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Building IP: SGMO Patent Application re "ENGINEERED TARGET SPECIFIC NUCLEASES"
ENGINEERED TARGET SPECIFIC NUCLEASES Described herein are engineered nucleases comprising mutations in the cleavage domain (e.g., FokI or homologue thereof) and/or DNA binding domain (zinc finger protein, TALE, single guide RNA) such that on-target specificity is increased.
1. A polynucleotide encoding a Flavobacterium okeanokoites (FokI) cleavage half-domain, wherein the engineered cleavage half-domain comprises one or more substitution mutations of a wild-type residue of the full length FokI cleavage domain as shown in SEQ ID NO:1, wherein the one or more substitution mutations are as follows: (i) the wild type Gln (Q) residue at position 481 is replaced with an Ala (A), Cys (C), Asp (D), Ser (S), or Glu (E) residue (Q481A, Q481C, Q481D, Q481S, Q481E); (ii) the wild-type Ser (S) residue at position 418 is replaced with a Glu (E) or Asp (D) residue (S418E); (iii) the wild-type Ile (I) residue at position 479 is replaced with a Gln (Q) or Thr (T) residue (I479Q or I479T); (iv) the wild-type Pro (P) residue at position 478 is replaced an Asp (D) residue (P478D); (v) the wild-type Lys (K) residue at position 525 is replaced with an Ala (A), Cys (C), Glu (E), Ile (I), Ser (S), Thr (T) or Val (V) residue (K525A, K525C, K525E, K2521, K525S, K5252T or K525V); (vi) the wild-type Arg (R) residue as position 416 is replaced with 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 with an Asp (D) residue (G480D); (viii) the wild-type Ser (S) residue a position 472 is replaced with an Asp (D) residue (S472D); (ix) the wild-type Asn (N) residue at position 476 is replaced with a Glu (E) residue or a Gly (G) residue (N476E, N476G); (x) the wild-type Asn (N) residue at position 527 is replaced with an Asp (D) residue (N527D); (xi) the wild-type Gln (Q) residue at position 531 is replaced with an Arg (R) or Thr (T) residue (Q531R or Q531T); (xii) the wild-type Arg (R) residue at position 422 is replaced with a His (H) residue (R422H); (xiii) the wild-type Ser (S) residue at position 446 is replaced with an Asp (D) residue (S446D); (xiv) the wild-type residue at position 448 is replaced with an Ala (A) residue (K448A); (xv) the wild-type His (H) residue at position 523 is replaced with a Glu (E) residue (H523E); (xvi) the wild-type Leu (L) residue at position 424 is replaced with a Phe (F) residue (L424F); and/or (xvi) the wild-type Asn (N) residue at position 542 is replaced with an Asp (D) residue (N541D). 2. The polynucleotide of claim 1, wherein the engineered comprises mutations as follows: the R416D, R416E, R416H, or R416N mutation and the R422H mutation; the R416D, R416E, R416H, or R416N mutation and the K448A mutation; the K448A and I479Q mutations; the K448A and Q481A mutations; and/or K448A mutation and the K525A, K525C, K525E, K2521, K525S, K5252T or K525V mutation. 3. The polynucleotide of claim 1, wherein the engineered cleavage half-domain further comprises an additional mutation at one or more of positions 432, 441, 483, 486, 487, 490, 496, 499, 527, 537, 538 and 559. 4. One or more polynucleotides encoding a heterodimer comprising a first engineered cleavage half-domain encoded by the polynucleotide of claim 1 and a second cleavage half-domain. 5. The one or more polynucleotides of claim 4, wherein the heterodimer comprises an artificial nuclease comprising an engineered cleavage half-domain and a DNA-binding domain. 6. The one or more polynucleotides of claim 5, wherein the DNA-binding domain comprises a zinc finger protein, a TALE-effector domain or a single guide RNA (sgRNA). 7. The one or more polynucleotides of claim 6, wherein the zinc finger protein comprises at least three zinc finger DNA-binding domains, wherein each zinc finger DNA-binding domain comprises two beta sheets, an alpha helix, a recognition helix region that binds to a nucleotide sequence, and further wherein one or more of the zinc finger DNA-binding domains comprise mutations in amino acid residues (-5), (-9) and/or (-14), numbered relative to the start of the alpha helix region. 8. An isolated cell comprising the polynucleotide of claim 1. 9. An isolated cell comprising the one or more polynucleotides of claim 4. 10. An isolated cell comprising the one or more polynucleotides of claim 5. 11. A method for cleaving genomic cellular chromatin in a region of interest, the method comprising: expressing one or more polynucleotides according to claim 5 in a cell, wherein the nuclease site-specifically cleaves a nucleotide sequence in the region of interest of the genomic cellular chromatin. 12. The method of claim 11, further comprising contacting the cell with a donor polynucleotide; wherein cleavage of the cellular chromatin facilitates homologous recombination between the donor polypeptide and the cellular chromatin. 13. A method of cleaving at least two target sites in genomic cellular chromatin, the method comprising: cleaving at least first and second target sites in genomic cellular chromatin, wherein each target site is cleaved using a composition comprising the one or more polynucleotides according to claim 5. 14. An isolated cell or cell line comprising at least one site-specific genomic modification made by the method of claim 11. 15. A composition comprising first and second polynucleotides of claim 4, wherein the artificial nuclease comprises a nuclease comprising first and second DNA-binding domains, wherein the first polynucleotide encodes the first nuclease and a second polynucleotide encodes the second nuclease and the ratio of the first and second nucleases is not one to one. 16. A zinc finger protein comprising at least three zinc finger DNA-binding domains, wherein each zinc finger DNA-binding domain comprises two beta sheets, an alpha helix, a recognition helix region that binds to a nucleotide sequence, and further wherein one or more of the zinc finger DNA-binding domains comprise mutations in amino acid residues (-5), (-9) and/or (-14), numbered relative to the start of the alpha helix region. 