Cas nucleases and related methods

Abstract

Provided herein are Cas nucleases (e.g., endonucleases, nickases, etc.) (and functional fragments, functional variants, and domains thereof), nucleic acid molecules encoding the same, and systems comprising the same. The disclosure further relates to methods of utilizing the Cas nucleases (e.g., endonucleases, nickases, etc.) (or nucleic acid molecules encoding the same), including, e.g., in methods of editing a nucleic acid molecule (e.g., a gene) and methods of treating diseases (e.g., genetic diseases).

Description

Attorney Docket No.62801.37WO01 CAS NUCLEASES AND RELATED METHODS RELATED APPLICATIONS

[0001] This application claims priority to U.S. Serial No.: 63 / 604,978, filed December 1, 2023, the entire contents of which is incorporated herein by reference. 1. FIELD

[0002] This disclosure relates to nucleases (e.g., endonucleases, nickases, etc.) (and functional fragments, functional variants, and domains thereof), nucleic acid molecules encoding the same, and systems comprising the same. The disclosure further relates to methods of utilizing the nucleases (e.g., endonucleases, nickases, etc.) (or nucleic acid molecules encoding the same), including, e.g., in methods of editing a nucleic acid molecule (e.g., a gene) and methods of treating diseases (e.g., genetic diseases). 2. BACKGROUND

[0003] CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR- associated protein) systems are adaptive immune systems of many prokaryotes (e.g., bacteria and archaea) that function to prevent infection (e.g., by phages, viruses, and other foreign genetic elements). Some classes of naturally occurring CRISPR-Cas systems comprise a CRISPR RNA (crRNA) and a Cas nuclease (e.g., endonuclease, nickase, etc.), wherein the crRNA directs the Cas nuclease (e.g., endonuclease, nickase, etc.) to a target nucleic acid molecule and mediates binding to the Cas nuclease (e.g., endonuclease, nickase, etc.), and the Cas nuclease (e.g., endonuclease, nickase, etc.) mediates cleavage of the target nucleic acid molecule (e.g., viral DNA). CRISPR- Cas systems have been adapted and modified for nucleic acid (e.g., gene) editing in e.g., eukaryotic cells. 3. SUMMARY

[0004] Provided herein are, inter alia, nucleases (e.g., endonucleases, nickases, etc.) (including novel nickases) and polynucleotides encoding the same; fusions and conjugates comprising a nuclease (e.g., endonuclease, nickase, etc.); methods of manufacturing; pharmaceutical compositions; and methods of use including, e.g., methods of editing a nucleic acid molecule (e.g., a gene) and methods of treating diseases (e.g., genetic diseases).Attorney Docket No.62801.37WO01

[0005] Accordingly, in one aspect, provided herein are recombinant nucleases that comprise an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any nuclease set forth in Table 1 or set forth in any one of SEQ ID NOS: 1-28.

[0006] In some embodiments, the nuclease has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, and / or 8) of the following properties (or engineered to have one or more of the following properties): (a) the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (e) DNA endonuclease activity; (f) DNA nickase activity; (g) RNA guided DNA endonuclease activity; and / or (h) RNA guided DNA nickase activity.

[0007] In some embodiments, the nuclease has one or more of the following properties: (a) the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) DNA endonuclease activity; and / or (c) RNA guided DNA endonuclease activity.

[0008] In some embodiments, the amino acid sequence of the nuclease comprises one or more amino acid variation that imparts one or more of the following properties on the nuclease: (a) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) DNA nickase activity; and / or (e) RNA guided DNA nickase activity.

[0009] In some embodiments, the nuclease has one or more of the following properties: (a) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) DNA endonuclease activity; (e) DNAAttorney Docket No.62801.37WO01 nickase activity; (f) RNA guided DNA endonuclease activity; and / or (g) RNA guided DNA nickase activity.

[0010] In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is a nickase.

[0011] In some embodiments, the amino acid sequence of the nuclease is less than about 1000, 950, 900, 850, or 800 amino acid acids in length.

[0012] In some embodiments, the amino acid sequence of the nuclease is from about 300- 1000, 300-950, 300-900, 300-850, 300-800, 300-750, 300-700, 300-650, 300-600, 300-550, 300- 500, 300-450, 300-400, 350-1000, 350-950, 350-900, 350-850, 350-800, 350-750, 350-700, 350- 650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-1000, 400-950, 400-900, 400-850, 400- 800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450, 450-1000, 450-950, 450- 900, 450-850, 450-800, 450-750, 450-700, 450-650, 450-600, 450-550, 450-500, 500-1000, 500- 950, 500-900, 500-850, 500-800, 500-750, 500-700, 500-650, 500-600, 500-550, 600-1000, 600- 950, 600-900, 600-850, 600-800, 600-750, 600-700, 600-650, 700-1000, 700-950, 700-900, 700- 850, 700-800, 700-750, 800-1000, 800-950, 800-900, or 800-850 amino acid acids in length.

[0013] In some embodiments, the nuclease recognizes a protospacer adjacent motif (PAM) sequence comprising the nucleotide sequence PAM sequence comprises or consists of 5′-TTR-3′, wherein R is A or G.

[0014] In some embodiments, the one or more amino acid variation (e.g., substitution, deletion, addition) reduces or eliminates the ability of the nuclease to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule.

[0015] In some embodiments, a nuclease comprising the one or more amino acid variation (e.g., substitution, deletion, addition) has the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule) and does not have the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule.

[0016] In some embodiments, a nuclease comprising the one or more amino acid variation (e.g., substitution, deletion, addition) is a nickase.

[0017] In some embodiments, the one or more amino acid variation (e.g., substitution, deletion, addition) alters the PAM nucleotide sequence recognized by the nuclease.

[0018] In some embodiments, the nuclease further comprises one or more heterologous moiety (e.g., a heterologous polypeptide). In some embodiments, the nuclease further comprises 2, 3, 4,Attorney Docket No.62801.37WO01 or 5 or more heterologous moieties. In some embodiments, the heterologous moiety is attached to the N-terminus, C-terminus, and / or internally between the N- and C-terminus of the nuclease. In some embodiments, the heterologous moiety (e.g., heterologous polypeptide) is directly attached to the nuclease. In some embodiments, the heterologous moiety (e.g., heterologous polypeptide) is indirectly attached to the nuclease. In some embodiments, the heterologous moiety (e.g., heterologous polypeptide) is indirectly attached to the nuclease via a linker. In some embodiments, the heterologous moiety is a peptide, polypeptide, carbohydrate, lipid, polymer, or small molecule. In some embodiments, the heterologous moiety is a nuclear localization signal (NLS), a tag, and / or a reporter gene.

[0019] In one aspect, provided herein are nucleases (e.g., nickases) (e.g., recombinant nuclease (e.g., recombinant nickase)) that comprise (a) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 1; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627,Attorney Docket No.62801.37WO01 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1; (b) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 2; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 15, 16, 17,18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2; (c) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 3; andAttorney Docket No.62801.37WO01 comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3; (d) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 4; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309,Attorney Docket No.62801.37WO01 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4; (e) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 5; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563, 564, 565, 566, 568, 569, 572, 573,Attorney Docket No.62801.37WO01 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5; (f) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 6; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772,Attorney Docket No.62801.37WO01 and / or 773, amino acid numbering relative to SEQ ID NO: 6; (g) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 7; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7; (h) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 8; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,Attorney Docket No.62801.37WO01 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8; (i) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 9; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504,Attorney Docket No.62801.37WO01 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9; (j) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 10; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 408, 409, 411, 412, 413, 415, 416, 417, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 441, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670,Attorney Docket No.62801.37WO01 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 734, 736, 737, 738, 742, 746, 747, 748, 749, 750, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, and / or 769, amino acid numbering relative to SEQ ID NO: 10; (k) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 11; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 74, 75, 76, 77, 78, 79, 94, 95, 96, 98, 101, 102, 103, 104, 105, 107, 108, 111, 118, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 155, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 266, 267, 268, 269, 270, 274, 275, 276, 280, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 299, 300, 304, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 334, 411, 412, 414, 415, 416, 418, 419, 420, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 492, 493, 494, 495, 496, 497, 498, 499, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 529, 530, 533, 544, 545, 546, 547, 548, 550, 551, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 611, 614, 615, 618, 619, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 634, 635, 636, 637, 638, 639, 640, 646, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 728, 730, 731, 732, 736, 740, 741, 742, 743, 744, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, and / or 763, amino acid numbering relative to SEQ ID NO: 11; (l) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 12; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17,Attorney Docket No.62801.37WO01 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 70, 71, 72, 87, 88, 89, 93, 96, 97, 98, 99, 100, 102, 103, 106, 113, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 150, 173, 174, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 405, 406, 408, 409, 410, 412, 413, 414, 423, 424, 425, 426, 427, 428, 429, 430, 431, 433, 434, 437, 438, 440, 442, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 496, 499, 500, 501, 502, 503, 504, 505, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 536, 537, 540, 551, 552, 553, 554, 555, 557, 558, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 587, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 620, 623, 624, 627, 628, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 643, 644, 645, 646, 647, 648, 649, 655, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 740, 741, 742, 746, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, and / or 769, amino acid numbering relative to SEQ ID NO: 12; (m) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 13; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 70, 84, 85, 86, 87, 88, 89, 104, 105, 106, 109, 112, 113, 114, 115, 116, 118, 119, 122, 129, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 166, 189, 190, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 274, 275, 276, 277, 278, 282, 283, 284, 288, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 307, 308, 310, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 416, 417, 419, 420, 421, 423, 424,Attorney Docket No.62801.37WO01 425, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 441, 442, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 501, 504, 505, 506, 507, 508, 509, 510, 511, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 541, 542, 545, 556, 557, 558, 559, 560, 562, 563, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 626, 629, 630, 633, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 649, 650, 651, 652, 653, 654, 655, 661, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 737, 738, 742, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, and / or 765, amino acid numbering relative to SEQ ID NO: 13; (n) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 14; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 27, 28, 31, 32, 35, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 79, 80, 81, 84, 90, 91, 92, 93, 94, 96, 97, 100, 107, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 145, 169, 170, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 253, 254, 255, 256, 257, 261, 262, 263, 267, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 287, 288, 290, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 396, 397, 399, 400, 401, 403, 404, 405, 419, 420, 421, 422, 423, 424, 425, 426, 427, 429, 430, 433, 434, 436, 438, 439, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 492, 495, 496, 497, 498, 499, 500, 501, 502, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 532, 533, 536, 547, 548, 549, 550, 551, 553, 554, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,Attorney Docket No.62801.37WO01 596, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 613, 616, 617, 620, 621, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 636, 637, 638, 639, 640, 641, 642, 648, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 728, 730, 731, 732, 736, 740, 741, 742, 743, 744, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, and / or 763, amino acid numbering relative to SEQ ID NO: 14; (o) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 15; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 89, 90, 91, 96, 97, 98, 99, 100, 102, 103, 106, 113, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 150, 173, 174, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 256, 257, 258, 259, 260, 264, 268, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 288, 289, 291, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 397, 398, 400, 401, 402, 404, 405, 406, 421, 422, 423, 424, 425, 426, 427, 428, 429, 431, 432, 435, 436, 438, 440, 441, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 494, 497, 498, 499, 500, 501, 502, 503, 504, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 534, 535, 538, 549, 550, 551, 552, 553, 555, 556, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 724, 726, 727, 728, 732, 736, 737, 738, 739, 740, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, and / or 758, amino acid numbering relative to SEQ ID NO: 15; (p) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forthAttorney Docket No.62801.37WO01 in SEQ ID NO: 16; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 33, 34, 37, 38, 41, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 87, 88, 89, 92, 105, 106, 107, 108, 109, 110, 111, 113, 114, 117, 124, 131, 132, 133, 134, 135, 136, 137, 138, 139, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 184, 185, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 269, 270, 271, 272, 273, 277, 278, 279, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 302, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 326, 327, 328, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 428, 429, 432, 433, 435, 440, 441, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 496, 499, 500, 501, 502, 503, 504, 505, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 536, 537, 540, 551, 552, 553, 554, 555, 557, 558, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 618, 621, 622, 625, 626, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 641, 642, 643, 644, 645, 646, 647, 653, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 725, 726, 727, 731, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, and / or 754, amino acid numbering relative to SEQ ID NO: 16; (q) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 17; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 29, 32, 33, 36, 37, 40, 59, 60, 62, 65, 66, 67, 68, 69, 70, 85, 86, 87, 90, 93, 94, 95, 96, 97, 99, 100, 103, 110, 117, 118, 119, 120, 121, 122, 123, 124, 125, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 147, 170, 171, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 253, 254, 255, 256, 257, 261, 262, 263, 267, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 286, 287, 289, 295, 296, 297, 298, 299, 300, 301, 302,Attorney Docket No.62801.37WO01 303, 304, 305, 306, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 320, 396, 397, 399, 400, 401, 403, 404, 405, 407, 408, 409, 410, 411, 412, 413, 414, 415, 417, 418, 421, 422, 424, 426, 427, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 482, 485, 486, 487, 488, 489, 490, 491, 492, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 522, 523, 526, 537, 538, 539, 540, 541, 543, 544, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 607, 610, 611, 614, 615, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 630, 631, 632, 633, 634, 635, 636, 642, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 723, 725, 726, 727, 731, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, and / or 753, amino acid numbering relative to SEQ ID NO: 17; (r) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 18; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 25, 28, 29, 32, 33, 36, 55, 56, 58, 59, 60, 63, 64, 65, 66, 67, 68, 83, 84, 85, 88, 91, 92, 93, 94, 95, 97, 98, 101, 108, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 145, 168, 169, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 253, 254, 255, 256, 257, 261, 262, 263, 267, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 286, 287, 291, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 397, 398, 400, 401, 402, 404, 405, 406, 409, 410, 411, 412, 413, 414, 415, 416, 417, 419, 420, 423, 424, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 483, 486, 487, 488, 489, 490, 491, 492, 493, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 523, 524, 527, 538, 539, 540, 541, 542, 544, 545, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,Attorney Docket No.62801.37WO01 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 608, 611, 612, 615, 616, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 631, 632, 633, 634, 635, 636, 637, 643, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 721, 722, 726, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, and / or 749, amino acid numbering relative to SEQ ID NO: 18; (s) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 19; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 94, 97, 98, 99, 100, 101, 103, 104, 107, 114, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 151, 175, 176, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 260, 261, 262, 263, 264, 268, 269, 270, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 293, 294, 296, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 617, 620, 621, 624, 625, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 640, 641, 642, 643, 644, 645, 646, 652, 659, 660, and / or 661, amino acid numbering relative to SEQ ID NO: 19; (t) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 20; and comprises an amino acid variation (e.g., substitution (e.g., an alanineAttorney Docket No.62801.37WO01 substitution)) at any one or more of amino acid positions 1, 2, 3, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 133, 134, 136, 137, 138, 140, 141, 142, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 163, 164, 166, 167, 168, 170, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 255, 266, 267, 268, 269, 270, 272, 273, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 334, 337, 338, 341, 342, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 357, 358, 359, 360, 361, 362, 363, 369, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 446, 448, 449, 450, 454, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, and / or 476, amino acid numbering relative to SEQ ID NO: 20; (u) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 21; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 88, 89, 90, 93, 110, 111, 112, 113, 114, 115, 116, 118, 119, 122, 129, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 166, 189, 190, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 274, 275, 276, 277, 278, 282, 283, 284, 288, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 306, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324, 325, 326, 327, 328, 329, 330, 331, 333, 334, 336, 412, 413, 415, 416, 417, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 431, 432, 435, 436, 438, 440, 441, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 496, 499, 500, 501, 502, 503, 504, 505, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522,Attorney Docket No.62801.37WO01 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 536, 537, 540, 551, 552, 553, 554, 555, 557, 558, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 620, 623, 624, 627, 628, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 643, 644, 645, 646, 647, 648, 649, 655, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 731, 732, 733, 737, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, and / or 761, amino acid numbering relative to SEQ ID NO: 21; (v) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 22; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 89, 90, 91, 94, 97, 98, 99, 100, 101, 103, 104, 107, 114, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 175, 176, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 259, 260, 261, 262, 263, 267, 268, 269, 273, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 291, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 319, 320, 321, 397, 398, 400, 401, 402, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 416, 417, 420, 421, 423, 428, 429, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 601, 604, 605, 608, 609, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 624, 625, 626, 627, 628, 629, 630, 636, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,Attorney Docket No.62801.37WO01 684, 685, 686, 687, 688, 689, 690, 691, 724, 725, 726, 730, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, and / or 754, amino acid numbering relative to SEQ ID NO: 22; (w) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 23; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 8, 11, 12, 15, 16, 19, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 64, 65, 66, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 82, 90, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 128, 151, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 240, 241, 242, 248, 249, 250, 251, 252, 256, 257, 258, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 287, 288, 290, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 396, 397, 399, 400, 401, 403, 404, 405, 413, 414, 415, 416, 417, 418, 419, 420, 421, 423, 424, 427, 428, 430, 435, 436, 438, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 489, 492, 493, 494, 495, 496, 497, 498, 499, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 529, 530, 533, 544, 545, 546, 547, 548, 550, 551, 554, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 730, 732, 733, 734, 738, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, and / or 758, amino acid numbering relative to SEQ ID NO: 23; (x) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 24; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 33, 36, 37, 40, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 86, 87, 88, 91, 104, 105, 106, 107, 108, 109, 110, 112, 113,Attorney Docket No.62801.37WO01 116, 122, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 160, 183, 184, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 304, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 334, 410, 411, 413, 414, 415, 417, 418, 419, 423, 424, 425, 426, 427, 428, 429, 430, 431, 433, 434, 437, 438, 440, 441, 442, 444, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 497, 500, 501, 502, 503, 506, 507, 508, 509, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 539, 540, 543, 554, 555, 556, 557, 558, 560, 561, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 622, 625, 626, 629, 630, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 645, 646, 647, 648, 649, 650, 651, 657, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 701, 702, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, and / or 765, amino acid numbering relative to SEQ ID NO: 24; (y) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 25; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 9, 10, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 38, 39, 40, 43, 46, 47, 48, 49, 50, 52, 53, 56, 63, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 101, 124, 125, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 208, 209, 210, 211, 212, 216, 217, 218, 222, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 240, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 259, 260, 261, 262, 263, 264, 265, 266, 268, 269, 270, 346, 347, 349, 350, 351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 369, 370, 372, 377, 378, 380, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,Attorney Docket No.62801.37WO01 417, 418, 419, 420, 425, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 469, 480, 481, 482, 483, 484, 486, 487, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 550, 553, 554, 557, 558, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 573, 574, 575, 576, 577, 578, 579, 585, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 673, 674, 675, 679, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, and / or 700, amino acid numbering relative to SEQ ID NO: 25; (z) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 26; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 3, 4, 6, 82, 83, 85, 86, 87, 89, 90, 91, 105, 106, 107, 108, 109, 110, 111, 112, 113, 115, 116, 119, 120, 122, 124, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 178, 181, 182, 183, 184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219, 222, 233, 234, 235, 236, 237, 239, 240, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 297, 300, 301, 304, 305, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 332, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 412, 414, 415, 416, 420, 424, 425, 426, 427, 428, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, and / or 450, amino acid numbering relative to SEQ ID NO: 26; (aa) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 27; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one orAttorney Docket No.62801.37WO01 more of amino acid positions 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 111, 112, 113, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 129, 138, 145, 146, 147, 148, 149, 150, 151, 152, 153, 155, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 176, 199, 200, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 286, 287, 288, 289, 290, 294, 295, 296, 300, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 319, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 349, 427, 428, 430, 431, 432, 434, 435, 436, 453, 454, 455, 456, 457, 458, 459, 460, 461, 463, 464, 467, 468, 470, 471, 472, 474, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 527, 530, 531, 532, 533, 536, 537, 538, 539, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 569, 570, 573, 584, 585, 586, 587, 588, 590, 591, 594, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 655, 658, 659, 662, 663, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 678, 679, 680, 681, 682, 683, 684, 690, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 766, 768, 769, 770, 774, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, and / or 798, amino acid numbering relative to SEQ ID NO: 27; or (bb) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 28; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 17, 18, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 102, 103, 104, 105, 106, 110, 111, 112, 116, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 162, 164, 240, 241, 243, 244, 245, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 259, 260, 263, 264, 266, 268, 269, 271, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,Attorney Docket No.62801.37WO01 307, 308, 309, 310, 311, 324, 327, 328, 329, 330, 331, 332, 333, 334, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 364, 365, 368, 379, 380, 381, 382, 383, 385, 386, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, and / or 404, amino acid numbering relative to SEQ ID NO: 28.

