Val Glu Phe Leu

35 40 45

Lys Glu Val Tyr Asp Tyr Asn Gly Lys His Leu Gly Gly Ser Arg Lys

50 55 60

Pro Asp Gly Ala Leu Tyr Thr Asn Gly Leu Lys Thr Asp Tyr Gly Ile

65 70 75 80

Ile Leu Asp Thr Lys Ala Tyr Lys Asp Gly Tyr Ser Leu Pro Ile Ser

85 90 95

Gln Ala Asp Glu Met Gln Arg Tyr Val Asp Glu Asn Asn Asn Asn Arg Asn

100 105 110

Ala Ile Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Asn Ser Ile

115 120 125

Leu Asp Phe Lys Phe Leu Phe Val Ser Gly Phe Phe Lys Gly Asp Tyr

130 135 140

Lys Lys Gln Leu Ala Arg Val Ser Asn Leu Thr Lys Arg Lys Gly Ala

145 150 155 160

Val Leu Ser Val Glu Gln Leu Leu Leu Gly Gly Glu Lys Ile Lys Asp

165 170 175

Gly Ser Leu Thr Leu Glu Asp Val Gly Asp Lys Phe Asn Asn Asp Glu

180 185 190

Ile Ile Phe

195

<210> 59

<211> 192

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 59

Gln Ile Val Lys Ser Ser Ile Glu Met Ser Lys Ala Asn Met Arg Asp

1 5 10 15

Asn Leu Gln Met Leu Pro His Asp Tyr Ile Glu Leu Ile Glu Ile Ser

20 25 30

Gln Asp Pro Tyr Gln Asn Arg Ile Phe Glu Met Lys Val Met Asp Leu

35 40 45

Phe Ile Asn Glu Tyr Gly Phe Ser Gly Ser His Leu Gly Gly Ser Arg

50 55 60

Lys Pro Asp Gly Ala Met Tyr Ala His Gly Phe Gly Val Ile Val Asp

65 70 75 80

Thr Lys Ala Tyr Lys Asp Gly Tyr Asn Leu Pro Ile Ser Gln Ala Asp

85 90 95

Glu Met Glu Arg Tyr Val Arg Glu Asn Ile Asp Arg Asn Glu His Val

100 105 110

Asn Ser Asn Arg Trp Trp Asn Ile Phe Pro Glu Asp Thr Asn Glu Tyr

115 120 125

Lys Phe Leu Phe Val Ser Gly Phe Phe Lys Gly Asn Phe Glu Lys Gln

130 135 140

Leu Glu Arg Ile Ser Ile Asp Thr Gly Val Gln Gly Gly Ala Leu Ser

145 150 155 160

Val Glu His Leu Leu Leu Gly Ala Glu Tyr Ile Lys Arg Gly Ile Leu

165 170 175

Thr Leu Tyr Asp Phe Lys Asn Ser Phe Leu Asn Lys Glu Ile Gln Phe

180 185 190

<210> 60

<211> 189

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 60

Lys Ser Ser Thr Glu Glu Leu Lys Ala Gln Leu Arg Thr Gln Leu Thr

1 5 10 15

Asn Ile Ser Leu Asp Tyr Leu Gln Leu Val Asp Ile Ser Thr Asp Ser

20 25 30

Lys Gln Asn Arg Leu Phe Glu Met Lys Val Met Asp Leu Phe Ile Asn

35 40 45

Glu Leu Asp Phe Lys Gly Ser His Leu Gly Gly Gly Arg Lys Pro Asp

50 55 60

Gly Ala Val Tyr Thr Thr Asn Tyr Gly Ile Ile Val Asp Thr Lys Ala

65 70 75 80

Tyr Lys Asp Gly Tyr Asn Leu Pro Ile Ser Gln Ala Asp Glu Met Glu

85 90 95

Arg Tyr Val Arg Glu Asn Ile Asp Arg Asn Lys Gly Ile Asn Pro Asn

100 105 110

Glu Trp Trp Thr Ile Phe Pro Ser Ser Ile Asn Asp Phe Thr Phe Leu

115 120 125

Phe Val Ser Gly Tyr Phe Lys Gly Asn Phe Glu Gly Gln Leu Gln Arg

130 135 140

Ile Ser Met Ser Thr Gly Ile Lys Gly Gly Ala Ile Gly Val Glu His