17. The zinc finger protein of claim 16, wherein the amino acids at (-5), (-9) and/or (-14) are mutated to an alanine (A), a leucine (L), a serine (S), an aspartic acid (N), glutamine (E), tyrosine (Y) and/or glutamine (Q) reside. 18. The zinc finger protein of claim 17, wherein the Arg (R) at position -5 is changed to a Tyr (Y), Asp (N), Glu (E), Leu (L), Gln (Q) or Ala (A) residue; the Arg (R) at position (-9) is replaced with Ser (S), Asp (N), or Glu (E); and/or the Arg (R) at position (-14) is replaced with Ser (S) or Gln (Q) residue. 19. A zinc finger nuclease comprising the zinc finger protein of claim 16 and a cleavage domain. 20. The zinc finger nuclease according to claim 19, wherein the cleavage domain comprises an engineered FokI cleavage domain. 21. The zinc finger nuclease according to claim 20, wherein the engineered FokI cleavage domain is a cleavage half-domain comprising one or more mutations in residues 416, 418, 421, 422, 424, 446, 448, 472, 478, 479, 480, 481, 525 or 542, wherein the amino acid residues are numbered relative to full length FokI wild-type cleavage domain as shown in SEQ ID NO:1. 22. The zinc finger nuclease according to claim 21, wherein the engineered FokI cleavage domain comprises one or more substitution mutations of a wild-type residue of the full length FokI cleavage domain as shown in SEQ ID NO:1, wherein the one or more substitution mutations are as follows: (i) the wild type Gln (Q) residue at position 481 is replaced with an Ala (A), Cys (C), Asp (D), Ser (S), or Glu (E) residue (Q481A, Q481C, Q481D, Q481S, Q481E); (ii) the wild-type Ser (S) residue at position 418 is replaced with a Glu (E) or Asp (D) residue (S418E); (iii) the wild-type Ile (I) residue at position 479 is replaced with a Gln (Q) or Thr (T) residue (I479Q or I479T); (iv) the wild-type Pro (P) residue at position 478 is replaced an Asp (D) residue (P478D); (v) the wild-type Lys (K) residue at position 525 is replaced with an Ala (A), Cys (C), Glu (E), Ile (I), Ser (S), Thr (T) or Val (V) residue (K525A, K525C, K525E, K2521, K525S, K5252T or K525V); (vi) the wild-type Arg (R) residue as position 416 is replaced with 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 with an Asp (D) residue (G480D); (viii) the wild-type Ser (S) residue a position 472 is replaced with an Asp (D) residue (S472D); (ix) the wild-type Asn (N) residue at position 476 is replaced with a Glu (E) residue or a Gly (G) residue (N476E, N476G); (x) the wild-type Asn (N) residue at position 527 is replaced with an Asp (D) residue (N527D); (xi) the wild-type Gln (Q) residue at position 531 is replaced with an Arg (R) or Thr (T) residue (Q531R or Q531T); (xii) the wild-type Arg (R) residue at position 422 is replaced with a His (H) residue (R422H); (xiii) the wild-type Ser (S) residue at position 446 is replaced with an Asp (D) residue (S446D); (xiv) the wild-type residue at position 448 is replaced with an Ala (A) residue (K448A); (xv) the wild-type His (H) residue at position 523 is replaced with a Glu (E) residue (H523E); (xvi) the wild-type Leu (L) residue at position 424 is replaced with a Phe (F) residue (L424F); and/or (xvi) the wild-type Asn (N) residue at position 542 is replaced with an Asp (D) residue (N541D). 23. The zinc finger nuclease according to claim 22, wherein the engineered comprises mutations as follows: the R416D, R416E, R416H, or R416N mutation and the R422H mutation; the R416D, R416E, R416H, or R416N mutation and the K448A mutation; the K448A and I479Q mutations; the K448A and Q481A mutations; and/or K448A mutation and the K525A, K525C, K525E, K2521, K525S, K5252T or K525V mutation. 24. The zinc finger nuclease according to claim 21, wherein the engineered cleavage half-domain further comprises an additional mutation at one or more of positions 432, 441, 483, 486, 487, 490, 496, 499, 527, 537, 538 and 559. 25. A zinc finger nuclease comprising a first zinc finger nuclease according to claim 19 and second zinc finger nuclease. 26. One or more polynucleotides encoding the zinc finger nuclease of claim 19. 27. An isolated cell comprising the one or more polynucleotides of claim 26. 28. A method for cleaving genomic cellular chromatin in a region of interest, the method comprising: expressing the one or more polynucleotides according to claim 26 in a cell, wherein the zinc finger nuclease is expressed in the cell and site-specifically cleaves a nucleotide sequence in the region of interest of the genomic cellular chromatin. 29. The method of claim 28, further comprising contacting the cell with a donor polynucleotide; wherein cleavage of the cellular chromatin facilitates homologous recombination between the donor polypeptide and the cellular chromatin. 30. An isolated cell or cell line comprising at least one site-specific genomic modification made by a zinc finger nuclease according to claim 19. 