[0020] In some embodiments, the nuclease has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, and / or 8) of the following properties (or engineered to have one or more of the following properties): (a) the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (e) DNA endonuclease activity; (f) DNA nickase activity; (g) RNA guided DNA endonuclease activity; and / or (h) RNA guided DNA nickase activity.

[0021] In some embodiments, the nuclease has one or more of the following properties: (a) the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) DNA endonuclease activity; and / or (c) RNA guided DNA endonuclease activity.

[0022] In some embodiments, the amino acid sequence of the nuclease comprises one or more amino acid variation that imparts one or more of the following properties on the nuclease: (a) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) DNA nickase activity; and / or (e) RNA guided DNA nickase activity.

[0023] In some embodiments, the nuclease has one or more of the following properties: (a) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a targetAttorney Docket No.62801.37WO01 double stranded nucleic acid (e.g., DNA) molecule; (d) DNA endonuclease activity; (e) DNA nickase activity; (f) RNA guided DNA endonuclease activity; and / or (g) RNA guided DNA nickase activity.

[0024] In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is a nickase.

[0025] In some embodiments, the amino acid sequence of the nuclease is less than about 1000, 950, 900, 850, or 800 amino acid acids in length.

[0026] In some embodiments, the amino acid sequence of the nuclease is from about 300- 1000, 300-950, 300-900, 300-850, 300-800, 300-750, 300-700, 300-650, 300-600, 300-550, 300- 500, 300-450, 300-400, 350-1000, 350-950, 350-900, 350-850, 350-800, 350-750, 350-700, 350- 650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-1000, 400-950, 400-900, 400-850, 400- 800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450, 450-1000, 450-950, 450- 900, 450-850, 450-800, 450-750, 450-700, 450-650, 450-600, 450-550, 450-500, 500-1000, 500- 950, 500-900, 500-850, 500-800, 500-750, 500-700, 500-650, 500-600, 500-550, 600-1000, 600- 950, 600-900, 600-850, 600-800, 600-750, 600-700, 600-650, 700-1000, 700-950, 700-900, 700- 850, 700-800, 700-750, 800-1000, 800-950, 800-900, or 800-850 amino acid acids in length.

[0027] In some embodiments, the nuclease recognizes a protospacer adjacent motif (PAM) sequence comprising the nucleotide sequence PAM sequence comprises or consists of 5′-TTR-3′, wherein R is A or G.

[0028] In some embodiments, the one or more amino acid variation (e.g., substitution, deletion, addition) reduces or eliminates the ability of the nuclease to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule.

[0029] In some embodiments, a nuclease comprising the one or more amino acid variation (e.g., substitution, deletion, addition) has the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule) and does not have the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule.

[0030] In some embodiments, a nuclease comprising the one or more amino acid variation (e.g., substitution, deletion, addition) is a nickase.

[0031] In some embodiments, the one or more amino acid variation (e.g., substitution, deletion, addition) alters the PAM nucleotide sequence recognized by the nuclease.

[0032] In some embodiments, the nuclease further comprises one or more heterologous moietyAttorney Docket No.62801.37WO01 (e.g., a heterologous polypeptide). In some embodiments, the nuclease further comprises 2, 3, 4, or 5 or more heterologous moieties. In some embodiments, the heterologous moiety is attached to the N-terminus, C-terminus, and / or internally between the N- and C-terminus of the nuclease. In some embodiments, the heterologous moiety (e.g., heterologous polypeptide) is directly attached to the nuclease. In some embodiments, the heterologous moiety (e.g., heterologous polypeptide) is indirectly attached to the nuclease. In some embodiments, the heterologous moiety (e.g., heterologous polypeptide) is indirectly attached to the nuclease via a linker. In some embodiments, the heterologous moiety is a peptide, polypeptide, carbohydrate, lipid, polymer, or small molecule. In some embodiments, the heterologous moiety is a nuclear localization signal (NLS), a tag, and / or a reporter gene.

[0033] In one aspect, provided herein are conjugates comprising a nuclease described herein and one or more heterologous moiety. In some embodiments, the heterologous moiety is a protein, peptide, small molecule, nucleic acid molecule (e.g., DNA, RNA, DNA / RNA hybrid molecule), carbohydrate, lipid, or synthetic polymer. In some embodiments, the heterologous moiety is operably connected to the N-terminus, C-terminus, and / or internally between the N- and C- terminus of the nuclease. In some embodiments, the heterologous moiety is directly operably connected to the nuclease. In some embodiments, the heterologous moiety is indirectly operably connected to the nuclease. In some embodiments, the heterologous moiety is indirectly operably connected to the nuclease via a linker.

[0034] In one aspect, provided herein are fusion proteins comprising a nuclease described herein and one or more heterologous polypeptide.

[0035] In some embodiments, the heterologous polypeptide is fused to the N-terminus, C- terminus, and / or internally between the N- and C-terminus of the nuclease. In some embodiments, the heterologous polypeptide is fused directly to the nuclease. In some embodiments, the heterologous polypeptide is fused indirectly to the nuclease. In some embodiments, the heterologous polypeptide is fused indirectly to the nuclease via a peptide linker.

[0036] In some embodiments, the heterologous polypeptide exhibits polymerase (e.g., reverse transcriptase) activity, nucleobase modifying activity (e.g., deaminase activity), methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavageAttorney Docket No.62801.37WO01 activity, or double-strand DNA cleavage activity, nucleic acid binding activity, or any combination of the foregoing.

[0037] In some embodiments, the amino acid sequence of the fusion protein is less than about 1500, 1300, 1200, 1100, 1000, 950, 900, 850, or 800 amino acid acids in length. In some embodiments, the amino acid sequence of the fusion protein is from about 300-1300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-950, 300-900, 300-850, 300-800, 300-750, 300- 700, 300-650, 300-600, 300-550, 300-500, 300-450, 300-400, 350-1350, 350-1400, 350-1350, 350-1200, 350-1100, 350-1000, 350-950, 350-900, 350-850, 350-800, 350-750, 350-700, 350- 650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-1400, 400-1400, 400-1400, 400-1200, 400-1100, 400-1000, 400-950, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450, 450-1450, 450-1400, 450-1450, 450-1200, 450-1100, 450-1000, 450- 950, 450-900, 450-850, 450-800, 450-750, 450-700, 450-650, 450, 600, 450-550, 450-500, 500- 1500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-950, 500-900, 500-850, 500-800, 500-700, 500-600, 600-1500, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-950, 600- 900, 600-850, 600-800, 600-700, 700-1500, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000, 700-950, 700-900, 700-850, 700-800, 800-1500, 800-1400, 800-1300, 800-1200, 800-1100, 800- 1000, 800-950, 800-900, or 800-850 amino acid acids in length.

[0038] In some embodiments, the heterologous polypeptide is a polymerase. In some embodiments, the polymerase has RNA-dependent DNA polymerase activity. In some embodiments, the polymerase is a reverse transcriptase (or a functional fragment or functional variant thereof). In some embodiments, the reverse transcriptase (or the functional fragment or functional variant thereof) is derived from a retrovirus or a retrotransposon.

[0039] In some embodiments, the heterologous polypeptide is a nucleobase editor. In some embodiments, the nucleobase editor is a deaminase (or a functional fragment or functional variant thereof). In some embodiments, the deaminase (or the functional fragment or functional variant thereof) exhibits adenosine deaminase activity and / or a or a cytidine deaminase activity. In some embodiments, the deaminase (or the functional fragment or functional variant thereof) comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a protein set forth in Table 3 or set forth in any one of SEQ ID NOS: 29-88. In some embodiments, the nucleobase editor is fused to an inhibitor of base excision repair (or a functional fragment orAttorney Docket No.62801.37WO01 functional variant thereof) (e.g., uracil glycosylase inhibitor (UGI), nuclease dead inosine specific nuclease (dISN)).

[0040] In one aspect, provided herein are systems for modifying a target nucleic acid (e.g., DNA) molecule, comprising: (a) a nuclease described herein, a conjugate described herein, a fusion protein described herein, a vector described herein, a carrier described herein, a cell (or population of cells) described herein, or a pharmaceutical composition described herein, and (b) a first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; a template RNA (e.g., as described herein)) or a nucleic acid (e.g., DNA) molecule encoding the first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; a template RNA (e.g., as described herein)).

[0041] In some embodiments, the system has one or more of the following characteristics: (a) the nuclease of the system is capable of binding to the first gRNA; (b) the nuclease of the system is capable of forming a break in a target nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (c) the nuclease of the system is capable of forming a single strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (d) the nuclease of the system is capable of forming a single strand break in the modified strand (as defined herein) of a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (e) the nuclease of the system is capable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (f) the nuclease of the system is incapable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (g) the endonuclease of the system is capable of forming a single strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule and is incapable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (h) the nuclease of the system is capable of forming a single strand break in in the modified strand (as defined herein) of a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule and is incapable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; and / or (i) is capable of mediating the insertion, deletion, or substitution of one or more nucleotides into / from a target nucleic acid (e.g., DNA) molecule (e.g., a target double stranded DNA molecule).

[0042] In some embodiments, the target nucleic acid molecule is a DNA molecule. In some embodiments, the target nucleic acid molecule is a double stranded DNA (dsDNA) molecule. In some embodiments, a portion of the nucleotide sequence of the non-edited strand (as definedAttorney Docket No.62801.37WO01 herein) of the target dsDNA molecule is complementary to at least a portion of the nucleotide sequence of the first gRNA. In some embodiments, the target nucleic acid molecule is within the genome of cell (e.g., a eukaryotic cell) (e.g., within a subject (e.g., a human subject), plant).

[0043] In some embodiments, (b) comprises the first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; or a template RNA (e.g., as described herein)). In some embodiments, (b) comprises the nucleic acid (e.g., DNA) molecule encoding the first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; or a template RNA (e.g., as described herein)).

[0044] In some embodiments, at least a portion of the nucleotide sequence of the first gRNA is complementary to a portion of the nucleotide sequence of the target nucleic acid molecule. In some embodiments, at least a portion of the nucleotide sequence of the first gRNA is complementary to a portion of the nucleotide sequence of the non-edited strand (as defined herein) of a dsDNA target nucleic acid molecule. In some embodiments, at least a portion of the nucleotide sequence of the first gRNA binds to a portion of the nucleotide sequence of the non-edited strand (as defined herein) of a dsDNA target nucleic acid molecule.

[0045] In some embodiments, the first gRNA comprises a crRNA (e.g., a single crRNA, a plurality of different crRNAs). In some embodiments, the first gRNA comprises a sgRNA (e.g., a single sgRNA, a plurality of different sgRNAs).

[0046] In some embodiments, the first gRNA comprises a crRNA (e.g., a single crRNA, a plurality of different crRNAs) and a tracrRNA (e.g., a single tracrRNA, a plurality of different tracrRNAs), wherein the crRNA and the tracrRNA are on separate RNA nucleic acid molecules (or encoded by separate nucleic acid (e.g., DNA) molecules).

[0047] In some embodiments, a portion (e.g., scaffold portion) of the first gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in Table 7.

[0048] In some embodiments, a portion (e.g., scaffold portion) of the first gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in any one of SEQ ID NOS: 243-315.

[0049] In some embodiments, the first gRNA comprises a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA,Attorney Docket No.62801.37WO01 a heterologous object sequence, and a 3' target homology domain. In some embodiments, the template RNA further comprises a sequence that binds a polymerase (e.g., a reverse transcriptase). In some embodiments, the template RNA comprises (e.g., from 5' to 3') a crRNA, a sequence that binds a polymerase (e.g., a reverse transcriptase), a heterologous object sequence, and a 3' target homology domain. In some embodiments, the first gRNA comprises a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a tracrRNA, a heterologous object sequence, and a 3' target homology domain. In some embodiments, the template RNA further comprises a sequence that binds a polymerase (e.g., a reverse transcriptase). In some embodiments, the template RNA comprises (e.g., from 5' to 3') a crRNA, a tracrRNA, a sequence that binds a polymerase (e.g., a reverse transcriptase), a heterologous object sequence, and a 3' target homology domain.

[0050] In some embodiments, the first gRNA comprises one or more nucleotide comprising one or more chemical modification (e.g., a base, ribose, and / or internucleotide linkage chemical modifications) (i.e., a modified nucleotide). In some embodiments, the modified nucleotide comprises a 2′-O-methyl (2′-OMe); 2′O-methoxyethyl (2′-O-MOE); 2′deoxy-2′-fluoro (2′-F); 2′- arabino-fluoro (2′-Ara-F); 2′-O-benzyl; 2′-O-methyl-4-pyridine (2-O-methyl-4-pyridine (2′-O- CH2Py(4)); 2′F-4′-Cα-OMe; or 2′,4′-di-Cα-OMe, 2ˈ-O-methyl-3ˈ-thioPACE, and / or S- constrained ethyl (cEt), or any combination of the foregoing. In some embodiments, the modified nucleotide comprises a chemically modified internucleotide (or internucleoside) linkage. In some embodiments, the modified internucleotide (or internucleoside) linkage comprises a phosphorothioate (e.g., a chiral phosphorothioate), a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, an alkyl (e.g., methyl) phosphonate (e.g., a 3ˈ-alkylene phosphonate, a chiral phosphonate), a phosphinate, a phosphoroamidate (e.g., a 3ˈ-amino phosphoroamidate, an aminoalkylphosphoramidate), a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, or a boranophosphate.