145 150 155 160

Leu Leu Leu Cys Ala Glu Tyr Tyr Lys Arg Gly Ile Leu Ser His Gln

165 170 175

Asp Ile Arg Asp Ser Phe Lys Asn Ala Glu Ile Glu Phe

180 185

<210> 61

<211> 192

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 61

Ile Lys Ser Asn Ile Ser Leu Leu Lys Asp Glu Leu Arg Gly Gln Ile

1 5 10 15

Ser His Ile Ser His Glu Tyr Leu Ser Leu Ile Asp Leu Ala Phe Asp

20 25 30

Ser Lys Gln Asn Arg Leu Phe Glu Met Lys Val Leu Glu Leu Leu Val

35 40 45

Asn Glu Tyr Gly Phe Lys Gly Arg His Leu Gly Gly Ser Arg Lys Pro

50 55 60

Asp Gly Ile Val Tyr Ser Thr Thr Leu Glu Asp Asn Phe Gly Ile Ile

65 70 75 80

Val Asp Thr Lys Ala Tyr Ser Glu Gly Tyr Ser Leu Pro Ile Ser Gln

85 90 95

Ala Asp Glu Met Glu Arg Tyr Val Arg Glu Asn Ser Asn Arg Asp Glu

100 105 110

Glu Val Asn Pro Asn Lys Trp Trp Glu Asn Phe Ser Glu Glu Val Lys

115 120 125

Lys Tyr Tyr Phe Val Phe Ile Ser Gly Ser Phe Lys Gly Lys Phe Glu

130 135 140

Glu Gln Leu Arg Arg Leu Ser Met Thr Thr Gly Val Asn Gly Ser Ala

145 150 155 160

Val Asn Val Val Asn Leu Leu Leu Gly Ala Glu Lys Ile Arg Ser Gly

165 170 175

Glu Met Thr Ile Glu Glu Glu Leu Glu Arg Ala Met Phe Asn Asn Ser Glu

180 185 190

<210> 62

<211> 186

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 62

Lys Leu Ala Lys Ser Ser Gln Ser Glu Thr Lys Glu Lys Leu Arg Glu

1 5 10 15

Lys Leu Arg Asn Leu Pro His Glu Tyr Leu Ser Leu Val Asp Leu Ala

20 25 30

Tyr Asp Ser Lys Gln Asn Arg Leu Phe Glu Met Lys Val Ile Glu Leu

35 40 45

Leu Thr Glu Glu Cys Gly Phe Gln Gly Leu His Leu Gly Gly Ser Arg

50 55 60

Arg Pro Asp Gly Val Leu Tyr Thr Ala Gly Leu Thr Asp Asn Tyr Gly

65 70 75 80

Ile Ile Leu Asp Thr Lys Ala Tyr Ser Ser Gly Tyr Ser Leu Pro Ile

85 90 95

Ala Gln Ala Asp Glu Met Glu Arg Tyr Val Arg Glu Asn Gln Thr Arg

100 105 110

Asp Glu Leu Val Asn Pro Asn Gln Trp Trp Glu Asn Phe Glu Asn Gly

115 120 125

Leu Gly Thr Phe Tyr Phe Leu Phe Val Ala Gly His Phe Asn Gly Asn

130 135 140

Val Gln Ala Gln Leu Glu Arg Ile Ser Arg Asn Thr Gly Val Leu Gly

145 150 155 160

Ala Ala Ala Ser Ile Ser Gln Leu Leu Leu Leu Ala Asp Ala Ile Arg

165 170 175

Gly Gly Arg Met Asp Arg Glu Arg Leu Arg

180 185

<210> 63

<211> 190

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 63

Lys Ile Ser Lys Thr Asn Ile Leu Glu Leu Lys Asp Lys Val Arg Asp

1 5 10 15

Lys Leu Lys Tyr Val Asp His Arg Tyr Leu Ala Leu Ile Asp Leu Ala

20 25 30

Tyr Asp Gly Thr Ala Asn Arg Asp Phe Glu Ile Gln Thr Ile Asp Leu

35 40 45

Leu Ile Asn Glu Leu Lys Phe Lys Gly Val Arg Leu Gly Glu Ser Arg

50 55 60

Lys Pro Asp Gly Ile Ile Ser Tyr Asn Ile Asn Gly Val Ile Ile Asp

65 70 75 80