31. A composition comprising first and second polynucleotides of claim 26, wherein the zinc finger nuclease comprises first and second zinc finger nucleases, wherein the first polynucleotide encodes the first zinc finger nuclease and a second polynucleotide encodes the second zinc finger nuclease and the ratio of the first and second zinc finger nucleases is not one to one. 32. A zinc finger nuclease that cleaves a T cell receptor constant region gene (TRAC) gene, the zinc finger nuclease comprising a first zinc finger nuclease comprising an ELD engineered FokI cleavage domain and a first zinc finger DNA-binding domain that binds to a target site in the TRAC gene and a second zinc finger nuclease comprising a KKR FokI cleavage domain and a second zinc finger DNA-binding domain, the first and second zinc finger DNA-binding domains comprising wherein (i) at least one of the ELD or KKR FokI cleavage domains further comprises a mutation in the FokI cleavage domain selected from the group consisting of: K393S, K394S, R398S, K400S, K402S, R416S, R422S, K427S, K434S, R439S, K441S, R447S, K448S, K469S, R487S, R495S, K497S, K506S, K516S, K525S, K529S, R534S, K559S, R569S, and R570S, numbered relative to wild-type FokI; and/or (ii) at least one of the first and second zinc finger DNA-binding domains comprises one or more mutations in amino acid residues (-5), (-9) and/or (-14), numbered relative to the start of the alpha helix region. 33. The zinc finger nuclease of claim 32, wherein the mutation in the ELD and/or KKR FokI cleavage domain is a R416S, R422S, R447S, K448S or K525S mutation. 34. The zinc finger nuclease of claim 32, wherein the amino acid at residue at position -5 is mutated in one, two or three fingers. 35. One or more polynucleotides encoding the zinc finger nuclease according to claim 45. 36. An isolated cell comprising the one or more polynucleotides of claim 35. 37. A method for cleaving a TCR alpha (TRAC) gene in a mammalian cell, the method comprising: expressing the one or more polynucleotides according to claim 35 in a cell, wherein the zinc finger nuclease is expressed in the cell and site-specifically cleaves to a nucleotide sequence in the TRAC gene. 38. The method of claim 37, further comprising contacting the cell with a donor polynucleotide; wherein cleavage of the cellular chromatin facilitates homologous recombination between the donor polypeptide and the cellular chromatin. 39. An isolated cell comprising at least one site-specific genomic modification in a TRAC gene made by a zinc finger nuclease according to claim 32. 40. A composition comprising first and second polynucleotides of claim 35, wherein the zinc finger nuclease comprises first and second zinc finger nucleases, wherein the first polynucleotide encodes the first zinc finger nuclease and a second polynucleotide encodes the second zinc finger nuclease and the ratio of the first and second zinc finger nucleases is not one to one. CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a divisional application of U.S. patent application Ser. No. 15/685,580, filed Aug. 24, 2017, which claims the benefit of U.S. Provisional Application No. 62/378,978, filed Aug. 24, 2016 and U.S. Provisional Application No. 62/443,981, filed Jan. 9, 2017, the disclosures of which are hereby incorporated by reference in their entireties. STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH [0002] Not applicable. TECHNICAL FIELD [0003] The present disclosure is in the fields of polypeptide and genome engineering and homologous recombination. BACKGROUND [0004] Artificial nucleases, such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), the CRISPR/Cas system with an engineered crRNA/tracr RNA (single guide RNA'), also referred to as RNA guided 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), comprise DNA binding domains (nucleotide or polypeptide) associated with or operably linked to cleavage domains, and have been used for targeted alteration of genomic sequences. For example, nucleases have been used to insert exogenous sequences, inactivate one or more endogenous genes, create organisms (e.g., crops) and cell lines with altered gene expression patterns, and the like. See, e.g., U.S. Pat. Nos. 9,255,250; 9,200,266; 9,045,763; 9,005,973; 8,956,828; 8,945,868; 8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 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; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960 and 20150056705. For instance, a pair of nucleases (e.g., zinc finger nucleases, TALENs, dCas-Fok fusions) may be used to cleave genomic sequences. Each member of the pair generally includes an engineered (non-naturally occurring) DNA-binding protein linked to one or more cleavage domains (or half-domains) of a nuclease. When the DNA-binding proteins bind to their target sites, the cleavage domains that are linked to those DNA binding proteins are positioned such that dimerization and subsequent cleavage of the genome can occur. [0005] Generally, intermolecular ion pairs (salt bridges) are essential for many DNA-protein interactions. Often, charged amino acid side chains (i.e. --NH3+, .dbd.NH2+) interact with the negatively charged phosphate groups of the DNA backbone to form a salt bridge. These ion pairs can be quite dynamic and can alternate between direct pairing of the two ions and pairing that is a `solvent-separated ion pair` when a solvent (e.g. a water molecule) is inserted between the two ions (Chen et al (2015) J Phys Chem Lett 6:2733-2737). [0006] In regards to zinc finger proteins, the specificity of a ZFP for a target DNA sequence is dependent upon sequence specific contacts between the zinc finger domains and specific DNA bases. In addition, the zinc finger domains also comprise amino acid residues that take part in non-specific ion pair interactions with the phosphates of the DNA backbone. Elrod-Erickson et al ((1996) Structure 4:1171) demonstrated through co-crystallization of a zinc finger protein and its cognate DNA target that there are specific amino acids capable of interacting with the phosphates on the DNA backbone through formation of hydrogen bonds. Zinc finger proteins that employ the well-known Zif268 backbone typically have an arginine as the amino terminal residue of their second strand of .beta.