[0051] In some embodiments, the first gRNA (e.g., the template RNA, crRNA) comprises a nucleic acid molecule comprising a toe-loop, hairpin, stem-loop, pseudoknot (e.g., a Mpknot1 moiety), aptamer, G-quadraplex, tRNA, riboswitch, or ribozyme. In some embodiments, the first gRNA (e.g., the template RNA, crRNA) wherein the nucleic acid molecule is a pseudoknot (e.g., a Mpknot1 moiety).

[0052] In some embodiments, the system further comprises a second gRNA (or a nucleic acidAttorney Docket No.62801.37WO01 (e.g., DNA) molecule encoding the gRNA) that directs the nuclease of the system to form a single strand break in the non-edited strand of a target dsDNA molecule.

[0053] In some embodiments, at least a portion of the nucleotide sequence of the second gRNA is complementary to a portion of the nucleotide sequence of the modified strand (as defined herein) of a dsDNA target nucleic acid molecule. In some embodiments, at least a portion of the nucleotide sequence of the second gRNA binds to a portion of the nucleotide sequence of the modified strand (as defined herein) of a dsDNA target nucleic acid molecule.

[0054] In some embodiments, the second gRNA is present on the same nucleic acid molecule as the first gRNA (or the nucleic acid (e.g., DNA) molecule encoding the second gRNA is present on the same nucleic acid (e.g., DNA) molecule encoding the first gRNA). In some embodiments, the second gRNA is present on a different nucleic acid molecule as the first gRNA (or the nucleic acid (e.g., DNA) molecule encoding the second gRNA is present on a different nucleic acid (e.g., DNA) molecule encoding the first gRNA).

[0055] In some embodiments, the second gRNA is a crRNA.

[0056] In some embodiments, a portion (e.g., scaffold portion) of the second gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in Table 7. In some embodiments, a portion (e.g., scaffold portion) of the second gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in any one of SEQ ID NOS: 243-315.

[0057] In some embodiments, the system further comprising a donor DNA template molecule (e.g., as defined herein).

[0058] In one aspect, provided herein are systems for modifying a dsDNA molecule, comprising: (a) a fusion protein described herein comprising a nuclease and a polymerase (e.g., a reverse transcriptase) or a nucleic acid molecule (e.g., a DNA, RNA molecule) encoding the fusion protein; and (b) a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a heterologous object sequence, and a 3' target homology domain; or a nucleic acid molecule (e.g., a DNA molecule) encoding the template RNA. In some embodiments, the template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a tracrRNA, a heterologous objectAttorney Docket No.62801.37WO01 sequence, and a 3ˈ target homology domain; or a nucleic acid molecule (e.g., a DNA molecule) encoding the template RNA.

[0059] In one aspect, provided herein are systems for modifying a dsDNA molecule, comprising: (a) the fusion protein described herein comprising a nuclease and a nucleobase editor or a nucleic acid molecule (e.g., a DNA, RNA molecule) encoding the fusion protein; and (b) a gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA); or a nucleic acid molecule (e.g., a DNA molecule) encoding the gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA).

[0060] In one aspect, provided herein are nucleic acid molecules encoding a nuclease described herein, a conjugate described herein, a fusion protein described herein, or a system described herein. In some embodiments, the nucleic acid molecule is a DNA or RNA (e.g., mRNA) molecule. In some embodiments, the nucleic acid molecule is codon optimized. In some embodiments, the nucleic acid molecule further comprises one or more transcription or translation regulatory elements (e.g., promoter, enhancer (e.g., cell or tissue specific transcription regulatory elements). In some embodiments, the nucleic acid molecule further encodes one or more gRNA (e.g., a crRNA, a tracrRNA, a sgRNA, a template RNA (e.g., as described herein)). In some embodiments, the gRNA is a crRNA.

[0061] In one aspect, provided herein are vectors comprising a nucleic acid molecule described herein. In some embodiments, the vector is a viral vector or a non-viral vector (e.g., plasmid, minicircle). In some embodiments, the vector is a viral vector (e.g., an adeno associated viral (AAV) vector, a lentiviral vector, an adenoviral vector). In some embodiments, the viral vector is an AAV vector.

[0062] In one aspect, described herein are carriers comprising a nuclease described herein, a conjugate described herein, a fusion protein described herein, s system described herein, a nucleic acid molecule described herein, a vector described herein. In some embodiments, the carrier is a nanoparticle, polymer, virus (e.g., a recombinant virus), virus like particle, virosome, fusosome, vesicle, or lipid-based carrier. In some embodiments, the carrier is a recombinant virus (e.g., an adeno associated virus (AAV), a lentivirus, an adenovirus). In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP), liposome, lipoplex, nanoliposome, an exosome, or a micelle. In some embodiments, the carrier further comprises one or more gRNA (e.g., a crRNA, a tracrRNA, a sgRNA, a template RNA (e.g., as described herein)).Attorney Docket No.62801.37WO01

[0063] In one aspect, provided herein are cells (or population of cells) comprising a nuclease described herein, a conjugate described herein, a fusion protein described herein, a system described herein, a nucleic acid molecule described herein, a vector described herein, or a carrier described herein. In some embodiments, the cell (or population of cells) is in vivo, ex vivo, or in vitro. In some embodiments, the cell (or population of cells) is a eukaryotic cell (e.g., an animal cell, a mammalian cell, a non-human primate cell, a primate cell, a human cell, a plant cell).

[0064] In one aspect, provided herein are pharmaceutical compositions comprising a nuclease described herein, a conjugate described herein, a fusion protein described herein, a system described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, or a cell (or population of cells) described herein, and a pharmaceutically acceptable excipient.

[0065] In one aspect, provided herein are kits comprising a nuclease described herein, a conjugate described herein, a fusion protein described herein, a system described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell (or population of cells) described herein, or a pharmaceutical composition described herein; and optionally instructions for using any one or more of the foregoing.

[0066] In one aspect, provided herein are reaction mixtures comprising (a) a cell and (b) a nuclease described herein, a conjugate described herein, a fusion protein described herein, a system described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell (or population of cells) described herein, or a pharmaceutical composition described herein.

[0067] In one aspect, provided herein are methods of delivering a nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, composition, pharmaceutical composition, or system to a cell, the method comprising, introducing into a cell a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein, to thereby deliver the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical composition, or system to the cell.

[0068] In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is euploid, is not immortalized, is part of a tissue, is part of an organism, is a primary cell, isAttorney Docket No.62801.37WO01 non-dividing, is haploid (e.g., a germline cell), is a non-cancerous polyploid cell, or is from a subject having a genetic disease. In some embodiments, the cell is a human cell. In some embodiments, the cell is in a plant cell. In some embodiments, the cell is in a subject (e.g., a human subject).

[0069] In one aspect, provided herein are methods of delivering a nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, , pharmaceutical composition, or system to a subject (e.g., human subject), the method comprising, administering to the subject a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein, to thereby deliver the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical composition, or system to the subject (e.g., human subject).

[0070] In one aspect, provided herein are methods of cleaving a target site in a target nucleic acid (e.g., DNA) molecule (e.g., a double stranded target nucleic acid sequence (e.g., dsDNA, (e.g., genomic dsDNA))), the method comprising contacting the cell with a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein, to thereby cleave the target site in the target nucleic acid (e.g., DNA) molecule.

[0071] In some embodiments, the nucleic acid molecule is in a cell (e.g., a eukaryotic cell).

[0072] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the cell is in a plant.

[0073] In one aspect, provided herein are methods of modifying a target site in a target nucleic acid (e.g., DNA) molecule (e.g., a double stranded target nucleic acid sequence (e.g., dsDNA, (e.g., genomic dsDNA))), the method comprising contacting the cell with a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein, to thereby modify the target site in the target nucleic acid (e.g., DNA) molecule.Attorney Docket No.62801.37WO01

[0074] In some embodiments, the nucleic acid molecule is in a cell (e.g., a eukaryotic cell).

[0075] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the cell is in a plant.

[0076] In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the genomic dsDNA in the cell. In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the target nucleic acid molecule. In some embodiments, the insertion comprises the insertion of from about 1-500, 1-400, 1-300, 1-200, 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site. In some embodiments, the deletion comprises the deletion of from about 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site.

[0077] In one aspect, provided herein are methods of modifying a target site in genomic dsDNA in a cell, the method comprising, contacting a target site in genomic dsDNA in a cell with a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein, to thereby modify the target site in the genomic DNA of the cell.

[0078] In some embodiments, the nucleic acid molecule is in a cell (e.g., a eukaryotic cell).

[0079] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the cell is in a plant.

[0080] In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the genomic dsDNA in the cell. In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the target nucleic acid molecule. In some embodiments, the insertion comprises the insertion of from about 1-500, 1-400, 1-300, 1-200, 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2Attorney Docket No.62801.37WO01 nucleotides at the target site. In some embodiments, the deletion comprises the deletion of from about 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site.

[0081] In one aspect, provided herein are methods of modifying a target site in a dsDNA molecule (e.g., genomic dsDNA (e.g., in a cell)), the method comprising: contacting a dsDNA molecule with (a) a fusion protein described herein comprising a nuclease and a polymerase (e.g., a reverse transcriptase) (or a nucleic acid molecule (e.g., a DNA, RNA, nucleic acid molecule) encoding the fusion protein), and (b) a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a heterologous object sequence, and a 3ˈ target homology domain, to thereby modify the target site in the dsDNA molecule (or a nucleic acid molecule (e.g., a DNA nucleic acid molecule) encoding the template RNA). In some embodiments, the template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5ˈ to 3ˈ) a crRNA, a tracrRNA, a heterologous object sequence, and a 3ˈ target homology domain, to thereby modify the target site in the dsDNA molecule (or a nucleic acid molecule (e.g., a DNA nucleic acid molecule) encoding the template RNA).

[0082] In some embodiments, the nucleic acid molecule is in a cell (e.g., a eukaryotic cell).

[0083] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the cell is in a plant.

[0084] In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the genomic dsDNA in the cell. In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the target nucleic acid molecule. In some embodiments, the insertion comprises the insertion of from about 1-500, 1-400, 1-300, 1-200, 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site. In some embodiments, the deletion comprises the deletion of from about 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site.

[0085] In one aspect, provided herein are methods of modifying a target site in a dsDNAAttorney Docket No.62801.37WO01 molecule (e.g., genomic dsDNA (e.g., in a cell)), the method comprising: contacting a dsDNA molecule with (a) a fusion protein described herein comprising a nuclease and a nucleobase editor (or a nucleic acid molecule (e.g., a DNA, RNA, nucleic acid molecule) encoding the fusion protein), and (b) a gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA); or a nucleic acid molecule (e.g., a DNA molecule) encoding the gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA), to thereby modify the target site in the dsDNA molecule (or a nucleic acid molecule (e.g., a DNA nucleic acid molecule) encoding the gRNA).

[0086] In some embodiments, the nucleic acid molecule is in a cell (e.g., a eukaryotic cell).

[0087] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is in vitro, ex vivo, or in vivo. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, the cell is in a plant.

[0088] In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the genomic dsDNA in the cell. In some embodiments, the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the target nucleic acid molecule. In some embodiments, the insertion comprises the insertion of from about 1-500, 1-400, 1-300, 1-200, 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site. In some embodiments, the deletion comprises the deletion of from about 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site.

[0089] In one aspect, provided herein are methods of treating or preventing a disease in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein, thereby treating or preventing the disease in the subject.

[0090] In some embodiments, the disease is associated with a genetic defect. In some embodiments, the gRNA of the system is capable of targeting the endonuclease to the site of the genetic defect. In some embodiments, the genetic defect comprises a duplication of a gene, deletion of a gene, or a mutation of a gene. In some embodiments, the administration results in theAttorney Docket No.62801.37WO01 correction of the genetic defect. In some embodiments, the subject is a human subject.

[0091] In one aspect, provided herein are nucleases described herein, conjugates described herein, fusion proteins described herein, nucleic acid molecules described herein, vectors described herein, carriers described herein, cells described herein, pharmaceutical compositions described herein, and system described herein for use in a method of treating or preventing a disease in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical, or system, thereby treating or preventing the disease in the subject.

[0092] In one aspect, provided herein are uses of a nuclease described herein, a conjugate described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, a carrier described herein, a cell described herein, a pharmaceutical composition described herein, or a system described herein in the manufacture of a medicament for use in a method of treating or preventing a disease in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical, or system, thereby treating or preventing the disease in the subject. 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] FIG. 1 shows an electropherogram, wherein the x-axis of the plot represents the size of the DNA fragments in base pairs (bp) and the y-axis represents the sample fluorescence intensity, measured in fluorescence intensity units (normalized FU). The A2 curve shows the electropherogram from the negative control, in which no gRNA was included in the reaction (see Example 4). The C2 and D2 curves display the electropherogram of a reaction containing all components: gRNA (scaffold SEQ ID NO: 312 and different target strand), purified CasNuc-1, and target dsDNA (see Example 4). 5. DETAILED DESCRIPTION

[0094] Typical CRISPR-Cas editing (e.g., gene editing) systems require a Cas nuclease to mediate cleavage of the target nucleic acid molecule. Cas nucleases vary in their ability to mediate target cleavage (e.g., in a cell) depending on e.g., the efficiency of target cleavage, their capability to mediate double and / or single strand breaks, protospacer adjacent motif (PAM) sequenceAttorney Docket No.62801.37WO01 requirements, the specificity of the PAM, etc. The delivery of Cas nucleases to cells in vitro and in vivo further varies based on the size of the Cas nuclease. For example, certain Cas9 nucleases are around 1400 amino acids in length, making standard viral vector-based delivery (e.g., AAV delivery) difficult. As such, a diverse set of Cas nucleases is useful to provide the ability to select a suitable Cas nuclease for each specific target nucleic acid molecule compatible with an efficient delivery system.

[0095] The inventors have, inter alia, identified Cas nucleases (e.g., Cas endonucleases) and discovered novel Cas nickases thereof that are significantly shorter in length (e.g., less than 800 amino acids) relative to other wild type Cas nucleases (e.g., Cas9). As such, the Cas nucleases (e.g., endonucleases, nickases, etc.) described herein can be used to modify target nucleic acid molecules, for example when used in nucleic acid editing systems (e.g., CRISPR-Cas systems). Accordingly, the current disclosure provides, inter alia, Cas nucleases (e.g., endonucleases, nickases, etc.) capable of cleaving target nucleic acid molecules (e.g., DNA, genes, genomic DNA) (e.g., in a cell, in a cell in a subject); as well as systems and methods of utilizing the same (e.g., methods of cleaving a nucleic acid molecule, methods of editing a nucleic acid molecule (e.g., genomic DNA), and methods of treating diseases (e.g., genetic diseases)). TABLE OF CONTENTS 5.1 Definitions 5.2 Cas Nucleases 5.2.1 Activity of Cas Nucleases 5.2.1.1 Endonuclease Activity 5.2.1.2 Nickase Activity 5.2.1.3 gRNA Binding Activity 5.2.1.4 Target Nucleic Acid Molecule Binding Activity 5.2.1.5 Target Nucleic Acid Editing Activity 5.2.1.6 Alteration of Activity 5.3 Fusion Proteins and Conjugates 5.3.1 Heterologous Proteins 5.3.1.1 Polymerases (e.g., Reverse Transcriptases (RTs)) 5.3.1.2 Nucleobase Editors 5.3.2 LinkersAttorney Docket No.62801.37WO01 ity of Systems ble Lipids s ntsAttorney Docket No.62801.37WO01 5.13 Methods of Use 5.13.1 Methods of Delivery 5.13.2 Methods of Cleaving a Target Nucleic Acid Molecule 5.13.3 Methods of Editing a Target Nucleic Acid Molecule 5.13.3.1 Methods of Editing a Target Nucleic Acid Molecule Utilizing an HDR-Based System 5.13.3.2 Methods of Editing a Target Nucleic Acid Molecule Utilizing an RT-Based System 5.13.3.3 Methods of Editing a Target Nucleic Acid Molecule Utilizing a Nucleobase Editor-Based System 5.13.4 Methods of Treating, Ameliorating, or Preventing a Disease 5.1 Definitions

[0096] The section headings used herein are for organizational purposes and do not limit the subject matter described.

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the general and detailed descriptions are exemplary and explanatory and are not restrictive of claimed subject matter.

[0098] In this application, the use of the singular includes the plural unless stated otherwise. For example, as used in the disclosure, the singular forms “a,” “an,” and “the” include plural referents unless the context dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0099] It is understood that aspects and embodiments described herein with “comprising” language, also otherwise include analogous aspects and embodiments described in terms of “consisting of” and “consisting essentially of.”

[0100] The term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B andAttorney Docket No.62801.37WO01 C; A (alone); B (alone); and C (alone).

[0101] As described herein, concentration ranges, percentage ranges, ratio ranges or integer ranges are understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0102] The term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as understood and / or determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., limitations of the measurement system. When particular values or compositions are provided in the disclosure, unless otherwise stated, the meaning of “about” is understood to be within an acceptable error range for that particular value or composition.

[0103] Where proteins are described herein, it is understood that polynucleotides (e.g., RNA or DNA nucleic acid molecules) encoding the proteins are also provided herein.

[0104] Where proteins, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, nucleic acid molecules, vectors, carriers, etc. are also provided herein.