Asn Lys Ala Tyr Ser Thr Gly Tyr Asn Leu Pro Ile Asn Gln Ala Asp

85 90 95

Glu Met Ile Arg Tyr Ile Glu Glu Asn Gln Thr Arg Asp Glu Lys Ile

100 105 110

Asn Ser Asn Lys Trp Trp Glu Ser Phe Asp Glu Lys Val Lys Asp Phe

115 120 125

Asn Tyr Leu Phe Val Ser Ser Phe Phe Lys Gly Asn Phe Lys Asn Asn

130 135 140

Leu Lys His Ile Ala Asn Arg Thr Gly Val Asn Gly Gly Ala Ile Asn

145 150 155 160

Val Glu Asn Leu Leu Tyr Phe Ala Glu Glu Leu Lys Ala Gly Arg Ile

165 170 175

Ser Tyr Leu Asp Ser Phe Lys Met Tyr Asn Asn Asp Glu Ile

180 185 190

<210> 64

<211> 190

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 64

Val Lys Ser Glu Val Ser Val Phe Lys Asp Tyr Leu Arg Thr His Leu

1 5 10 15

Thr His Val Asp His Arg Tyr Leu Ile Leu Val Asp Leu Gly Phe Asp

20 25 30

Gly Ser Ser Asp Arg Asp Tyr Glu Met Lys Thr Ala Glu Leu Phe Thr

35 40 45

Ala Glu Leu Gly Phe Met Gly Ala Arg Leu Gly Asp Thr Arg Lys Pro

50 55 60

Asp Val Cys Val Tyr His Gly Ala Asn Gly Leu Ile Ile Asp Asn Lys

65 70 75 80

Ala Tyr Gly Lys Gly Tyr Ser Leu Pro Ile Lys Gln Ala Asp Glu Ile

85 90 95

Tyr Arg Tyr Ile Glu Glu Asn Lys Glu Arg Asp Ala Arg Leu Asn Pro

100 105 110

Asn Gln Trp Trp Lys Val Phe Asp Glu Ser Val Thr His Phe Arg Phe

115 120 125

Ala Phe Ile Ser Gly Ser Phe Thr Gly Gly Phe Lys Asp Arg Ile Glu

130 135 140

Leu Ile Ser Met Arg Ser Gly Ile Cys Gly Ala Ala Val Asn Ser Val

145 150 155 160

Asn Leu Leu Leu Met Ala Glu Glu Leu Lys Ser Gly Arg Leu Asp Tyr

165 170 175

Glu Glu Trp Phe Gln Tyr Phe Asp Asn Asp Glu Ile Ser Phe

180 185 190

<210> 65

<211> 192

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 65

Gln Glu Leu Lys Asp Glu Gln Ala Glu Lys Arg Lys Ala Lys Phe Leu

1 5 10 15

Lys Glu Thr Asn Leu Pro Met Lys Tyr Ile Glu Leu Leu Asp Ile Ala

20 25 30

Tyr Asp Gly Lys Arg Asn Arg Asp Phe Glu Ile Val Thr Met Glu Leu

35 40 45

Phe Arg Asn Val Tyr Arg Leu His Ser Lys Leu Leu Gly Gly Gly Arg

50 55 60

Lys Pro Asp Gly Leu Leu Tyr Gln Asp Arg Phe Gly Val Ile Val Asp

65 70 75 80

Thr Lys Ala Tyr Gly Lys Gly Tyr Ser Lys Ser Ile Asn Gln Ala Asp

85 90 95

Glu Met Ile Arg Tyr Ile Glu Asp Asn Lys Arg Arg Asp Glu Asn Arg

100 105 110

Asn Pro Ile Lys Trp Trp Glu Ala Phe Pro Asp Thr Ile Pro Glu Phe

115 120 125

Tyr Phe Met Trp Val Ser Ser Lys Phe Ile Gly Lys Phe Gln Glu Gln

130 135 140

Leu Asp Tyr Thr Ser Asn Glu Thr Gln Ile Lys Gly Ala Ala Leu Asn

145 150 155 160

Val Glu Gln Leu Leu Leu Gly Ala Asp Leu Val Leu Lys Gly Gln Leu

165 170 175

His Ile Ser Asp Leu Pro Ser Tyr Phe Gln Asn Lys Glu Ile Glu Phe

180 185 190

<210> 66

<211> 189

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 66