-sheet, which is also the second position carboxyl-terminal to the second invariant cysteine (see FIG. 5A). This position can be referred to as (-5) within each zinc finger domain, as it is 5.sup.th residue preceding the start of the .alpha.-helix (FIG. 5A). The arginine at this position can interact with a phosphate on the DNA backbone via formation of a charged hydrogen bond with its side-chain guanidinium group. Zinc finger proteins in the Zif268 backbone also frequently have a lysine at a position that is 4 residues amino-terminal to the first invariant cysteine. This position can be referred to as (-14) within each finger, as it is 14.sup.th residue preceding the start of the .alpha.-helix for zinc fingers with two residues between the zinc coordinating cysteine residues (FIG. 5A). The lysine can interact with a phosphate on the DNA backbone via formation of a water-mediated charged hydrogen bond with its side-chain amino group. Since phosphate groups are found all along the DNA backbone, this type of interaction between the zinc finger and a DNA molecule is generally considered to be non-sequence specific (J. Miller, Massachusetts Institute of Technology Ph.D. Thesis, 2002). [0007] Recent studies have hypothesized that non-specific phosphate contacting side chains in some nucleases may also account for some amount of non-specificity cleavage activity of those nucleases (Kleinstiver et al, (2016) Nature 529(7587):490-5; Guilinger et al (2014) Nat Meth: 429-435). Researchers have proposed that these nucleases may possess `excess DNA-binding energy`, meaning that the nucleases may have a greater affinity for their DNA target than is required to substantially bind and cleave the target site. Thus, attempts were made to decrease the cationic charges in the TALE DNA binding domain (Guilinger, ibid) or the Cas9 DNA binding domain (Kleinstiver, ibid) to lower the DNA-binding energy of these nucleases, which resulted in increased cleavage specificity in vitro. However, additional studies (Sternberg et al (2015) Nature 527(7576):110-113) also suggest a role in proper folding and activation of the Cas9 nuclease domain for some of the cationic amino acids that were mutated in the Kleinstiver study of the Cas9 DNA binding domain. Thus, the exact role of these amino acids in Cas9 activity is not known. [0008] For optimal cleavage specificity by a sequence-selective (artificial) nuclease, it is desirable to arrange conditions so that on-target binding and activity is not saturating. Under saturating conditions--by definition--an excess of nuclease is used over what is necessary to achieve complete on-target activity. This excess provides no on-target benefit but can nonetheless result in increased cleavage at off-target sites. For monomeric nucleases, saturating conditions may be readily avoided by performing a simple dose response study to identify and avoid the saturating plateau on a titration curve. However, for a dimeric nuclease such as ZFN, TALEN or dCas-Fok, identifying and avoiding saturating conditions may be more complicated if the binding affinities of the individual monomers are dissimilar. In such cases, a dose response study using a simple 1:1 nuclease ratio will only reveal the saturation point of the weaker binding monomer. Under such a scenario, if, for example, monomer affinities differ by a factor of 10, then at the saturation point identified in a 1:1 titration study the higher affinity monomer will be present at a concentration that is 10-fold higher than it needs to be. The resulting excess of the higher affinity monomer can in turn lead to increased off-target activity without providing any beneficial increase in cleavage at the intended target, potentially leading to a decreased specificity overall for any given nuclease pair. [0009] To decrease off-target cleavage events, engineered obligate heterodimeric cleavage half-domains have been developed. See, e.g., U.S. Pat. Nos. 7,914,796; 8,034,598; 8,962,281 and 8,623,618; U.S. Patent Publication Nos. 20080131962 and 20120040398. These obligate heterodimers dimerize and cleave their targets only when the differing engineered cleavage domains are positioned at the appropriate target site by the ZFPs, thereby reducing and/or eliminating monomeric off-target cleavage. [0010] However, there remains a need for additional methods and compositions to engineered nuclease cleavage systems to decrease off-target cleavage activity. SUMMARY [0011] The present disclosure provides methods and compositions to increase the specificity of a nuclease (e.g., nuclease pair) for its intended target relative to other unintended cleavage sites, also known as off-target sites. Thus, described herein are artificial nucleases (e.g., zinc finger nucleases (ZFNs), TALENs, CRISPR/Cas nucleases) comprising mutations in one or more of the DNA binding domain regions (e.g., the backbone of a zinc finger protein or TALE) and/or one or more mutations in a FokI nuclease cleavage domain or cleavage half domain. Further, described herein are methods to increase specificity of cleavage activity by using these novel nucleases (e.g., ZFNs, TALENs, etc.) and/or through independent titration of the engineered cleavage half-domain partners of a nuclease complex. When used individually or in combination, the methods and compositions of the invention provide surprising and unexpected increases in targeting specificity via reductions in off-target cleavage activity. The disclosure also provides methods of using these compositions for targeted cleavage of cellular chromatin in a region of interest and/or integration of a transgene via targeted integration at a predetermined region of interest in cells. [0012] Thus, in one aspect, described herein is an engineered nuclease cleavage half domain comprising one or more mutations as compared to a parental (e.g., wild-type) cleavage domain from which these mutants are derived. In certain embodiments, the one or more mutations are one or more of the mutations shown in any of the appended Tables and Figures, including any combination of these mutants with each other and with other mutants (such as dimerization and/or catalytic domain mutants 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 to non-positively charged residues (e.g., mutations of K and R residues (e.g., mutated to S); N residues (e.g., to D), and Q residues (e.g., to E); mutations to residues that are predicted to be close to the DNA backbone based on molecular modeling and that show variation in FokI homologs (FIGS. 1 and 17); and/or mutations at other residues (e.g., U.S. 