[0105] Where proteins, nucleic acid molecules, etc. are described herein, it is understood that recombinant forms of the proteins, nucleic acid molecules, etc. are also provided herein.

[0106] Where proteins or sets of proteins are described herein, it is understood that both proteins comprising the primary structure are provided herein as well as proteins folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are provided herein.

[0107] As used herein, the term “administering” refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of the therapeutic agent that is metabolized or altered within the body of the subject to produce the therapeutic agent in vivo) (e.g., systems comprising nucleases (e.g., endonucleases) for introducing variations into a target nucleic acid) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. Therapeutic agents include agents whose effect is intended to be preventative (i.e., prophylactic), such as agents for modifying target nucleic acids (e.g., systems comprising nucleases for introducing a variation into a target nucleic acid). The term administering includes self-administration by the subject and administration to the subject by another.Attorney Docket No.62801.37WO01

[0108] As used herein, the term “bicyclic sugar” refers to a modified sugar (e.g., ribose) moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure. In some embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In some embodiments, the furanosyl sugar moiety is a ribosyl moiety.

[0109] As used herein, the term “bicyclic nucleoside” (“BNA”) is a nucleoside comprising a bicyclic sugar.

[0110] As used herein, the term “cell” or “cells” refers to both the subject cell or cells and progeny thereof. For example, in some embodiments, described herein are methods of modifying genomic DNA in a target cell ex vivo or in vitro and subsequently administering the target cell to the subject. Such methods would include administration of the target cell and / or administration of progeny thereof comprising the subject modification.

[0111] As used herein, the term “crRNA” refers to an RNA molecule (e.g., a gRNA or part of a gRNA molecule) that is capable of binding a protospacer in a target nucleic acid (e.g., DNA) molecule. In some embodiments, the crRNA comprises a scaffold portion that is not specific for a target nucleic acid molecule and a spacer portion that is specific for targeting to a target nucleic acid molecule. In some embodiments, the crRNA is capable of interacting with a nuclease (e.g., endonuclease) (e.g., a nuclease described herein (e.g., an endonuclease described herein)). In some embodiments, when the target nucleic acid (e.g., DNA) molecule is double stranded, the crRNA is capable of binding a protospacer within the strand opposite of the strand that contains a PAM. In some embodiments, the crRNA is sufficient for Cas nuclease to mediate nuclease activity. In some embodiments, the crRNA must be paired with a tracrRNA (e.g., as separate molecules or as a single gRNA) for a Cas nuclease to mediate nuclease activity.

[0112] As used herein, the term “disease” refers to an abnormal condition that impairs physiological function. The term encompasses any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology. The term disease includes infection (e.g., a viral, bacterial, fungal, protozoal infection).

[0113] As used herein, the term “donor template nucleic acid molecule” refers to a nucleic acid molecule that contains a donor region comprising a nucleic acid sequence of interest (e.g., contains a nucleotide variation of interest (e.g., a substitution, addition, deletion, inversions, etc.))Attorney Docket No.62801.37WO01 and two homology arms each comprising a nucleotide sequence of sufficient homology to the nucleotide sequence of the region flanking the target cleavage site of a nuclease (e.g., endonuclease) described herein (also referred to herein as homology arms). Each of the homology arms flank the donor region, such that the donor region is between the two homology arms. In some embodiments, the donor template nucleic acid molecule is a donor DNA template nucleic acid molecule. In some embodiments, the donor template nucleic acid molecule is an RNA template molecule. In some embodiments, the donor template nucleic acid molecule is double stranded. In some embodiments, the donor template nucleic acid molecule is single stranded. In some embodiments, the donor template nucleic acid molecule can be utilized in a system described herein (e.g., an HDR based system described herein), wherein the molecular machinery of the cell can utilize the exogenous donor template nucleic acid in repairing and / or resolving a cleavage site in a target nucleic acid molecule mediated by a nuclease (e.g., an endonuclease) (or functional fragment, functional variant, or domain thereof) (e.g., of the system).

[0114] The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably and refer to macromolecules including multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.

[0115] As used herein, the term “domain” refers to a structure of a biomolecule (e.g., a protein, nucleic acid (e.g., DNA, RNA)) molecule) that contributes to a specified function of the biomolecule (e.g., a protein, nucleic acid (e.g., DNA, RNA)). A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct non-contiguous regions (e.g., non- contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, a nuclease domain (e.g., an endonuclease domain), a DNA binding domain, a reverse transcriptase domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain. In some embodiments, a domain (e.g., a Cas domain) can comprise two or more smaller domains (e.g., a DNA binding domain and a nuclease domain).

[0116] As used herein, the term “editing” with reference to a nucleic acid molecule (e.g., a target nucleic acid (e.g., DNA) molecule (e.g., a double stranded target nucleic acid sequence (e.g., dsDNA, (e.g., genomic dsDNA))) refers to the introduction of a variation (as defined herein) (also referred to as an edit herein) in the nucleic acid molecule or a non-sequence based alteration to the nucleic acid molecule, including, without limitation epigenetic modifications (including, but not limited to, e.g., methylation, demethylation, acetylation, deacetylation, phosphorylation,Attorney Docket No.62801.37WO01 dephosphorylation, histone alteration (e.g., histone acetylation, deacetylation methylation, phosphorylation, ubiquitination and sumoylation), alteration to chromatin structure), nucleotide modifications (e.g., a nucleotide modification described herein) (e.g., mediated at least in part by a fusion protein described herein). In some embodiments, the variation or edit comprises a substitution, addition, deletion, or inversion.

[0117] As used herein, the term “edited strand” with reference to a double stranded nucleic acid molecule (e.g., a dsDNA molecule) refers to the strand of the double stranded nucleic acid molecule that is edited by e.g., a nuclease, system, etc. described herein. Likewise, as used herein, the term “non-edited strand” with reference to a double stranded nucleic acid molecule (e.g., a dsDNA molecule) refers to the strand of the double stranded nucleic acid molecule that is not edited by e.g., a nuclease, system, etc. described herein. For example, in some embodiments, an additional gRNA is utilized in s system described herein that targets the non-edited strand for nicking by a Cas nuclease (e.g., a Cas nuclease described herein). Without wishing to be bound by theory, it is thought that the in some embodiments, the addition of a nick in the non-edited strand biases mismatch DNA repair in favor of the edited sequence. See, e.g., Scholefield, J., Harrison, P.T. Prime editing – an update on the field. Gene Ther 28, 396–401 (2021). https: / / doi.org / 10.1038 / s41434-021-00263-9, the entire contents of which are incorporated by reference herein for all purposes.

[0118] As used herein, the term “functional fragment” in reference to a protein refers to a fragment of a reference protein that retains at least one particular function. Not all functions of the reference protein need be retained by a functional fragment of the protein. In some instances, one or more functions are selectively reduced or eliminated. In some embodiments, the reference protein is a wild type protein. For example, a functional fragment of a polymerase, reverse transcriptase, or nuclease can refer to a fragment of said protein that retains activity. In some embodiments, the functional fragment comprises one or more domains (e.g., 1, 2, 3, or more) of the reference protein.

[0119] As used herein, the term “functional variant” in reference to a protein refers to a protein that comprises at least one but not more than 20%, not more than 15%, not more than 12%, no more than 10%, no more than 8% amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference protein, wherein the protein retains at least one particular function of the reference protein. Not all functions of the reference protein (e.g.,Attorney Docket No.62801.37WO01 wild type) need be retained by the functional variant of the protein. In some instances, one or more functions are selectively altered, reduced or eliminated (e.g., nuclease (e.g., endonuclease) activity). In some embodiments, the reference protein is a wild type protein. In some embodiments, the functional variant comprises one or more domains (e.g., 1, 2, 3, or more) of the reference protein.

[0120] As used herein, the term “functional fragment or variant thereof” and the like with reference to an agent (e.g., a protein) should be understood to include (a) functional variants, (b) functional fragments, and (c) functional fragments and functional variants.

[0121] As used herein, the term “fuse” and grammatical equivalents thereof refers to the operable connection of at least a first polypeptide to a second polypeptide, wherein the first and second polypeptides are not naturally found operably connected together. For example, the first and second polypeptides are derived from different proteins and / or are from different organisms. The term fuse encompasses both a direct connection of the at least two polypeptides through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker).

[0122] As used herein, the term “fusion protein” and grammatical equivalents thereof refer to a protein that comprises at least one polypeptide operably connected to another polypeptide, wherein the first and second polypeptides are not naturally found operably connected together. For example, the first and second polypeptides of the fusion protein are each derived from different proteins and / or are from heterologous organisms. For the sake of clarity, it will be understood that neither the first nor second polypeptide is required to be a full-length protein (e.g., a full-length naturally occurring protein). For example, the first and / or second polypeptide can comprise or consist of fragments (e.g., functional fragments or domains of full-length proteins (e.g., engineered, naturally occurring). The at least two polypeptides of the fusion protein can be directly operably connected through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker). Thus, the term fusion polypeptide encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A – Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A – peptide linker – Polypeptide B).

[0123] As used herein, the term “guide RNA” or “gRNA” refers to an RNA molecule that can associate with a nuclease (e.g., endonuclease) (e.g., a nuclease described herein (e.g., an endonuclease described herein)) to direct the nuclease (e.g., endonuclease) (e.g., a nucleaseAttorney Docket No.62801.37WO01 described herein (e.g., an endonuclease described herein)) to a target nucleic acid molecule (e.g., within a gene (e.g., within a cell)). In some embodiments, the gRNA comprises or consists of a crRNA. In some embodiments, the gRNA comprises a crRNA and a tracrRNA. As described throughout, in embodiments, wherein the gRNA comprises a crRNA and a tracrRNA, the crRNA and tracrRNA may be part of the same larger RNA molecule (e.g., a sgRNA) or separate RNA molecules.

[0124] As used herein, the term “heterologous,” when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a protein comprising a “heterologous moiety” means a protein that is joined to a moiety (e.g., small molecule, protein, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the protein in nature.

[0125] As used herein, the term “heterologous object sequence” refers to an RNA molecule that encodes a desired edit (e.g., substitution, addition, deletion of one or more nucleotides) of a target nucleic acid (e.g., DNA) sequence (e.g., a gene) that can be utilized as a template strand by a polymerase (e.g., a reverse transcriptase) (e.g., described herein) to polymerize the desired nucleic acid sequence (e.g., DNA sequence (e.g., gene sequence)) (i.e., to polymerize sequence complementary to the edit template). In some embodiments, the edit template is part of a template gRNA (e.g., described herein).

[0126] It is clear from the disclosure, but for the sake of clarity, it is to be understood that the use of the term “heterologous protein” (e.g., any heterologous protein described herein) includes the full-length protein, as well as less than the full-length protein, including, e.g., functional fragments, functional variants, and domains of the full-length protein.

[0127] As used herein, the term “isolated” with reference to a biomolecule (e.g., a protein or polynucleotide) refers to a biomolecule (e.g., a protein or polynucleotide) that is substantially free of other cellular components with which it is associated in the natural state.

[0128] As used herein, the term “tracrRNA” refers to an RNA molecule (e.g., part of a gRNA (e.g., a sgRNA)) that mediates binding of a gRNA to a nuclease (e.g., an endonuclease described herein (e.g., a nickase described herein)).

[0129] As used herein, the term “translatable RNA” refers to any RNA that encodes at least one polypeptide and can be translated to produce the encoded protein in vitro, in vivo, in situ or ex vivo. A translatable RNA may be an mRNA or a circular RNA encoding a polypeptide.Attorney Docket No.62801.37WO01

[0130] As used herein, the terms “agent” and “moiety” are used interchangeably herein and refer to any macro or micro molecule that can be operably connected to another macro or micro molecule (e.g., a protein (e.g., a nuclease (or a functional fragment, functional variant, or domain thereof)) or a nucleic acid molecule encoding the protein (e.g., nuclease (e.g., endonuclease))). Exemplary moieties include, but are not limited small molecules, proteins, polynucleotides (e.g., DNA, RNA), carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG).

[0131] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single-stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non- natural, or altered internucleotide linkage, including a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan appreciates that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U).

[0132] As used herein, the term “nucleobase editor” refers to an agent (e.g., a biomolecule (e.g., a protein (or a functional fragment, functional variant, or domain thereof))) that can mediate nucleobase editing activity.

[0133] As used herein, the term “nucleobase editing activity” refers to the ability of an agent (e.g., a biomolecule (e.g., a protein (or a functional fragment, functional variant, or domain thereof))) to chemically alter a nucleobase within a polynucleotide. In some embodiments, the nucleobase editing activity is cytidine deaminase activity, e.g., converting a target C·G to Τ·Α. In some embodiments, the nucleobase editing activity is adenosine deaminase activity, e.g., converting Α·Τ to G·C. In some embodiment, the nucleobase editing activity is cytidine deaminaseAttorney Docket No.62801.37WO01 activity and adenosine deaminase activity, e.g., converting Α·Τ to G·C.

[0134] As used herein, the term “operably connected” refers to the linkage of two moieties in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) such that both polypeptides are functional (e.g., an in-frame fusion protein comprising a nuclease described herein (e.g., an endonuclease described herein). Or for example, a transcription regulatory polynucleotide e.g., a promoter, enhancer, or other expression control element operably linked to a polynucleotide that encodes a protein to affect the transcription of the polynucleotide that encodes the protein. The term “operably connected” also refers to the conjugation of a moiety to e.g., a polynucleotide or polypeptide (e.g., the conjugation of a PEG polymer to a protein).

[0135] As used herein, the term “PAM” or “protospacer adjacent motif” refers to a short nucleic acid molecule (usually about 2-6 base pairs in length) that follows the nucleic acid region targeted for cleavage by a nuclease (e.g., an endonuclease) (e.g., a nuclease described herein (e.g., of a system described herein)). In some embodiments, the PAM is required for a nuclease (e.g., an endonuclease) (e.g., a nuclease described herein (e.g., of a system described herein)) to cleave the target nucleic acid molecule.

[0136] Determination of “percent identity” between two sequences (e.g., protein (amino acid sequences) or polynucleotide (nucleic acid sequences)), as used herein, can be accomplished using a mathematical algorithm. For example, a specific, non-limiting example of an algorithm utilized for the comparison of two sequences is described in Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such algorithm(s) is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches are performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. For gapped alignment comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform searches which detect distantAttorney Docket No.62801.37WO01 relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is described in Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) and is a part of the GCG sequence alignment software package. When comparing amino acid sequences with the ALIGN program, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0137] As used herein, the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more).

[0138] As used herein, the term “pharmaceutical composition” refers to a composition that is suitable for administration to an animal, e.g., a human subject, and comprises an agent (e.g., therapeutic agent) and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier or diluent” means a substance intended for use in contact with the tissues of human beings and / or non-human animals, and without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit / risk ratio.

[0139] As used herein, the terms “protein” and “polypeptide” refer to a polymer of at least 2 (e.g., at least 5) amino acids linked by a peptide bond. The term “polypeptide” does not denote a specific length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2-50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” and “protein” are used interchangeably herein. In some embodiments, a protein is folded into its three-dimensional structure. Where proteins are contemplated herein, it should be understood that proteins comprising the primary structure are provided herein as well as proteins folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are provided herein.

[0140] As used herein, the term “prophylactic treatment” and the like refers to a treatmentAttorney Docket No.62801.37WO01 administered to a subject for the purpose of decreasing the risk of developing pathology in a subject who does not exhibit signs of a disease or exhibits only early signs of a disease.

[0141] The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered internucleotide linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule.

[0142] As used herein, the term “sgRNA” refers to a gRNA molecule that comprises both a crRNA and a tracrRNA. The components of the sgRNA may be arranged in any suitable order and any component may be operably connected to the adjacent component(s) directly or indirectly (e.g., via a nucleotide linker).

[0143] As used herein, the term “signal peptide” or “signal sequence” refers to a sequence that can direct the transport or localization of a protein, such as a nuclease (e.g., an endonuclease), to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence peptide and the nucleic acid sequence encoding the signal peptide. Thus, references to a signal peptide in the context of a nucleic acid refers to the nucleic acid sequence encoding the signal peptide. Exemplary signal sequences include for example, nuclear localization signal and nuclear export signal.

[0144] As used herein, the term “subject” includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject is a human. In some embodiments, the method subject is a non-human mammal. In some embodiments, the subject is a non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).

[0145] As used herein, the term “template RNA” refers to gRNA molecule that comprises a crRNA, a heterologous object sequence, and a 3' target homology domain. In some embodiments,Attorney Docket No.62801.37WO01 the template RNA further comprises a tracrRNA. In some embodiments, the template RNA further comprises an RNA sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein described herein). The components of the template RNA may be arranged in any suitable order and any component may be operably connected to the adjacent component(s) directly or indirectly (e.g., via a nucleotide linker). In some embodiments, the template RNA comprises from 5' to 3' a crRNA, a heterologous object sequence, and a 3' target homology domain. In some embodiments, the template RNA comprises from 5' to 3' a crRNA, a sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein described herein), a heterologous object sequence, and a 3' target homology domain. In some embodiments, the template RNA comprises from 5′ to 3′ a crRNA, a tracrRNA, a heterologous object sequence, and a 3′ target homology domain. In some embodiments, the template RNA comprises from 5′ to 3′ a crRNA, a tracrRNA, a sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein described herein), a heterologous object sequence, and a 3′ target homology domain. In some embodiments, the template RNA is part of a system (e.g., a reverse transcriptase-based system) described herein.