Lys Val Ser Lys Thr Asn Ile Leu Glu Leu Lys Asp Asn Thr Arg Glu

1 5 10 15

Lys Leu Val Tyr Leu Asp His Arg Tyr Leu Ser Leu Phe Asp Leu Ala

20 25 30

Tyr Asp Asp Lys Ala Ser Arg Asp Phe Glu Ile Gln Thr Ile Asp Leu

35 40 45

Leu Ile Asn Glu Leu Gln Phe Lys Gly Leu Arg Leu Gly Glu Arg Arg

50 55 60

Lys Pro Asp Gly Ile Ile Tyr Gly Val Asn Gly Val Ile Ile Asp Asn

65 70 75 80

Lys Ala Tyr Ser Lys Gly Tyr Asn Leu Pro Ile Arg Gln Ala Asp Glu

85 90 95

Met Ile Arg Tyr Ile Gln Glu Asn Gln Ser Arg Asp Glu Lys Leu Asn

100 105 110

Pro Asn Lys Trp Trp Glu Asn Phe Glu Glu Glu Thr Ser Lys Phe Asn

115 120 125

Tyr Leu Phe Ile Ser Ser Lys Phe Ile Ser Gly Phe Lys Lys Asn Leu

130 135 140

Gln Tyr Ile Ala Asp Arg Thr Gly Val Asn Gly Gly Ala Ile Asn Val

145 150 155 160

Glu Asn Leu Leu Cys Phe Ala Glu Met Leu Lys Ser Gly Lys Leu Glu

165 170 175

Tyr Asn Asp Phe Phe Asn Gln Tyr Asn Asn Asp Glu Ile

180 185

<210> 67

<211> 185

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

polypeptide

<400> 67

Ile Glu Glu Gln Lys Ala Ile Phe Leu Gln Lys Thr Lys Leu Pro Leu

1 5 10 15

Ser Tyr Ile Glu Leu Leu Glu Ile Ala Arg Asp Gly Lys Arg Ser Arg

20 25 30

Asp Phe Glu Phe Ile Thr Met Glu Leu Phe Lys Asn Ile Tyr Lys Ile

35 40 45

Asn Ala Arg Ile Leu Gly Gly Ala Arg Lys Pro Asp Gly Val Leu Tyr

50 55 60

Met Pro Glu Phe Gly Val Ile Val Asp Thr Lys Ala Tyr Ala Asp Gly

65 70 75 80

Tyr Ser Lys Ser Ile Ala Gln Ala Asp Glu Met Ile Arg Tyr Ile Glu

85 90 95

Asp Asn Lys Arg Arg Asp Pro Ser Arg Asn Ser Thr Lys Trp Trp Glu

100 105 110

His Phe Pro Thr Ser Ile Asn Asn Phe Tyr Phe Leu Trp Val Ser Ser

115 120 125

Val Phe Val Asn Lys Phe His Glu Gln Leu Ser Tyr Thr Ala Gln Glu

130 135 140

Thr Gln Thr Val Gly Ala Ala Leu Ser Val Glu Gln Leu Leu Leu Gly

145 150 155 160

Ala Asp Ser Val Leu Lys Gly Asn Leu Thr Thr Glu Lys Phe Ile Asp

165 170 175

Ser Phe Lys Asn Gln Glu Ile Val Phe

180 185

<210> 68

<211> 9

<212> PRT/PROTEIN

<213> Unknown

<220>

<223> Description of the unknown sequence:

The sequence of the peptide motif of the "LAGLIDADG" family

<400> 68

Leu Ala Gly Leu Ile Asp Ala Asp Gly

fifteen

<210> 69

<211> 4

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 69

Asn Glu Leu Asp

one

<210> 70

<211> 4

<212> PRT/PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

peptide

<400> 70

Cys Lys Lys Arg

one

<210> 71

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 71

tgtcaggatt gggttccg 18

<210> 72

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic

oligonucleotide

<400> 72

ctcctgaaag tggccggg 18

<---

Claims (40)