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 criteria. The initial promising mutations were positively charged residues predicted to be close to the DNA backbone when FokI is bound to DNA. The cleavage domains described herein may include one, two, three, four, five or more of the mutations described herein and may further include additional known mutations. Therefore, mutations of the invention do not include specific mutations disclosed in U.S. Pat. No. 8,623,618 (e.g., N527D, S418P, K448M, Q531R, etc.) when used alone; however, provided here are novel mutants that can be used in combination with the mutants of U.S. Pat. No. 8,623,618. Nickase mutants wherein one of the catalytic nuclease domains in a dimer pair comprises one or more mutations rendering it catalytically inactive (see U.S. Pat. Nos. 8,703,489; 9,200,266; and 9,631,186) may also be used in combination with any of the mutants described herein. Nickases can be ZFN nickases, TALEN nickases and CRISPR/dCas systems. [0014] In certain embodiments, the engineered cleavage half domains are derived from FokI or FokI homologues and comprise a mutation in one or more of amino acid residues 416, 422, 447, 448, and/or 525, numbered relative to the wild-type full length FokI as shown in SEQ ID NO:1 or corresponding residues in FokI homologues (see, FIG. 17). In other embodiments, the cleavage half domains derived from FokI comprises a mutation in 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. The mutations may include mutations to residues found in natural restriction enzymes homologous to FokI at the corresponding positions (FIG. 17). In certain embodiments, the mutations are substitutions, for example substitution of the wild-type residue with any different amino acid, for example 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 appended Tables and Figures. In certain embodiments, the FokI nuclease domain comprises a mutation at one or more of 416, 422, 447, 479 and/or 525 (numbered relative to wild-type, SEQ ID NO:1). The nuclease domains may also comprise 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, e.g., U.S. Pat. No. 8,623,618. In still further embodiments, the cleavage domain includes mutations at 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 certain embodiments, the variant cleavage domains described herein include mutations to the residues involved in nuclease dimerization (dimerization domain mutations), and one or more additional mutations; for example to phosphate contact 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 of the dimerization domain, for example in amino acid residues that may participate in phosphate contact. In a preferred embodiment, the mutation at positions 416, 422, 447, 448 and/or 525 comprise replacement of a positively charged amino acid with an uncharged or a negatively charged amino acid. In other embodiments, mutations at positions 446, 472 and/or 478 (and optionally additional residues for example in the dimerization or catalytic domains) are made. [0015] In other embodiments, the engineered cleavage half domain comprises mutations in the dimerization domain, for example, amino acid residues 490, 537, 538, 499, 496 and 486 in addition to the mutations described herein. In a preferred embodiment, the invention provides fusion proteins wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type Gln (Q) residue at position 486 is replaced with a Glu (E) residue, the wild-type Ile (I) residue at position 499 is replaced with a Leu (L) residue and the wild-type Asn (N) residue at position 496 is replaced with an Asp (D) or a Glu (E) residue ("ELD" or "ELE") in addition to one or more mutations described herein. In another embodiment, the engineered cleavage half domains are derived from a wild-type FokI or FokI homologue cleavage half domain and comprise mutations in the amino acid residues 490, 538 and 537, numbered relative to wild-type FokI (SEQ ID NO:1) in addition to the one or more mutations at amino acid residues 416, 422, 447, 448, or 525. In a preferred embodiment, the invention provides a fusion protein, wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue, the wild-type Ile (I) residue at position 538 is replaced with a Lys (K) residue, and the wild-type His (H) residue at position 537 is replaced with a Lys (K) residue or an Arg (R) residue ("KKK" or "KKR") (see U.S. Pat. No. 8,962,281, incorporated by reference herein) in addition to one or more mutations described herein. [0016] In another embodiment, the engineered cleavage half domains are derived from a wild-type FokI cleavage half domain or homologues thereof and comprise mutations in the amino acid residues 490, and 538, numbered relative to wild-type FokI in addition to the one or more mutations at amino acid residues 416, 422, 447, 448, or 525. In a preferred embodiment, the invention provides a fusion protein, wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type Glu (E) residue at position 