[0146] As used herein, the term “therapeutically effective amount” of an agent (e.g., therapeutic agent) refers to any amount of the agent (e.g., therapeutic agent) that, when used alone or in combination with another therapeutic agent, improves a disease condition, e.g., protects a subject against the onset of a disease (or infection); improves a symptom of disease or infection, e.g., decreases severity of disease or infection symptoms, decreases frequency or duration of disease or infection symptoms, increases disease or infection symptom-free periods; prevents or reduces impairment or disability due to the disease or infection; or promotes disease (or infection) regression. The ability of a therapeutic agent to improve a disease condition can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0147] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease and / or symptom(s) associated therewith or obtaining a desired pharmacologic and / or physiologic effect. It will be appreciated that, although not precluded, treating a disease does not require that the disease, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, theAttorney Docket No.62801.37WO01 effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and / or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a compositions as described herein.

[0148] As used herein, the term terms “variant” or “variation” with reference to a nucleic acid molecule (e.g., a nucleic acid molecule encoding a nuclease as described herein), refer to a nucleic acid molecule that comprises at least one substitution, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid molecule. As used herein, the term “variant” or “variation” with reference to a protein refers to a peptide or protein (e.g., nucleases (e.g., endonucleases) described herein) that comprises at least one substitution, inversion, addition, or deletion of an amino acid residue compared to a reference protein.

[0149] As used herein, the term “3' target homology domain” refers to an RNA molecule that is capable of hybridizing to the 3' end of a single stranded nucleic acid flap (the 3' target sequence) created after induction of a single strand break (i.e., a nick) in a target double stranded nucleic acid (e.g., DNA) molecule (e.g., by a nuclease (e.g., endonuclease) described herein (or a fusion protein comprising the same)). The hybridization of the 3' target homology domain to the 3' target sequence creates a duplex that can be utilized as a substrate by a polymerase (e.g., a reverse transcriptase) (e.g., described herein) for polymerization of a nucleic acid (e.g., DNA) molecule (e.g., utilizing the heterologous object sequence). In some embodiments, the 3' target homology domain is part of a template RNA (e.g., described herein). 5.2 Cas Nucleases

[0150] Provided herein are, inter alia, nucleases (e.g., endonucleases, nickases, etc.) (and functional fragments, functional variants, and domains thereof), useful in, inter alia, modifying (e.g., editing) a nucleic acid molecule (e.g., DNA, gene, genome (e.g., within a cell, e.g., within a cell in a subject (e.g., a mammalian subject, e.g., a human subject))) (e.g., in vivo, ex vivo, or in vitro). In some embodiments, the nuclease (e.g., endonuclease, nickase) is non-naturally occurring. The amino acid sequence of exemplary nucleases (e.g., endonucleases) of the disclosure is set forth in Table 1 and in SEQ ID NOS: 1-28.Attorney Docket No.62801.37WO01 Table 1. The Amino Acid Sequence of Nucleases (e.g., Endonucleases). Description Amino Acid Sequence SEQ ID NOAttorney Docket No.62801.37WO01 HEFNKNKGENEISKTFIFKTKCGKNDITSLWVPAMEEYCTYYNRVSKWICDN LTEMRIGDLAQYIDNHGSAYYSAVTDITKKDLPLYKIFKKGFSGLCADNALY CAIAKLNPEGYDGNMFGLSETYYRRQGYIANVFGNYRTKMNAGLKVGCAKWKAttorney Docket No.62801.37WO01 KNDELFVHLTSPIDFEKEVCEIKNAVGVDVNIKHNMLATSIKDDGNVKGYIN LYKELVNDCDFISTCNEDEFDLYRQMSESVNFGILETDSLFERVVNQSKGGC LNNKFIRRELAMQKVFDNITKTNKDQNIVDYVNYVKMLRAKYKAYFILKEKYAttorney Docket No.62801.37WO01 KAIHFADIKDKFAQLCNNNDVNVVFGPSAFTSQMDSETHSLYYVEKETNGKN GKTGKKFVLADKKSVRRRQETHINGLNADFNAARNLEYIASNPELLERMTKR TKSGKDMYNTPSWNIRQEFKKNLSVRTINTFRELGNVKYGKAttorney Docket No.62801.37WO01 QLIKTQTIFEVVNKKIESETDFENLITYFKNRETPNDEKIKRLELLFDYYTK HKNEINEEIEKHAVESLKSFNGCRRNGNRKTMTVQMQKMLLKKHGLTSYILH LVLDKKPYDINLMGNRQTVKVDNNGNRVDLVDISSKHGYDLTFEVKGKTLFFAttorney Docket No.62801.37WO01 GFVDDSTLSKDGMDKRRFESPFNNTDCAKQIKSKLDNVLQDIEGCRKNIITY VYKIFETNGYVTIGLENLDNSNFEYHKPLCSIKSLLNYHKLLNKTIEQVKEN NSVADLIEKDYYIFTFDENKNIIDAKFSDKGFYKINKSWFINQLIKVLHFASAttorney Docket No.62801.37WO01 DGTLKGYLNIYKKLLLDTELTSLLHKQDFDDMKELSHNVCFGPIEYNFLLSR ILDLDAYEKKVEDRITHSMKEMLKTETDERNKMYLGSVIKMRALLKVYISTK NRYHKEQQSYDESMGFTDTSTASKDTMDKRRFENPFSETETGKKLNNDLSALAttorney Docket No.62801.37WO01 KNPKTKKLVIMDKDKVRPIQEKYKLNGLNADFNSARNIAYIVENEILRNSFL KEETKKYTYNTPLFTPRLKSSEKIITELKKLGMTTVI TIGEAASRICQKKLQGKAKYVKHCLSAEWRDQPLYKIFCKGFNGMDADNLII

[0151] In some embodiments, the amino acid sequence of the Cas nuclease (e.g.,Attorney Docket No.62801.37WO01 endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any polypeptide set forth in Table 1 or set forth in any one of SEQ ID NOS: 1-28. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any polypeptide set forth in Table 1 or set forth in any one of SEQ ID NOS: 1-28. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any polypeptide set forth in Table 1 or set forth in any one of SEQ ID NOS: 1-28.

[0152] In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a polypeptide set forth in Table 1. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a polypeptide set forth in Table 1. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a polypeptide set forth in Table 1.

[0153] In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises 1 or more but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%) amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functionalAttorney Docket No.62801.37WO01 variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of from about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 1-040, 10-30, 10-20, 50-200, 50-150, 50-100, 50-90, 50-80, 50-70, or 50-60 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0154] In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises 1 or more but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g.,Attorney Docket No.62801.37WO01 endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of a polypeptide set forth in Table 1, and further comprises or consists of from about 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-200, 10- 150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 1-040, 10-30, 10-20, 50-200, 50-150, 50-100, 50- 90, 50-80, 50-70, or 50-60 amino acid substitutions.

[0155] In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOS: 1-28. In some embodiments, the amino acid sequence of Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOS: 1-28. In some embodiments, the amino acid sequence of Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOS: 1-28.

[0156] In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises 1 or more but less than 20% (e.g., less than 15%, less than 12%, less than 10%, less than 8%) amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or theAttorney Docket No.62801.37WO01 functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid variations (e.g., substitutions, additions, deletions, etc.). In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of from about 1-100, 1-90, 1-80, 1-70, 1- 60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 1-040, 10-30, 10-20, 50-200, 50-150, 50-100, 50-90, 50-80, 50-70, or 50-60 amino acid variations (e.g., substitutions, additions, deletions, etc.).

[0157] In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises 1 or more but less than 15% (less than 12%, less than 10%, less than 8%), amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment, functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid substitutions. In some embodiments, the amino acid sequence of the Cas nuclease (e.g., endonuclease) (or the functional fragment,Attorney Docket No.62801.37WO01 functional variant, or domain thereof) comprises or consists of the amino acid sequence of any one of SEQ ID NOS: 1-28, and further comprises or consists of from about 1-100, 1-90, 1-80, 1-70, 1- 60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 10-200, 10-150, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 1-40, 10-30, 10-20, 50-200, 50-150, 50-100, 50-90, 50-80, 50-70, or 50-60 amino acid substitutions.

[0158] In some embodiments, the amino acid sequence of the nuclease is less than about 1000, 950, 900, 850, or 800 amino acid acids in length. In some embodiments, the amino acid sequence of the nuclease is from about 500-1000, 500-950, 500-900, 500-850, 500-800, 600-1000, 600-950, 600-900, 600-850, 600-800, 700-1000, 700-950, 700-900, 700-850, 700-800, 800-1000, 800-950, 800-900, or 800-850 amino acid acids in length. In some embodiments, the amino acid sequence of the nuclease is from about 300-1000, 300-950, 300-900, 300-850, 300-800, 300-750, 300-700, 300-650, 300-600, 300-550, 300-500, 300-450, 300-400, 350-1000, 350-950, 350-900, 350-850, 350-800, 350-750, 350-700, 350-650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-1000, 400-950, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450, 450-1000, 450-950, 450-900, 450-850, 450-800, 450-750, 450-700, 450-650, 450-600, 450-550, 450-500, 500-1000, 500-950, 500-900, 500-850, 500-800, 500-750, 500-700, 500-650, 500-600, 500-550, 600-1000, 600-950, 600-900, 600-850, 600-800, 600-750, 600-700, 600-650, 700-1000, 700-950, 700-900, 700-850, 700-800, 700-750, 800-1000, 800-950, 800-900, or 800- 850 amino acid acids in length.

[0159] In some embodiments, the amino acid sequence of the nuclease is less than about 1000, 950, 900, 850, or 800 amino acid acids in length. In some embodiments, the amino acid sequence of the nuclease is from about 500-1000, 500-950, 500-900, 500-850, 500-800, 600-1000, 600-950, 600-900, 600-850, 600-800, 700-1000, 700-950, 700-900, 700-850, 700-800, 800-1000, 800-950, 800-900, or 800-850 amino acid acids in length.

[0160] In some embodiments, the nuclease has a molecular weight of about 65kDa-90kDa, 65kDa-85kDa, about 65kDa-80kDa, about 65kDa-75kDa, about 65kDa-70kDa, 70kDa-90kDa, 70kDa-85kDa, about 70kDa-80kDa, about 70kDa-75kDa, 75kDa-90kDa, 75kDa-85kDa, about 75kDa-80kDa, 80kDa-90kDa, or 85kDa-90kDa.

[0161] Provided herein are, inter alia, Cas nickases (and functional fragments, functional variants, and domains thereof), useful in, inter alia, modifying (e.g., editing) a nucleic acid molecule (e.g., DNA, gene, genome (e.g., within a cell, e.g., within a cell in a subject (e.g., aAttorney Docket No.62801.37WO01 mammalian subject, e.g., a human subject))) (e.g., in vivo, ex vivo, or in vitro). In some embodiments, the nickase is non-naturally occurring. In some embodiments, the Cas nickase comprises one or more amino acid substitution (e.g., substitution of a non-alanine amino acid residue with an alanine) relative to one of the Cas nucleases (e.g., endonucleases, nickases, etc.) described herein (e.g., CasNuc-1-18 (SEQ ID NOS: 1-28)). In some embodiments, the Cas nickase comprises one or more substitution of a non-alanine amino acid residue with an alanine relative to one of the Cas nucleases (e.g., endonucleases, nickases, etc.) described herein (e.g., CasNuc-1-18 (SEQ ID NOS: 1-28)). The positions of exemplary substitutions (e.g., alanine substitutions) in the amino acid sequence of Cas nickases described herein relative to the parental Cas nuclease (e.g., endonuclease, nickase, etc.) are set forth in Table 2. Table 2. The Amino Acid Sequence of Nickases. Position of Amino Acid (e.g., Alanine) Substitution Description (Amino Acid Numberin Relative to the Parental Cas nuclease (e endonuclease, 8,Attorney Docket No.62801.37WO01 2, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 5, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 8 319 320 322 323 324 325 326 327 328 329 330 331 333 409 410 412 1, 2,Attorney Docket No.62801.37WO01 0, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 7, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 9 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 2,Attorney Docket No.62801.37WO01 9, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 6, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 8 619 622 623 625 626 627 628 629 630 631 632 633 634 635 636 638 2, 6,Attorney Docket No.62801.37WO01 2, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 9, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770 5 6 7 9 10 11 12 13 14 15 16 17 18 19 32 35 36 39 40 43 62 63 65 66 2,Attorney Docket No.62801.37WO01 4, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 6, 267, 268, 269, 270, 274, 275, 276, 280, 285, 286, 287, 288, 289, 290, 291, 292, 3 294 299 300 304 310 311 312 313 314 315 316 317 318 319 320 321 2,Attorney Docket No.62801.37WO01 6, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 4, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 1 482 483 484 485 486 487 488 501 504 505 506 507 508 509 510 511Attorney Docket No.62801.37WO01 2, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 598, 600, 1, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 5 626 627 628 629 630 631 632 633 634 635 636 638 639 640 641 642 8,Attorney Docket No.62801.37WO01 6, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 3 2 5 6 7 8 9 10 11 12 25 28 29 32 33 36 55 56 58 59 60 63 64 65 66 67, 2,Attorney Docket No.62801.37WO01 9, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 255, 266, 267, 268, 9, 270, 272, 273, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 8 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 6,Attorney Docket No.62801.37WO01 0, 621, 622, 624, 625, 626, 627, 628, 629, 630, 636, 643, 644, 645, 646, 647, 648, 9, 650, 651, 652, 653, 654, 655, 656, 657, 669, 670, 671, 672, 673, 674, 675, 676, 7 678 679 680 681 682 683 684 685 686 687 688 689 690 691 724 725 1, 7,Attorney Docket No.62801.37WO01 , 10, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 38, 39, 40, 43, 46, 47, 48, 49, 50, 52, 53, 56, 3, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 5 96 97 101 124 125 181 182 183 184 185 186 187 188 189 190 191 192 3,Attorney Docket No.62801.37WO01 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 640, 641, 642, 643 644 645 646 647 648 649 650 651 652 655 658 659 662 663 665 666 8,at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 1, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,Attorney Docket No.62801.37WO01 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1.

[0163] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO:Attorney Docket No.62801.37WO01 1; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or 99% identical the amino acid sequence set forth in SEQ ID NO: 1.

[0164] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1.

[0165] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 1, andAttorney Docket No.62801.37WO01 comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1.

[0166] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320,Attorney Docket No.62801.37WO01 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1; and other than alanine at any one or more of the above recited amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 1.

[0167] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489,Attorney Docket No.62801.37WO01 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715, 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1.

[0168] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 2, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624,Attorney Docket No.62801.37WO01 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2.

[0169] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,Attorney Docket No.62801.37WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 2.

[0170] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2.

[0171] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 2, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95,Attorney Docket No.62801.37WO01 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2.

[0172] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,Attorney Docket No.62801.37WO01 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 2.

[0173] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,Attorney Docket No.62801.37WO01 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2.

[0174] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 3, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722,Attorney Docket No.62801.37WO01 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3.

[0175] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 3.

[0176] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13,Attorney Docket No.62801.37WO01 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3.

[0177] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 3, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332,Attorney Docket No.62801.37WO01 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3.

[0178] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607,Attorney Docket No.62801.37WO01 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 3.

[0179] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771,Attorney Docket No.62801.37WO01 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3.

[0180] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 4, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4.

[0181] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91,Attorney Docket No.62801.37WO01 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 4.

[0182] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320,Attorney Docket No.62801.37WO01 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4.

[0183] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 4, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562,Attorney Docket No.62801.37WO01 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4.

[0184] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, aminoAttorney Docket No.62801.37WO01 acid numbering relative to SEQ ID NO: 4; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 4.

[0185] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4.

[0186] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 5, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any oneAttorney Docket No.62801.37WO01 or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563, 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5.

[0187] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422,Attorney Docket No.62801.37WO01 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563, 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 5.

[0188] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551,Attorney Docket No.62801.37WO01 562, 563, 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5.

[0189] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 5, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563, 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700,Attorney Docket No.62801.37WO01 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5.

[0190] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563, 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 5.

[0191] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, and comprises an alanine at any one or more of amino acid positionsAttorney Docket No.62801.37WO01 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563, 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5.

[0192] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 6, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306,Attorney Docket No.62801.37WO01 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6.

[0193] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574,Attorney Docket No.62801.37WO01 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 6.

[0194] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696,Attorney Docket No.62801.37WO01 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6.

[0195] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 6, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6.

[0196] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126,Attorney Docket No.62801.37WO01 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 6.

[0197] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411,Attorney Docket No.62801.37WO01 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6.

[0198] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 7, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563,Attorney Docket No.62801.37WO01 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7.