1. An engineered Fok I cleavage half-domain, wherein the engineered cleavage half-domain contains one or more of the following mutations in the form of substitutions: (i) the wild-type Gln (Q) residue at position 481 is replaced by an Ala (A), Cys (C), Asp (D), Ser (S) or Glu (E) residue (Q481A, Q481C, Q481D, Q481S or Q481E) ; (ii) the wild-type Ser (S) residue at position 418 is replaced by a Glu (E) or Asp (D) residue (S418E or S418D); (iii) the wild-type Ile (I) residue at position 479 is replaced by a Gln (Q) or Thr (T) residue (I479Q or I479T); (iv) the wild-type Pro (P) residue at position 478 is replaced by an Asp (D) residue (P478D); (v) the wild-type Lys (K) residue at position 525 is replaced by an Ala (A), Cys (C), Glu (E), Ile (I), Ser (S), Thr (T) or Val (V) residue ( K525A, K525C, K525E, K525I, K525S, K525T or K525V); (vi) the wild-type Arg (R) residue at position 416 is replaced by an Asp (D), Glu (E), His (H) or Asn (N) residue (R416D, R416E, R416H or R416N); (vii) the wild-type Gly (G) residue at position 480 is replaced by an Asp (D) residue (G480D); (viii) the wild-type Ser (S) residue at position 472 is replaced by an Asp (D) residue (S472D); (ix) the wild-type Asn (N) residue at position 476 is replaced by a Glu (E) or Gly (G) residue (N476E or N476G); (x) the wild-type Asn (N) residue at position 527 is replaced by an Asp (D) residue (N527D); (xi) the wild-type Gln (Q) residue at position 531 is replaced by an Arg (R) or Thr (T) residue (Q531R or Q531T); (xii) the wild-type Arg (R) residue at position 422 is replaced by a His (H) residue (R422H); (xiii) the wild-type Ser (S) residue at position 446 is replaced by an Asp (D) residue (S446D); (xiv) the wild-type residue at position 448 is replaced by an Ala (A) residue (K448A); (xv) the wild-type His (H) residue at position 523 is replaced by a Glu (E) residue (H523E); (xvi) the wild-type Leu (L) residue at position 424 is replaced by a Phe (F) residue (L424F); and/or (xvii) the wild-type Asn (N) residue at position 542 is replaced by an Asp (D) residue (N542D), moreover, the numbering of amino acid residues corresponds to the full-length wild-type Fok I cleavage domain, as shown in SEQ ID NO: 1, and moreover, in addition to one or more mutations in the form of substitutions (i) - (xvii) the engineered cleavage domain in the case optionally further contains an additional amino acid mutation at one or more of positions 432, 441, 483, 486, 487, 490, 496, 499, 527, 537, 538 and 559. 2. Constructed cleavage half-domain according to claim 1, containing the following mutations: R416E and R422H mutations; mutations R416D and R422H; mutations R416E and K448A; mutations K448A and I479Q; mutations K448A and Q481A; or mutations K448A and K525A. 3. The engineered cleavage half-domain of claim 1 wherein the wild-type residue at position 481 is replaced by Ala (A). 4. Heterodimer containing (i) a first protein containing the engineered FokI cleavage half-domain according to any one of paragraphs. 1-3, and (ii) a second protein containing a wild-type or engineered FokI cleavage half-domain, wherein the heterodimer site-specifically cleaves the nucleotide sequence. 5. An artificial nuclease containing a designed cleavage half-domain according to any one of paragraphs. 1-3 and a DNA binding domain, wherein the DNA binding domain comprises a zinc finger protein (ZFP), a TALE effector domain, or a single guide RNA (sRNA). 6. The artificial nuclease of claim 5, wherein the DNA binding domain is ZFP and the artificial nuclease is a zinc finger nuclease (ZFN) comprising first and second ZFNs. 7. The artificial nuclease of claim 6 wherein said first or second ZFN zinc finger protein contains one or more mutations at positions -14, -9 and -5. 8. Polynucleotide for obtaining an artificial nuclease, encoding an artificial nuclease according to any one of paragraphs. 5-7. 9. Composition for delivering polynucleotides to a target cell, containing the first and second polynucleotides encoding the heterodimer according to claim 4. 10. An isolated cell comprising the composition of claim 9 wherein the heterodimer site-specifically cleaves a nucleotide sequence in the cell. 11. A method for cleavage of genomic cellular chromatin in a region of interest, comprising: expression of an artificial nuclease containing a designed cleavage domain according to any one of paragraphs. 5-7, in a cage wherein the nuclease site-specifically cleaves a nucleotide sequence in a region of interest in the genomic cell chromatin. 12. The method of claim 11, further comprising bringing the cell into contact with a donor polynucleotide; wherein cleavage of the cellular chromatin facilitates homologous recombination between the donor polypeptide and the cellular chromatin. 13. The method of claim 11, wherein the artificial nuclease is a ZFN comprising a first and a second ZFN expressed from the first and second polynucleotides, and wherein the ratio of the first and second ZFN is not one to one and optionally is a 1:2 ratio , 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10, 1:20 or 1:30. 14. A method for cleaving at least two target sites in genomic cell chromatin, comprising cleavage of at least the first and second target site in the genomic cellular chromatin, each target site being cleaved using a composition containing two or more artificial nucleases according to any one of paragraphs. 5-7. 15. Dedicated cell to obtain an artificial nuclease, containing an artificial nuclease according to any one of paragraphs. 5-7. 16. A cell line for obtaining a heterodimer containing a composition according to claim 9. 17. Composition for site-specific cleavage of a nucleotide sequence in a cell, containing the first and second polynucleotides encoding the first and second ZFN according to claim 7 or claim 8, respectively, and the ratio of the first and second ZFN is not one to one, optionally represents the ratio 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10, 1:20 or 1:30.

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