490 is replaced with a Lys (K) residue, and the wild-type Ile (I) residue at position 538 is replaced with a Lys (K) residue ("KK") in addition to one or more mutations at positions 416, 422, 447, 448, or 525. In a preferred embodiment, the invention provides a fusion protein, wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type Gln (Q) residue at position 486 is replaced with an Glu (E) residue, and the wild-type Ile (I) residue at position 499 is replaced with a Leu (L) residue ("EL") (See U.S. Pat. No. 8,034,598, incorporated by reference herein) in addition to one or more mutations at positions 416, 422, 447, 448, or 525. [0017] In one aspect, the invention provides fusion molecules wherein the engineered cleavage half-domain comprises a polypeptide in which the wild-type amino acid residue 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 FokI catalytic domain are mutated. In some embodiments, the one or more mutations alter the wild type amino acid from a positively charged residue to a neutral residue or a negatively charged residue. In any of these embodiments, the mutants described may also be made in a FokI domain comprising one or more additional mutations. In preferred embodiments, these additional mutations are in the dimerization domain, e.g. 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 yet another aspect, any of the engineered cleavage half domains described above may be incorporated into artificial nucleases, for example by associating them with a DNA-binding domain, including but not limited to zinc finger nucleases, TALENs, CRISPR/Cas nucleases, and the like. The zinc finger proteins of the zinc finger nucleases may comprise non-canonical zinc-coordinating residues (e.g. CCHC rather than the canonical C2H2 configuration, see U.S. Pat. No. 9,234,187). [0019] In another aspect, fusion molecules comprising a DNA binding domain and an engineered FokI or homologue thereof cleavage half-domain as described herein that produce an artificial nuclease are provided. In certain embodiments, the DNA-binding domain of the fusion molecule is a zinc finger binding domain (e.g., an engineered zinc finger binding domain). In other embodiments, the DNA-binding domain is a TALE DNA-binding domain. In still further embodiments, the DNA binding domain comprises a DNA binding molecule (e.g. 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 only capable of cleaving only one strand of a double-stranded DNA molecule, forming a nickase (see U.S. Pat. No. 9,200,266). [0020] The methods and compositions of the invention also include mutations to one or more amino acids within the DNA binding domain outside the residues that recognize the nucleotides of the target sequence (e.g., one or more mutations to the `ZFP backbone` (outside the DNA recognition helix region) or to the `TALE backbone` (outside of the RVDs)) that can interact non-specifically with phosphates on the DNA backbone. Thus, in certain embodiments, the invention includes mutations of cationic amino acid residues in the ZFP backbone that are not required for nucleotide target specificity. In some embodiments, these mutations in the ZFP backbone comprise mutating a cationic amino acid residue to a neutral or anionic amino acid residue. In some embodiments, these mutations in the ZFP backbone comprise mutating a polar amino acid residue to a neutral or non-polar amino acid residue. In preferred embodiments, mutations at made at position (-5), (-9) and/or position (-14) relative to the DNA binding helix. In some embodiments, a zinc finger may comprise one or more mutations at (-5), (-9) and/or (-14). In further embodiments, one or more zinc fingers in a multi-finger zinc finger protein may comprise mutations in (-5), (-9) and/or (-14). In some embodiments, the amino acids at (-5), (-9) and/or (-14) (e.g. an arginine (R) or lysine (K)) are mutated to an alanine (A), leucine (L), Ser (S), Asp (N), Glu (E), Tyr (Y) and/or glutamine (Q). [0021] In another aspect, polynucleotides encoding any of the engineered cleavage half-domains or fusion proteins as described herein are provided. [0022] In yet another aspect, cells comprising any of the nucleases, polypeptides (e.g., fusion molecules or fusion polypeptides) and/or polynucleotides as described herein are also provided. In one embodiment, the cells comprise a pair of fusion polypeptides, one fusion polypeptide comprising, in addition to one or more mutations in 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 one fusion polypeptide comprising, 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, a KKK or KKR cleavage half-domain (see U.S. Pat. No. 8,962,281). [0023] In any of these fusion polypeptides described herein, the ZFP partners may further comprise mutations in the zinc finger DNA binding domain in the (-5), (-9) and/or (-14) positions. In some embodiments, the Arg (R) at position -5 is changed to a Tyr (Y), Asp (N), Glu (E), Leu (L), Gln (Q), or Ala (A). In other embodiments, the Arg (R) at position (-9) is replaced with Ser (S), Asp (N), or Glu (E). In further embodiments, the Arg (R) at position (-14) is replaced with Ser (S) or Gln (Q). In other embodiments, the fusion polypeptides can comprise mutations in the zinc finger DNA binding domain where the amino acids at the (-5), (-9) and/or (-14) positions are changed to any of the above listed amino acids in any combination. [0024] Also provided herein are cells that have been modified by the polypeptides and/or polynucleotides of the invention. In some embodiments, the cells comprise a nuclease-mediated insertion of a transgene, or a nuclease-mediated knock out of a gene. The modified cells, and any cells derived from the modified cells do not necessarily comprise the nucleases of the invention more