[0199] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 57, 59, 60, 61, 62, 63, 64, 65, 66, 67, 88, 92, 106, 107, 108, 109, 110, 111, 112, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 159, 160, 161, 162, 163, 164, 165, 167, 168, 182, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 211, 214, 216, 218, 227, 230, 232, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 285, 313, 314, 315, 316, 317, 318, 319, 324, 325, 326, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 347, 348, 349, 350, 351, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 373, 374, 375, 376, 377, 378, 379, 381, 383, 384, 385, 387, 388, 389, 390, 391, 392, 393, 394, 447, 448, 451, 454, 455, 458, 459, 461, 462, 463, 466, 467, 485, 486, 514, 515, 516, 517, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 555, 556, 557, 558, 560, 561, 562, 564, 565, 569, 570, 571, 572, 573, 574, 577, 578, 580, 581, 582, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 631, 632, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 663, 664, 665, 666, 667, 668, 669, 671, 672, 673, 674, 675, 676, 677, 683, 685, 736, 737, and / or 738, amino acid numbering relative to SEQ ID NO: 7; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forthAttorney Docket No.62801.37WO01 in SEQ ID NO: 7.

[0200] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7.

[0201] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 7, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134,Attorney Docket No.62801.37WO01 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7.

[0202] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516,Attorney Docket No.62801.37WO01 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 7.

[0203] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635,Attorney Docket No.62801.37WO01 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7.

[0204] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 8, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8.

[0205] In some embodiments, the nickase comprises or consists of the amino acid sequenceAttorney Docket No.62801.37WO01 set forth in SEQ ID NO: 8, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 8.

[0206] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177,Attorney Docket No.62801.37WO01 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8.

[0207] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 8, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,Attorney Docket No.62801.37WO01 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8.

[0208] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,Attorney Docket No.62801.37WO01 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 8.

[0209] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8.

[0210] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,Attorney Docket No.62801.37WO01 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 9, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9.

[0211] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290,Attorney Docket No.62801.37WO01 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 9.

[0212] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502,Attorney Docket No.62801.37WO01 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9.

[0213] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 9, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647,Attorney Docket No.62801.37WO01 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9.

[0214] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9; and other than the alanine at any one or more of the foregoing amino acid positions is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 9.Attorney Docket No.62801.37WO01

[0215] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, and comprises an alanine at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9.

[0216] In some embodiments, the nickase comprises or consists of an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 10, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243,Attorney Docket No.62801.37WO01 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 408, 409, 411, 412, 413, 415, 416, 417, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 441, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 734, 736, 737, 738, 742, 746, 747, 748, 749, 750, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, and / or 769, amino acid numbering relative to SEQ ID NO: 10.

[0217] In some embodiments, the nickase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, and comprises an amino acid variation (e.g., substitution (e.g., substitution with an alanine)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 408, 409, 411, 412, 413, 415, 416, 417, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 441, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,Attorney Docket No.62801.37WO01 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 734, 736, 737, 738, 742, 746, 747, 748, 749, 750, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, and / or 769, amino acid numbering relative to SEQ ID NO: 10; and other than the one or more amino acid variation is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical the amino acid sequence set forth in SEQ ID NO: 10.

[0218] In some embodim...

Claims

Attorney Docket No.62801.37WO01 CLAIMS What Is Claimed Is:

1. A recombinant nuclease that comprises an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any nuclease set forth in Table 1 or set forth in any one of SEQ ID NOS: 1-28.

2. A nuclease (e.g., nickase) (e.g., recombinant nuclease (e.g., recombinant nickase)) that comprises (a) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 1; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 176, 177, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 263, 264, 265, 266, 267, 271, 272, 273, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 398, 399, 401, 402, 403, 405, 406, 407, 414, 415, 416, 417, 418, 419, 420, 421, 422, 424, 425, 428, 429, 431, 432, 433, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 598, 601, 602, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 621, 622, 623, 624, 625, 626, 627, 633, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 711, 713, 714, 715,Attorney Docket No.62801.37WO01 719, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, and / or 746, amino acid numbering relative to SEQ ID NO: 1; (b) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 2; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 37, 40, 41, 44, 45, 48, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 94, 95, 96, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 112, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 303, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 444, 445, 447, 448, 449, 451, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 504, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 544, 545, 548, 559, 560, 561, 562, 563, 565, 566, 568, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 630, 633, 634, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 653, 654, 655, 656, 657, 658, 659, 665, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 708, 709, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 774, 775, 776, 777, and / or 778, amino acid numbering relative to SEQ ID NO: 2; (c) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 3; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17,Attorney Docket No.62801.37WO01 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 86, 87, 88, 89, 90, 91, 106, 107, 108, 111, 114, 115, 116, 117, 118, 120, 121, 124, 131, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 168, 192, 193, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 277, 278, 279, 280, 281, 285, 286, 287, 291, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 310, 311, 313, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 424, 425, 427, 428, 429, 431, 432, 433, 443, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 457, 458, 460, 461, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 514, 517, 518, 519, 520, 521, 522, 523, 524, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 554, 555, 558, 569, 570, 571, 572, 573, 575, 576, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 636, 639, 640, 643, 644, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 659, 660, 661, 662, 663, 664, 665, 671, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 758, 760, 761, 762, 766, 770, 771, 772, 773, 774, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, and / or 793, amino acid numbering relative to SEQ ID NO: 3; (d) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 4; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 150, 154, 178, 179, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323,Attorney Docket No.62801.37WO01 324, 325, 326, 327, 329, 410, 411, 413, 414, 415, 417, 418, 419, 429, 430, 431, 432, 433, 434, 435, 436, 437, 439, 440, 443, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 500, 503, 504, 505, 506, 507, 508, 509, 510, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 544, 555, 556, 557, 558, 559, 561, 562, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 625, 628, 629, 632, 633, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 648, 649, 650, 651, 652, 653, 654, 660, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 741, 743, 744, 745, 749, 753, 754, 755, 756, 757, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, and / or 776, amino acid numbering relative to SEQ ID NO: 4; (e) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 5; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 76, 77, 78, 79, 80, 81, 96, 97, 98, 101, 104, 105, 106, 107, 108, 110, 111, 114, 121, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 158, 182, 183, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 304, 305, 307, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 415, 416, 418, 419, 420, 422, 423, 424, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 447, 450, 451, 453, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 507, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 547, 548, 551, 562, 563,Attorney Docket No.62801.37WO01 564, 565, 566, 568, 569, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 629, 632, 633, 636, 637, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 652, 653, 654, 655, 656, 657, 658, 664, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 740, 742, 743, 744, 748, 752, 753, 754, 755, 756, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 5; (f) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 6; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699,Attorney Docket No.62801.37WO01 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 6; (g) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 7; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 738, 740, 741, 742, 746, 750, 751, 752, 753, 754, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, and / or 773, amino acid numbering relative to SEQ ID NO: 7; (h) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 8; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 71, 72, 73, 74, 75, 76, 91, 92, 93, 96, 99,Attorney Docket No.62801.37WO01 100, 101, 102, 103, 105, 106, 109, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 133, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 153, 177, 178, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 258, 259, 260, 261, 262, 266, 270, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 289, 290, 292, 298, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 398, 399, 401, 402, 403, 405, 406, 407, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 435, 438, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 491, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 597, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and / or 770, amino acid numbering relative to SEQ ID NO: 8; (i) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 9; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 77, 78, 79, 80, 81, 82, 97, 98, 100, 103, 104, 105, 106, 107, 109, 110, 113, 120, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 157, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 265, 266, 267, 268, 269, 273, 274, 275, 279, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 298, 299, 301, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 331, 407, 408, 410, 411, 412, 414, 415, 416, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 445, 447, 448, 449, 450, 451, 452,Attorney Docket No.62801.37WO01 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 498, 501, 502, 503, 504, 505, 506, 507, 508, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 538, 539, 542, 553, 554, 555, 556, 557, 559, 560, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 595, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 628, 631, 632, 635, 636, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 651, 652, 653, 654, 655, 656, 657, 663, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 747, 748, 752, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, and / or 775, amino acid numbering relative to SEQ ID NO: 9; (j) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 10; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 92, 95, 98, 99, 100, 101, 102, 104, 105, 108, 115, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 176, 177, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 261, 262, 263, 264, 265, 269, 270, 271, 275, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 294, 295, 297, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 327, 408, 409, 411, 412, 413, 415, 416, 417, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 441, 442, 444, 445, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 494, 495, 496, 497, 498, 499, 500, 501, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 531, 532, 535, 546, 547, 548, 549, 550, 552, 553, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,Attorney Docket No.62801.37WO01 595, 597, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 734, 736, 737, 738, 742, 746, 747, 748, 749, 750, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, and / or 769, amino acid numbering relative to SEQ ID NO: 10; (k) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 11; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 74, 75, 76, 77, 78, 79, 94, 95, 96, 98, 101, 102, 103, 104, 105, 107, 108, 111, 118, 125, 126, 127, 128, 129, 130, 131, 132, 133, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 155, 180, 181, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 266, 267, 268, 269, 270, 274, 275, 276, 280, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 299, 300, 304, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 334, 411, 412, 414, 415, 416, 418, 419, 420, 425, 426, 427, 428, 429, 430, 431, 432, 433, 435, 436, 439, 440, 442, 444, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 492, 493, 494, 495, 496, 497, 498, 499, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 529, 530, 533, 544, 545, 546, 547, 548, 550, 551, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 611, 614, 615, 618, 619, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 634, 635, 636, 637, 638, 639, 640, 646, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 728, 730, 731, 732, 736, 740, 741, 742, 743, 744, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, and / or 763, amino acid numbering relative to SEQ ID NO: 11;Attorney Docket No.62801.37WO01 (l) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 12; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 70, 71, 72, 87, 88, 89, 93, 96, 97, 98, 99, 100, 102, 103, 106, 113, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 150, 173, 174, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 263, 264, 265, 266, 267, 271, 272, 273, 277, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 296, 297, 299, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 405, 406, 408, 409, 410, 412, 413, 414, 423, 424, 425, 426, 427, 428, 429, 430, 431, 433, 434, 437, 438, 440, 442, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 496, 499, 500, 501, 502, 503, 504, 505, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 536, 537, 540, 551, 552, 553, 554, 555, 557, 558, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 587, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 620, 623, 624, 627, 628, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 643, 644, 645, 646, 647, 648, 649, 655, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 740, 741, 742, 746, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, and / or 769, amino acid numbering relative to SEQ ID NO: 12; (m) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 13; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 70, 84, 85, 86, 87, 88, 89, 104, 105, 106, 109, 112, 113, 114, 115, 116, 118, 119, 122, 129, 136, 137, 138, 139, 140, 141, 142, 143,Attorney Docket No.62801.37WO01 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 166, 189, 190, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 274, 275, 276, 277, 278, 282, 283, 284, 288, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 307, 308, 310, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 340, 416, 417, 419, 420, 421, 423, 424, 425, 427, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 441, 442, 444, 446, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 501, 504, 505, 506, 507, 508, 509, 510, 511, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 541, 542, 545, 556, 557, 558, 559, 560, 562, 563, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 626, 629, 630, 633, 634, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 649, 650, 651, 652, 653, 654, 655, 661, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 737, 738, 742, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, and / or 765, amino acid numbering relative to SEQ ID NO: 13; (n) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 14; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 27, 28, 31, 32, 35, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 79, 80, 81, 84, 90, 91, 92, 93, 94, 96, 97, 100, 107, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 145, 169, 170, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 253, 254, 255, 256, 257, 261, 262, 263, 267, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 287, 288, 290, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 396, 397, 399, 400, 401, 403, 404, 405, 419, 420, 421, 422, 423, 424, 425, 426, 427, 429, 430, 433, 434, 436, 438, 439, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,Attorney Docket No.62801.37WO01 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 492, 495, 496, 497, 498, 499, 500, 501, 502, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 532, 533, 536, 547, 548, 549, 550, 551, 553, 554, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 613, 616, 617, 620, 621, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 636, 637, 638, 639, 640, 641, 642, 648, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 728, 730, 731, 732, 736, 740, 741, 742, 743, 744, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, and / or 763, amino acid numbering relative to SEQ ID NO: 14; (o) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 15; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 34, 37, 38, 41, 42, 45, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 89, 90, 91, 96, 97, 98, 99, 100, 102, 103, 106, 113, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 150, 173, 174, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 256, 257, 258, 259, 260, 264, 268, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 288, 289, 291, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 397, 398, 400, 401, 402, 404, 405, 406, 421, 422, 423, 424, 425, 426, 427, 428, 429, 431, 432, 435, 436, 438, 440, 441, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 494, 497, 498, 499, 500, 501, 502, 503, 504, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 534, 535, 538, 549, 550, 551, 552, 553, 555, 556, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 598, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 615, 618, 619, 622, 623, 625, 626,Attorney Docket No.62801.37WO01 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 638, 639, 640, 641, 642, 643, 644, 650, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 724, 726, 727, 728, 732, 736, 737, 738, 739, 740, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, and / or 758, amino acid numbering relative to SEQ ID NO: 15; (p) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 16; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 33, 34, 37, 38, 41, 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 87, 88, 89, 92, 105, 106, 107, 108, 109, 110, 111, 113, 114, 117, 124, 131, 132, 133, 134, 135, 136, 137, 138, 139, 141, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 161, 184, 185, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 269, 270, 271, 272, 273, 277, 278, 279, 284, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 302, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 322, 323, 324, 325, 326, 327, 328, 330, 331, 333, 409, 410, 412, 413, 414, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 428, 429, 432, 433, 435, 440, 441, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 496, 499, 500, 501, 502, 503, 504, 505, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 536, 537, 540, 551, 552, 553, 554, 555, 557, 558, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 618, 621, 622, 625, 626, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 641, 642, 643, 644, 645, 646, 647, 653, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 725, 726, 727, 731, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, and / or 754, amino acid numbering relative to SEQ ID NO: 16;Attorney Docket No.62801.37WO01 (q) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 17; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 29, 32, 33, 36, 37, 40, 59, 60, 62, 65, 66, 67, 68, 69, 70, 85, 86, 87, 90, 93, 94, 95, 96, 97, 99, 100, 103, 110, 117, 118, 119, 120, 121, 122, 123, 124, 125, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 147, 170, 171, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 253, 254, 255, 256, 257, 261, 262, 263, 267, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 286, 287, 289, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 311, 312, 313, 314, 315, 317, 318, 320, 396, 397, 399, 400, 401, 403, 404, 405, 407, 408, 409, 410, 411, 412, 413, 414, 415, 417, 418, 421, 422, 424, 426, 427, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 482, 485, 486, 487, 488, 489, 490, 491, 492, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 522, 523, 526, 537, 538, 539, 540, 541, 543, 544, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 607, 610, 611, 614, 615, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 630, 631, 632, 633, 634, 635, 636, 642, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 723, 725, 726, 727, 731, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, and / or 753, amino acid numbering relative to SEQ ID NO: 17; (r) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 18; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 25, 28, 29, 32, 33, 36, 55, 56, 58, 59, 60, 63, 64, 65, 66, 67, 68, 83, 84, 85, 88, 91, 92, 93, 94, 95, 97, 98, 101, 108, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132,Attorney Docket No.62801.37WO01 133, 134, 135, 136, 137, 138, 139, 140, 141, 145, 168, 169, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 253, 254, 255, 256, 257, 261, 262, 263, 267, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 286, 287, 291, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 321, 397, 398, 400, 401, 402, 404, 405, 406, 409, 410, 411, 412, 413, 414, 415, 416, 417, 419, 420, 423, 424, 426, 428, 429, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 483, 486, 487, 488, 489, 490, 491, 492, 493, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 523, 524, 527, 538, 539, 540, 541, 542, 544, 545, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 608, 611, 612, 615, 616, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 631, 632, 633, 634, 635, 636, 637, 643, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 721, 722, 726, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, and / or 749, amino acid numbering relative to SEQ ID NO: 18; (s) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 19; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 90, 91, 94, 97, 98, 99, 100, 101, 103, 104, 107, 114, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 151, 175, 176, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 260, 261, 262, 263, 264, 268, 269, 270, 274, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 293, 294, 296, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 326, 403, 404, 406, 407, 408, 410, 411, 412, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 436, 437, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451,Attorney Docket No.62801.37WO01 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 493, 496, 497, 498, 499, 500, 501, 502, 503, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 533, 534, 537, 548, 549, 550, 551, 552, 554, 555, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 599, 600, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 617, 620, 621, 624, 625, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 640, 641, 642, 643, 644, 645, 646, 652, 659, 660, and / or 661, amino acid numbering relative to SEQ ID NO: 19; (t) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 20; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 3, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 133, 134, 136, 137, 138, 140, 141, 142, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 163, 164, 166, 167, 168, 170, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 214, 215, 216, 217, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 252, 255, 266, 267, 268, 269, 270, 272, 273, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 334, 337, 338, 341, 342, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 357, 358, 359, 360, 361, 362, 363, 369, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 446, 448, 449, 450, 454, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, and / or 476, amino acid numbering relative to SEQ ID NO: 20; (u) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease setAttorney Docket No.62801.37WO01 forth in SEQ ID NO: 21; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 88, 89, 90, 93, 110, 111, 112, 113, 114, 115, 116, 118, 119, 122, 129, 136, 137, 138, 139, 140, 141, 142, 143, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 166, 189, 190, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 274, 275, 276, 277, 278, 282, 283, 284, 288, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 306, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 324, 325, 326, 327, 328, 329, 330, 331, 333, 334, 336, 412, 413, 415, 416, 417, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 431, 432, 435, 436, 438, 440, 441, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 496, 499, 500, 501, 502, 503, 504, 505, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 536, 537, 540, 551, 552, 553, 554, 555, 557, 558, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 620, 623, 624, 627, 628, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 643, 644, 645, 646, 647, 648, 649, 655, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 731, 732, 733, 737, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, and / or 761, amino acid numbering relative to SEQ ID NO: 21; (v) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 22; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 2, 3, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 32, 35, 36, 39, 40, 43, 62, 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 89, 90, 91, 94, 97, 98, 99, 100, 101, 103, 104, 107, 114, 122, 123, 124, 125, 126, 127, 128, 129, 130, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 152, 175, 176, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,Attorney Docket No.62801.37WO01 252, 253, 259, 260, 261, 262, 263, 267, 268, 269, 273, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 291, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 319, 320, 321, 397, 398, 400, 401, 402, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 416, 417, 420, 421, 423, 428, 429, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 476, 479, 480, 481, 482, 483, 484, 485, 486, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 516, 517, 520, 531, 532, 533, 534, 535, 537, 538, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 601, 604, 605, 608, 609, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 624, 625, 626, 627, 628, 629, 630, 636, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 724, 725, 726, 730, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, and / or 754, amino acid numbering relative to SEQ ID NO: 22; (w) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 23; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 8, 11, 12, 15, 16, 19, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 64, 65, 66, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 82, 90, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 128, 151, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 240, 241, 242, 248, 249, 250, 251, 252, 256, 257, 258, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 287, 288, 290, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 396, 397, 399, 400, 401, 403, 404, 405, 413, 414, 415, 416, 417, 418, 419, 420, 421, 423, 424, 427, 428, 430, 435, 436, 438, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 489, 492, 493, 494, 495, 496, 497, 498, 499, 501, 502, 503, 504,Attorney Docket No.62801.37WO01 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 529, 530, 533, 544, 545, 546, 547, 548, 550, 551, 554, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 614, 617, 618, 621, 622, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 637, 638, 639, 640, 641, 642, 643, 649, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 730, 732, 733, 734, 738, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, and / or 758, amino acid numbering relative to SEQ ID NO: 23; (x) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 24; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 32, 33, 36, 37, 40, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 86, 87, 88, 91, 104, 105, 106, 107, 108, 109, 110, 112, 113, 116, 122, 129, 130, 131, 132, 133, 134, 135, 136, 137, 139, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 160, 183, 184, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 270, 271, 272, 273, 274, 278, 279, 280, 285, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 301, 302, 304, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 334, 410, 411, 413, 414, 415, 417, 418, 419, 423, 424, 425, 426, 427, 428, 429, 430, 431, 433, 434, 437, 438, 440, 441, 442, 444, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 497, 500, 501, 502, 503, 506, 507, 508, 509, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 539, 540, 543, 554, 555, 556, 557, 558, 560, 561, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 622, 625, 626, 629, 630, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 645, 646, 647, 648, 649, 650, 651, 657, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673,Attorney Docket No.62801.37WO01 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 701, 702, 735, 737, 738, 739, 743, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, and / or 765, amino acid numbering relative to SEQ ID NO: 24; (y) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 25; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 9, 10, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 38, 39, 40, 43, 46, 47, 48, 49, 50, 52, 53, 56, 63, 71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 101, 124, 125, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 208, 209, 210, 211, 212, 216, 217, 218, 222, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 240, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 259, 260, 261, 262, 263, 264, 265, 266, 268, 269, 270, 346, 347, 349, 350, 351, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 369, 370, 372, 377, 378, 380, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 425, 428, 429, 430, 431, 432, 433, 434, 435, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 465, 466, 469, 480, 481, 482, 483, 484, 486, 487, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 550, 553, 554, 557, 558, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 573, 574, 575, 576, 577, 578, 579, 585, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 673, 674, 675, 679, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, and / or 700, amino acid numbering relative to SEQ ID NO: 25; (z) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 26; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 1, 2, 3, 4, 6, 82, 83, 85, 86, 87,Attorney Docket No.62801.37WO01 89, 90, 91, 105, 106, 107, 108, 109, 110, 111, 112, 113, 115, 116, 119, 120, 122, 124, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 178, 181, 182, 183, 184, 185, 186, 187, 188, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 218, 219, 222, 233, 234, 235, 236, 237, 239, 240, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 297, 300, 301, 304, 305, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 332, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 412, 414, 415, 416, 420, 424, 425, 426, 427, 428, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, and / or 450, amino acid numbering relative to SEQ ID NO: 26; (aa) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 27; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 35, 38, 39, 42, 43, 46, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 111, 112, 113, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 129, 138, 145, 146, 147, 148, 149, 150, 151, 152, 153, 155, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 176, 199, 200, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 286, 287, 288, 289, 290, 294, 295, 296, 300, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 316, 317, 319, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 349, 427, 428, 430, 431, 432, 434, 435, 436, 453, 454, 455, 456, 457, 458, 459, 460, 461, 463, 464, 467, 468, 470, 471, 472, 474, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 527, 530, 531, 532, 533, 536, 537, 538, 539, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 569, 570, 573, 584, 585, 586, 587, 588, 590, 591, 594, 597, 598, 599, 600, 601, 602, 603, 604,Attorney Docket No.62801.37WO01 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 655, 658, 659, 662, 663, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 678, 679, 680, 681, 682, 683, 684, 690, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 766, 768, 769, 770, 774, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, and / or 798, amino acid numbering relative to SEQ ID NO: 27; or (bb) an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the nuclease set forth in SEQ ID NO: 28; and comprises an amino acid variation (e.g., substitution (e.g., an alanine substitution)) at any one or more of amino acid positions 17, 18, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 102, 103, 104, 105, 106, 110, 111, 112, 116, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 134, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 152, 153, 154, 155, 156, 157, 158, 159, 161, 162, 164, 240, 241, 243, 244, 245, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 259, 260, 263, 264, 266, 268, 269, 271, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 324, 327, 328, 329, 330, 331, 332, 333, 334, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 364, 365, 368, 379, 380, 381, 382, 383, 385, 386, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, and / or 404, amino acid numbering relative to SEQ ID NO:

28.

3. The nuclease of claim 1 or 2, having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, and / or 8) of the following properties (or engineered to have one or more of the following properties): (a) the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule;Attorney Docket No.62801.37WO01 (d) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (e) DNA endonuclease activity; (f) DNA nickase activity; (g) RNA guided DNA endonuclease activity; and / or (h) RNA guided DNA nickase activity.

4. The nuclease of claim 1 or 3, having one or more of the following properties: (a) the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) DNA endonuclease activity; and / or (c) RNA guided DNA endonuclease activity.

5. The nuclease of claim 1, 3, or 4, wherein the amino acid sequence of the nuclease comprises one or more amino acid variation that imparts one or more of the following properties on the nuclease: (a) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (c) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) DNA nickase activity; and / or (e) RNA guided DNA nickase activity.

6. The nuclease of any one of the preceding claims, wherein the nuclease is an endonuclease.

7. The nuclease of claim 2, having one or more of the following properties: (a) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (b) the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule;Attorney Docket No.62801.37WO01 (c) the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule and the inability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule; (d) DNA endonuclease activity; (e) DNA nickase activity; (f) RNA guided DNA endonuclease activity; and / or (g) RNA guided DNA nickase activity.

8. The nuclease of any one of the preceding claims, wherein the nuclease is an endonuclease.

9. The nuclease of any one of the preceding claims, wherein the nuclease is a nickase.

10. The nuclease of any one of the preceding claims, wherein the amino acid sequence of the nuclease is less than about 1000, 950, 900, 850, or 800 amino acid acids in length.

11. The nuclease of any one of the preceding claims, wherein the amino acid sequence of the nuclease is from about 300-1000, 300-950, 300-900, 300-850, 300-800, 300-750, 300-700, 300- 650, 300-600, 300-550, 300-500, 300-450, 300-400, 350-1000, 350-950, 350-900, 350-850, 350- 800, 350-750, 350-700, 350-650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-1000, 400- 950, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400- 450, 450-1000, 450-950, 450-900, 450-850, 450-800, 450-750, 450-700, 450-650, 450-600, 450- 550, 450-500, 500-1000, 500-950, 500-900, 500-850, 500-800, 500-750, 500-700, 500-650, 500- 600, 500-550, 600-1000, 600-950, 600-900, 600-850, 600-800, 600-750, 600-700, 600-650, 700- 1000, 700-950, 700-900, 700-850, 700-800, 700-750, 800-1000, 800-950, 800-900, or 800-850 amino acid acids in length.

12. The nuclease of any one of the preceding claims, wherein the nuclease recognizes a protospacer adjacent motif (PAM) sequence comprising the nucleotide sequence PAM sequence comprises or consists of 5′-TTR-3′, wherein R is A or G.

13. The nuclease of any one of the preceding claims, wherein the amino acid sequence of the nuclease comprises one or more amino acid variation (e.g., substitution, deletion, addition).

14. The nuclease of claim 13, wherein the one or more amino acid variation (e.g., substitution, deletion, addition) reduces the nuclease activity of the nuclease by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,Attorney Docket No.62801.37WO01 95%, 96%, 97%, 98%, 99% or 100% relative to the nuclease lacking the one or more amino acid variation (e.g., substitution, deletion, addition).

15. The nuclease of any one of claims 13-14, wherein the one or more amino acid variation (e.g., substitution, deletion, addition) reduces or eliminates the ability of the nuclease to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule.

16. The nuclease of any one of claims 13-15, wherein a nuclease comprising the one or more amino acid variation (e.g., substitution, deletion, addition) has the ability to mediate a single strand break in a target double stranded nucleic acid (e.g., DNA) molecule) and does not have the ability to mediate a double strand break in a target double stranded nucleic acid (e.g., DNA) molecule.

17. The nuclease of any one of claims 13-16, wherein a nuclease comprising the one or more amino acid variation (e.g., substitution, deletion, addition) is a nickase.

18. The nuclease of any one of claims 13-17, wherein the one or more amino acid variation (e.g., substitution, deletion, addition) alters the PAM nucleotide sequence recognized by the nuclease.

19. The nuclease of any one of the preceding claims, further comprising one or more heterologous moiety (e.g., a heterologous polypeptide).

20. The nuclease of any one of the preceding claims, further comprising 2, 3, 4, or 5 or more heterologous moieties.

21. The nuclease of claim 19 or 20, wherein the heterologous moiety is attached to the N- terminus, C-terminus, and / or internally between the N- and C-terminus of the nuclease.

22. The nuclease of any one of claims 19-21, wherein the heterologous moiety (e.g., heterologous polypeptide) is directly attached to the nuclease.

23. The nuclease of any one of claims 19-22, wherein the heterologous moiety (e.g., heterologous polypeptide) is indirectly attached to the nuclease.

24. The nuclease of any one of claims 19-23, wherein the heterologous moiety (e.g., heterologous polypeptide) is indirectly attached to the nuclease via a linker.

25. The nuclease of any one of claims 19-24, wherein the heterologous moiety is a peptide, polypeptide, carbohydrate, lipid, polymer, or small molecule.

26. The nuclease of any one of claims 19-25, wherein the heterologous moiety is a nuclear localization signal (NLS), a tag, and / or a reporter gene.Attorney Docket No.62801.37WO01 27. A conjugate comprising the nuclease of any one of the preceding claims and one or more heterologous moiety.

28. The conjugate of claim 27, wherein the heterologous moiety is a protein, peptide, small molecule, nucleic acid molecule (e.g., DNA, RNA, DNA / RNA hybrid molecule), carbohydrate, lipid, or synthetic polymer.

29. The conjugate of any one of claims 27-28, wherein the heterologous moiety is operably connected to the N-terminus, C-terminus, and / or internally between the N- and C-terminus of the nuclease.

30. The conjugate of any one of claims 27-29, wherein the heterologous moiety is directly operably connected to the nuclease.

31. The conjugate of any one of claims 27-30, wherein the heterologous moiety is indirectly operably connected to the nuclease.

32. The conjugate of any one of claims 27-31, wherein the heterologous moiety is indirectly operably connected to the nuclease via a linker.

33. A fusion protein comprising the nuclease of any one of claims 1-26 and one or more heterologous polypeptide.

34. The fusion protein of claim 33, wherein the heterologous polypeptide is fused to the N- terminus, C-terminus, and / or internally between the N- and C-terminus of the nuclease.

35. The fusion protein of claim 33 or 34, wherein the heterologous polypeptide is fused directly to the nuclease.

36. The fusion protein of any one of claims 33-35, wherein the heterologous polypeptide is fused indirectly to the nuclease.

37. The fusion protein of any one of claims 33-36, wherein the heterologous polypeptide is fused indirectly to the nuclease via a peptide linker.

38. The fusion protein of any one of claims 33-37, wherein the heterologous polypeptide exhibits polymerase (e.g., reverse transcriptase) activity, nucleobase modifying activity (e.g., deaminase activity), methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, or double-strand DNA cleavage activity, nucleic acid binding activity, or any combination of the foregoing.Attorney Docket No.62801.37WO01 39. The fusion protein of any one of claims 33-38, wherein the amino acid sequence of the fusion protein is less than about 1500, 1300, 1200, 1100, 1000, 950, 900, 850, or 800 amino acid acids in length.

40. The fusion protein of any one of claims 33-39, wherein the amino acid sequence of the fusion protein is from about 300-1300, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000, 300-950, 300-900, 300-850, 300-800, 300-750, 300-700, 300-650, 300-600, 300-550, 300-500, 300-450, 300-400, 350-1350, 350-1400, 350-1350, 350-1200, 350-1100, 350-1000, 350-950, 350-900, 350-850, 350-800, 350-750, 350-700, 350-650, 350-600, 350-550, 350-500, 350-450, 350-400, 400-1400, 400-1400, 400-1400, 400-1200, 400-1100, 400-1000, 400-950, 400-900, 400-850, 400-800, 400-750, 400-700, 400-650, 400-600, 400-550, 400-500, 400-450, 450-1450, 450-1400, 450-1450, 450-1200, 450-1100, 450-1000, 450-950, 450-900, 450-850, 450-800, 450- 750, 450-700, 450-650, 450, 600, 450-550, 450-500, 500-1500, 500-1400, 500-1300, 500-1200, 500-1100, 500-1000, 500-950, 500-900, 500-850, 500-800, 500-700, 500-600, 600-1500, 600- 1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-950, 600-900, 600-850, 600-800, 600-700, 700-1500, 700-1400, 700-1300, 700-1200, 700-1100, 700-1000, 700-950, 700-900, 700-850, 700-800, 800-1500, 800-1400, 800-1300, 800-1200, 800-1100, 800-1000, 800-950, 800-900, or 800-850 amino acid acids in length.

41. The fusion protein of any one of claims 33-40, wherein the heterologous polypeptide is a polymerase.

42. The fusion protein of claim 41, wherein the polymerase has RNA-dependent DNA polymerase activity.

43. The fusion protein of claim 42, wherein the polymerase is a reverse transcriptase (or a functional fragment or functional variant thereof).

44. The fusion protein of claim 43, wherein the reverse transcriptase (or the functional fragment or functional variant thereof) is derived from a retrovirus or a retrotransposon.

45. The fusion protein of claim 33-40, wherein the heterologous polypeptide is a nucleobase editor.

46. The fusion protein of claim 45, wherein the nucleobase editor is a deaminase (or a functional fragment or functional variant thereof).Attorney Docket No.62801.37WO01 47. The fusion protein of claim 46, wherein the deaminase (or the functional fragment or functional variant thereof) exhibits adenosine deaminase activity and / or a or a cytidine deaminase activity.

48. The fusion protein of claim 46 or 47, wherein the deaminase (or the functional fragment or functional variant thereof) comprises an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of a protein set forth in Table 3 or set forth in any one of SEQ ID NOS: 29-88.

49. The fusion protein of any one of claims 45-48, wherein the nucleobase editor is fused to an inhibitor of base excision repair (or a functional fragment or functional variant thereof) (e.g., uracil glycosylase inhibitor (UGI), nuclease dead inosine specific nuclease (dISN)).

50. A nucleic acid molecule encoding the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, or the fusion protein of any one of claims 33-49.

51. The nucleic acid molecule of claim 50, wherein the nucleic acid molecule is a DNA or RNA (e.g., mRNA) molecule.

52. The nucleic acid molecule of any one of claims 50-51, wherein the nucleic acid molecule is codon optimized.

53. The nucleic acid molecule of any one of claims 50-52, further comprising one or more transcription or translation regulatory elements (e.g., promoter, enhancer (e.g., cell or tissue specific transcription regulatory elements).

54. The nucleic acid molecule of any one of claims 50-53, further encoding one or more gRNA (e.g., a crRNA, a tracrRNA, a sgRNA, a template RNA (e.g., as described herein)).