than transiently, but the genomic modifications mediated by such nucleases remain. [0025] In yet another aspect, methods for targeted cleavage of cellular chromatin in a region of interest; methods of causing homologous recombination to occur in a cell; methods of treating infection; and/or methods of treating disease are provided. These methods maybe practiced in vitro, ex vivo or in vivo or a combination thereof. The methods involve cleaving cellular chromatin at a predetermined region of interest in cells by expressing a pair of fusion polypeptides as described herein (i.e., a pair of fusion polypeptides in which one or both fusion polypeptide(s) comprises the engineered cleavage half-domains as described herein). In certain embodiments, the targeted cleavage of the on-target site is increased by at least 50 to 200% (or any value therebetween) or more, including 50%-60% (or any value therebetween), 60%-70% (or any value therebetween), 70%-80% (or any value therebetween), 80%-90% (or any value therebetween, 90% to 200% (or any value therebetween), as compared to cleavage domains without the mutations as described herein. Similarly, using the methods and compositions as described herein, off-target site cleavage is reduced by 1-100 or more-fold, including but not limited to 1-50-fold (or any value therebetween). [0026] The engineered cleavage half domains described herein can be used in methods for targeted cleavage of cellular chromatin in a region of interest and/or homologous recombination at a predetermined region of interest in cells. Cells include cultured cells, cell lines, cells in an organism, cells that have been removed from an organism for treatment in cases where the cells and/or their descendants will be returned to the organism after treatment, and cells removed from an organism, modified using the fusion molecules of the invention, and then returned to the organism in a method of treatment (cell therapy). A region of interest in cellular chromatin can be, for example, a genomic sequence or portion thereof. Compositions include fusion molecules or polynucleotides encoding fusion molecules that comprise a DNA binding molecule (e.g., an engineered zinc finger or 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, e.g., by delivering the fusion molecule to the cell as a polypeptide, or by delivering a polynucleotide encoding the fusion molecule to a cell, wherein the polynucleotide, if DNA, is transcribed and is translated, to generate the fusion molecule. Further, if the polynucleotide is an mRNA encoding the fusion molecule, following delivery of the mRNA to the cell, the mRNA is translated, thus generating the fusion molecule. [0028] In other aspects of the invention are provided methods and compositions for increasing engineered nuclease specificity. In one aspect, methods are provided for increasing overall on-target cleavage specificity by decreasing off-target cleavage activity. In some embodiments, the engineered cleavage half-domain partners of an engineered nuclease complex are used to contact a cell, where each partner of the complex is given in a ratio to the other partner other than one to one. In some embodiments, the ratio of the two partners (half cleavage domains) is given at a 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10 or 1:20 ratio, or any value therebetween. In other embodiments, the ratio of the two partners is greater than 1:30. In other embodiments, the two partners are deployed at a ratio that is chosen to be different from 1:1. In some aspects, each partner is delivered to the cell as an mRNA or is delivered in a viral or non-viral vector where different quantities of mRNA or vector encoding each partner are delivered. In further embodiments, each partner of the nuclease complex may be comprised on a single viral or non-viral vector, but is deliberately expressed such that one partner is expressed at a higher or lower value that the other, ultimately delivering the cell a ratio of cleavage half domains that is other than one to one. In some embodiments, each cleavage half domain is expressed using different promoters with different expression efficiencies. In other embodiments, the two cleavage domains are delivered to the cell using a viral or non-viral vector where both are expressed from the same open reading frame, but the genes encoding the two partners are separated by a sequence (e.g. self-cleaving 2A sequence or IRES) that results in the 3' partner being expressed at a lower rate, such that the ratios of the two partners are 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:9, 1:10 or 1:20 ratio, or any value therebetween. In other embodiments, the two partners are deployed at a ratio that is chosen to be different from 1:1. [0029] Also provided are methods to decrease off-target nuclease activity when two or more nuclease complexes are used. For example, the invention provides methods for varying the ratio of DNA binding molecules when two or more nuclease complexes are used. In some embodiments, the DNA binding molecules are polypeptide DNA binding domains (e.g., ZFNs, TALENs, dCas-Fok, megaTALs, meganucleases), while in others, the DNA binding molecules are guide RNAs for use with RNA-guided nucleases. In preferred embodiments, the ratio of the 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 ratio, or any value therebetween. In other embodiments, the two DNA binding molecules are deployed at a ratio that is chosen to be different from 1:1. In some aspects, the non-1:1 ratio is achieved by altering the ratio of guide RNAs used to transfect a cell. In other aspects, the ratio is altered by changing the ratio of each Cas9 protein-guide RNA complex used to treat the cells of interest. In a still further aspect, the altered ratio is achieved by using differing ratios of DNAs encoding the guide RNAs (viral or non-viral) for treatment of the cells, or by using promoters with different expression strengths to differentially express the DNA binding molecules inside the cells. Off-target events can be reduced by 2 to 1000-fold (or any amount therebetween) or more, including but not limited to, reduction by at least 10, 50, 60, 70, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000-fold (of any value therebetween) or more. [0030] Accordingly, in another aspect, a method for cleaving cellular chromatin in a region of interest can comprise (a) selecting a first sequence in the region of interest; (b) engineering a first DNA-binding molecule to specifically bind to the first sequence; (c) expressing a first fusion molecule in the cell, the first fusion molecule comprising the first DNA-binding molecule (e.g., zinc finger, TALE, sgRNA), and a cleavage domain (or half-domain); and (d) expressing a second fusion protein in the cell, the second fusion molecule comprising 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, and further wherein the first fusion molecule binds to the first sequence, and the second fusion molecule binds to a second sequence located between 2 and 50 nucleotides from the first sequence, such that an active nuclease complex can form and cellular chromatin is cleaved in the region of interest. In certain embodiments, both fusion molecules comprise a linker as described herein between the DNA binding domain and the catalytic nuclease domain. [0031] Also provided are methods of altering a region of cellular chromatin, for example to introduce targeted mutations. In certain embodiments, methods of altering cellular chromatin comprise introducing into the cell one or more targeted nucleases to create a double-stranded break in cellular chromatin at a predetermined site, and a donor polynucleotide, having homology to the nucleotide sequence of the cellular chromatin in the region of the break. Cellular DNA repair processes are activated by the presence of the double-stranded break and the donor polynucleotide is used as a template for repair of the break, resulting in the introduction of all or part of the nucleotide sequence of the donor into the cellular chromatin. Thus, a sequence in cellular chromatin can be altered and, in certain embodiments, can be converted into a sequence present in a donor polynucleotide. [0032] Targeted alterations include, but are not limited to, point mutations (i.e., conversion of a single base pair to a different base pair), substitutions (i.e., conversion of a plurality of base pairs to a different sequence of identical length), insertions or one or more base pairs, deletions of one or more base pairs and any combination of the aforementioned sequence alterations. Alterations can also include conversion of base pairs that are part of a coding sequence such that the encoded amino acid is altered. [0033] The donor polynucleotide can be DNA or RNA, can be linear or circular, and can be single-stranded or double-stranded. It can be delivered to the cell as naked nucleic acid, as a complex with one or more delivery agents (e.g., liposomes, nanoparticles, poloxamers) or contained in a viral delivery vehicle, such as, for example, an adenovirus, lentivirus or an Adeno-Associated Virus (AAV). Donor sequences can range in length from 10 to 1,000 nucleotides (or any integral value of nucleotides therebetween) or longer. In some embodiments, the donor comprises a full-length gene flanked by regions of homology with the targeted cleavage site. In some embodiments, the donor lacks homologous regions and is integrated into a target locus through homology independent mechanism (i.e. NHEJ). In other embodiments, the donor comprises a smaller piece of nucleic acid flanked by homologous regions for use in the cell (i.e. for gene correction). In some embodiments, the donor comprises a gene encoding a functional or structural component such as a shRNA, RNAi, miRNA or the like. In other embodiments, the donor comprises sequences encoding a regulatory element that binds to and/or modulates expression of a gene of interest. In other embodiments, the donor is a regulatory protein of interest (e.g. ZFP TFs, TALE TFs or a CRISPR/Cas TF) that binds to and/or modulates expression of a gene of interest. [0034] For any of the aforementioned methods, the cellular chromatin can be in a chromosome, episome or organellar genome. Cellular chromatin can be present 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] In yet another aspect, cells comprising any of the polypeptides (e.g., fusion molecules) and/or polynucleotides as described herein are also provided. In one embodiment, the cells comprise a pair of fusion molecules, each comprising a cleavage domain as disclosed herein. Cells include cultured cells, cells in an organism and cells that have been removed from an organism for treatment in cases where the cells and/or their descendants will be returned to the organism after treatment. A region of interest in cellular chromatin can be, for example, a genomic sequence or portion thereof. [0036] In another aspect, described 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; ancillary reagents; and optionally instructions and suitable containers. The kit may also include one or more nucleases or polynucleotides encoding such nucleases. [0037] These and other aspects will be readily apparent to the skilled artisan in light of disclosure as a whole. |
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Msg # | Subject | Author | Recs | Date Posted |
156913 | Re: Building IP: SGMO Patent Application re "ENGINEERED TARGET SPECIFIC NUCLEASES" | Chilly | 17 | 4/1/2021 8:46:34 AM |