55. The nucleic acid molecule of claim 54, wherein the gRNA is a crRNA.

56. A vector comprising the nucleic acid molecule of any one of claims 50-55.

57. The vector of claim 56, wherein the vector is a viral vector or a non-viral vector (e.g., plasmid, minicircle).

58. The vector of any one of claims 56-57, wherein the vector is a viral vector (e.g., an adeno associated viral (AAV) vector, a lentiviral vector, an adenoviral vector).

59. The vector of any one of claims 58, wherein the viral vector is an AAV vector.Attorney Docket No.62801.37WO01 60. A carrier comprising the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, the fusion protein of any one of claims 33-49, the nucleic acid molecule of any one of claims 50-55, or the vector of any one of claims 56-59.

61. The carrier of claim 60, wherein the carrier is a nanoparticle, polymer, virus (e.g., a recombinant virus), virus like particle, virosome, fusosome, vesicle, or lipid-based carrier.

62. The carrier of any one of claims 60-61, wherein the carrier is a recombinant virus (e.g., an adeno associated virus (AAV), a lentivirus, an adenovirus).

63. The carrier of any one of claims 60-62, wherein the carrier is a lipid-based carrier.

64. The carrier of claim 63, wherein the lipid-based carrier is a lipid nanoparticle (LNP), liposome, lipoplex, nanoliposome, an exosome, or a micelle.

65. The carrier of any one of claims 60-64, further comprising one or more gRNA (e.g., a crRNA, a tracrRNA, a sgRNA, a template RNA (e.g., as described herein)).

66. A cell (or population of cells) comprising the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, the fusion protein of any one of claims 33-49, the nucleic acid molecule of any one of claims 50-55, the vector of any one of claims 56-59, or the carrier of any one of claims 60-65.

67. The cell (or population of cells) of claim 66, wherein the cell (or population of cells) is in vivo, ex vivo, or in vitro.

68. The cell (or population of cells) of claim 66 or 67, wherein the cell (or population of cells) is a eukaryotic cell (e.g., an animal cell, a mammalian cell, a non-human primate cell, a primate cell, a human cell, a plant cell).

69. A pharmaceutical composition comprising the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, the fusion protein of any one of claims 33-49, the nucleic acid molecule of any one of claims 50-55, the vector of any one of claims 56-59, the carrier of any one of claims 60-65, or the cell (or population of cells) of any one of claims 66-68, and a pharmaceutically acceptable excipient.

70. A kit comprising the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, the fusion protein of any one of claims 33-49, the nucleic acid molecule of any one of claims 50-55, the vector of any one of claims 56-59, the carrier of any one of claims 60-65, the cell (or population of cells) of any one of claims 66-68, or the pharmaceutical composition of claim 69; and optionally instructions for using any one or more of the foregoing.Attorney Docket No.62801.37WO01 71. A reaction mixture comprising (a) a cell and (b) the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, the fusion protein of any one of claims 33-49, the nucleic acid molecule of any one of claims 50-55, the vector of any one of claims 56-59, the carrier of any one of claims 60-65, or the pharmaceutical composition of claim 69.

72. A system for modifying a target nucleic acid (e.g., DNA) molecule, comprising: (a) the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, the fusion protein of any one of claims 33-49, the nucleic acid molecule of any one of claims 50-55, the vector of any one of claims 56-59, the carrier of any one of claims 60-65, the cell (or population of cells) of any one of claims 66-68, or the pharmaceutical composition of claim 69, and (b) a first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; a template RNA (e.g., as described herein)) or a nucleic acid (e.g., DNA) molecule encoding the first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; a template RNA (e.g., as described herein)).

73. The system of claim 72, having one or more of the following characteristics: (a) the nuclease of the system is capable of binding to the first gRNA; (b) the nuclease of the system is capable of forming a break in a target nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (c) the nuclease of the system is capable of forming a single strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (d) the nuclease of the system is capable of forming a single strand break in the modified strand (as defined herein) of a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (e) the nuclease of the system is capable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (f) the nuclease of the system is incapable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (g) the endonuclease of the system is capable of forming a single strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule and is incapable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; (h) the nuclease of the system is capable of forming a single strand break in in the modified strand (as defined herein) of a target double stranded nucleic acid (e.g., DNA (e.g.,Attorney Docket No.62801.37WO01 dsDNA)) molecule and is incapable of forming a double strand break in a target double stranded nucleic acid (e.g., DNA (e.g., dsDNA)) molecule; and / or (i) is capable of mediating the insertion, deletion, or substitution of one or more nucleotides into / from a target nucleic acid (e.g., DNA) molecule (e.g., a target double stranded DNA molecule).

74. The system of any one of claims 72-73, wherein the target nucleic acid molecule is a DNA molecule.

75. The system of any one of claims 72-74, wherein the target nucleic acid molecule is a double stranded DNA (dsDNA) molecule.

76. The system of any one of claims 72-75, wherein a portion of the nucleotide sequence of the non-edited strand (as defined herein) of the target dsDNA molecule is complementary to at least a portion of the nucleotide sequence of the first gRNA.

77. The system of any one of claims 72-76, wherein the target nucleic acid molecule is within the genome of cell (e.g., a eukaryotic cell) (e.g., within a subject (e.g., a human subject), plant).

78. The system of any one of claims 72-77, wherein (b) comprises the first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; or a template RNA (e.g., as described herein)).

79. The system of any one of claims 72-78, wherein (b) comprises the nucleic acid (e.g., DNA) molecule encoding the first gRNA (e.g., a crRNA; a crRNA and a tracrRNA; a sgRNA; or a template RNA (e.g., as described herein)).

80. The system of any one of claims 72-79, wherein at least a portion of the nucleotide sequence of the first gRNA is complementary to a portion of the nucleotide sequence of the target nucleic acid molecule.

81. The system of any one of claims 72-80, wherein at least a portion of the nucleotide sequence of the first gRNA is complementary to a portion of the nucleotide sequence of the non- edited strand (as defined herein) of a dsDNA target nucleic acid molecule.

82. The system of any one of claims 72-81, wherein at least a portion of the nucleotide sequence of the first gRNA binds to a portion of the nucleotide sequence of the non-edited strand (as defined herein) of a dsDNA target nucleic acid molecule.

83. The system of any one of claims 72-82, wherein the first gRNA comprises a crRNA (e.g., a single crRNA, a plurality of different crRNAs).Attorney Docket No.62801.37WO01 84. The system of any one of claims 72-83, wherein the first gRNA comprises a sgRNA (e.g., a single sgRNA, a plurality of different sgRNAs).

85. The system of any one of claims 72-84, wherein the first gRNA comprises a crRNA (e.g., a single crRNA, a plurality of different crRNAs) and a tracrRNA (e.g., a single tracrRNA, a plurality of different tracrRNAs), wherein the crRNA and the tracrRNA are on separate RNA nucleic acid molecules (or encoded by separate nucleic acid (e.g., DNA) molecules).

86. The system of any one of claims 72-85, wherein a portion (e.g., scaffold portion) of the first gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in Table 7.

87. The system of any one of claims 72-85, wherein a portion (e.g., scaffold portion) of the first gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in any one of SEQ ID NOS: 243-315.

88. The system of any one of claims 72-87, wherein the first gRNA comprises a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a heterologous object sequence, and a 3' target homology domain.

89. The system of any one of claims 72-88, wherein the template RNA further comprises a sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein of any one of claims 41-44).

90. The system of any one of claims 72-89, wherein the template RNA comprises (e.g., from 5' to 3') a crRNA, a sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein of any one of claims 41-44), a heterologous object sequence, and a 3' target homology domain.

91. The system of any one of claims 72-90, wherein the first gRNA comprises a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a tracrRNA, a heterologous object sequence, and a 3' target homology domain.

92. The system of any one of claims 72-91, wherein the template RNA further comprises a sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein of any one of claims 41-44).Attorney Docket No.62801.37WO01 93. The system of any one of claims 72-92, wherein the template RNA comprises (e.g., from 5' to 3') a crRNA, a tracrRNA, a sequence that binds a polymerase (e.g., a reverse transcriptase, e.g., of a fusion protein of any one of claims 41-44), a heterologous object sequence, and a 3' target homology domain.

94. The system of any one of claims 72-93, wherein the first gRNA comprises one or more nucleotide comprising one or more chemical modification (e.g., a base, ribose, and / or internucleotide linkage chemical modifications) (i.e., a modified nucleotide).

95. Thesystem of claim 94, wherein the modified nucleotide comprises a 2′-O-methyl (2′- OMe); 2′O-methoxyethyl (2′-O-MOE); 2′deoxy-2′-fluoro (2′-F); 2′-arabino-fluoro (2′-Ara-F); 2′- O-benzyl; 2′-O-methyl-4-pyridine (2-O-methyl-4-pyridine (2′-O-CH2Py(4)); 2′F-4′-Cα-OMe; or 2′,4′-di-Cα-OMe, 2ˈ-O-methyl-3ˈ-thioPACE, and / or S-constrained ethyl (cEt), or any combination of the foregoing.

96. The system of any one of claims 92-95, wherein the modified nucleotide comprises a chemically modified internucleotide (or internucleoside) linkage.

97. The system of any one of claims 94-96, wherein the modified internucleotide (or internucleoside) linkage comprises a phosphorothioate (e.g., a chiral phosphorothioate), a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, an alkyl (e.g., methyl) phosphonate (e.g., a 3ˈ-alkylene phosphonate, a chiral phosphonate), a phosphinate, a phosphoroamidate (e.g., a 3ˈ-amino phosphoroamidate, an aminoalkylphosphoramidate), a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, or a boranophosphate.

98. The system of any one of claims 72-97, wherein the first gRNA (e.g., the template RNA, crRNA) comprises a nucleic acid molecule comprising a toe-loop, hairpin, stem-loop, pseudoknot (e.g., a Mpknot1 moiety), aptamer, G-quadraplex, tRNA, riboswitch, or ribozyme.

99. The system of any one of claims 72-98, wherein the first gRNA (e.g., the template RNA, crRNA) wherein the nucleic acid molecule is a pseudoknot (e.g., a Mpknot1 moiety).

100. The system of any one of claims 72-99, further comprising a second gRNA (or a nucleic acid (e.g., DNA) molecule encoding the gRNA) that directs the nuclease of the system to form a single strand break in the non-edited strand of a target dsDNA molecule.Attorney Docket No.62801.37WO01 101. The system of claim 100, wherein at least a portion of the nucleotide sequence of the second gRNA is complementary to a portion of the nucleotide sequence of the modified strand (as defined herein) of a dsDNA target nucleic acid molecule.

102. The system of any one of claims 98-101, wherein at least a portion of the nucleotide sequence of the second gRNA binds to a portion of the nucleotide sequence of the modified strand (as defined herein) of a dsDNA target nucleic acid molecule.

103. The system of any one of claims 98-102, wherein the second gRNA is present on the same nucleic acid molecule as the first gRNA (or the nucleic acid (e.g., DNA) molecule encoding the second gRNA is present on the same nucleic acid (e.g., DNA) molecule encoding the first gRNA).

104. The system of any one of claims 98-103, wherein the second gRNA is present on a different nucleic acid molecule as the first gRNA (or the nucleic acid (e.g., DNA) molecule encoding the second gRNA is present on a different nucleic acid (e.g., DNA) molecule encoding the first gRNA).

105. The system of any one of claims 98-104, wherein the second gRNA is a crRNA.

106. The system of any one of claims 72-105, wherein a portion (e.g., scaffold portion) of the second gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in Table 7.

107. The system of any one of claims 72-106, wherein a portion (e.g., scaffold portion) of the second gRNA (e.g., crRNA) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of a gRNA scaffold set forth in any one of SEQ ID NOS: 243-315.

108. The system of any one of claims 72-107, further comprising a donor DNA template molecule (e.g., as defined herein).

109. A system for modifying a dsDNA molecule, comprising: (a) the fusion protein of any one of claims 41-44 or a nucleic acid molecule (e.g., a DNA, RNA molecule) encoding the fusion protein of any one of claims 41-44; and (b) a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a heterologous object sequence, and a 3' targetAttorney Docket No.62801.37WO01 homology domain; or a nucleic acid molecule (e.g., a DNA molecule) encoding the template RNA.

110. The system of claim 109, wherein the template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a tracrRNA, a heterologous object sequence, and a 3ˈ target homology domain; or a nucleic acid molecule (e.g., a DNA molecule) encoding the template RNA.

111. A system for modifying a dsDNA molecule, comprising: (a) the fusion protein of any one of claims 45-49 or a nucleic acid molecule (e.g., a DNA, RNA molecule) encoding the fusion protein of any one of claims 45-49; and (b) a gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA); or a nucleic acid molecule (e.g., a DNA molecule) encoding the gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA).

112. A nucleic acid molecule encoding the system of any one of claims 72-111.

113. The nucleic acid molecule of claim 112, wherein the nucleic acid molecule is a DNA or RNA (e.g., mRNA) molecule.

114. The nucleic acid molecule of claim 112 or 113, wherein the nucleic acid molecule is codon optimized.

115. The nucleic acid molecule of any one of claims 112-114, further comprising one or more transcription or translation regulatory elements (e.g., promoter, enhancer (e.g., cell or tissue specific transcription regulatory elements).

116. A vector comprising the nucleic acid molecule of any one of claims 112-115.

117. The vector of claim 116, wherein the vector is a viral vector or a non-viral vector (e.g., plasmid, minicircle).

118. The vector of claim 116 or 117, wherein the vector is a viral vector (e.g., an adeno associated viral (AAV) vector, a lentiviral vector, an adenoviral vector).

119. A carrier comprising the system of any one of claims 72-111, the nucleic acid molecule of any one of claims 112-115, and / or the vector of any one of claims 116-118.

120. The carrier of claim 119, wherein the carrier is a nanoparticle, polymer, virus (e.g., a recombinant virus), virus like particle, virosome, fusosome, vesicle, or lipid-based carrier.

121. The carrier of any one of claims 119 or 120, wherein the carrier is a recombinant virus (e.g., an adeno associated virus (AAV), a lentivirus, an adenovirus).Attorney Docket No.62801.37WO01 122. The carrier of claim 119 or 120, wherein the carrier is a nanoparticle.

123. The carrier of any one of claims 119, 120, or 122, wherein the carrier is a lipid-based carrier.

124. The carrier of claim 123, wherein the lipid-based carrier is a lipid nanoparticle (LNP), liposome, lipoplex, nanoliposome, an exosome, or a micelle.

125. The carrier of any one of claims 119-124, further comprising one or more gRNA (e.g., a crRNA, a tracrRNA, a sgRNA, a template RNA (e.g., as described herein)).

126. A reaction mixture comprising (a) a cell (e.g., comprising a target nucleic acid molecule) or a target nucleic acid molecule; and (b) the system of any one of claims 72-111, the nucleic acid molecule of any one of claims 112-115, the vector of any one of claims 116-118, and / or the carrier of any one of claims 119-125.

127. A cell comprising the system of any one of claims 72-111, the nucleic acid molecule of any one of claims 112-115, the vector of any one of claims 116-118, the carrier of any one of claims 119-125, and / or the reaction mixture of claim 126.

128. The cell (or population of cells) of claim 127, wherein the cell (or population of cells) is in vivo, ex vivo, or in vitro.

129. The cell (or population of cells) of claim 127 or 128, wherein the cell (or population of cells) is a eukaryotic cell (e.g., an animal cell, a mammalian cell, a non-human primate cell, a primate cell, a human cell, a plant cell).

130. A pharmaceutical composition comprising the system of any one of claims 72-111, the nucleic acid molecule of any one of claims 112-115, the vector of any one of claims 116-118, the carrier of any one of claims 119-125, the reaction mixture of claim 126, and / or the cell of any one of claims 127-129, and a pharmaceutically acceptable excipient.

131. A kit comprising the system of any one of claims 72-111, the nucleic acid molecule of any one of claims 112-115, the vector of any one of claims 116-118, the carrier of any one of claims 119-125, the reaction mixture of claim 126, the cell of any one of claims 127-129, and / or the pharmaceutical composition of claim 130; and optionally instructions for using any one or more of the foregoing.

132. A method of delivering a nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, composition, pharmaceutical composition, or system to a cell, the method comprising, introducing into a cell the nuclease of any one of claims 1-26, the conjugate of anyAttorney Docket No.62801.37WO01 one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72-111, to thereby deliver the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical composition, or system to the cell.

133. The method of any one of claims 132, wherein the cell is in vitro, ex vivo, or in vivo.

134. The method of any one of claims 132-133, wherein the cell is euploid, is not immortalized, is part of a tissue, is part of an organism, is a primary cell, is non-dividing, is haploid (e.g., a germline cell), is a non-cancerous polyploid cell, or is from a subject having a genetic disease.

135. The method of any one of claims 132-134, wherein the cell is a human cell.

136. The method of any one of claims 132-135, wherein the cell is in a plant cell.

137. The method of any one of claims 132-136, wherein the cell is in a subject (e.g., a human subject).

138. A method of delivering a nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, , pharmaceutical composition, or system to a subject (e.g., human subject), the method comprising, administering to the subject the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72- 111, to thereby deliver the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical composition, or system to the subject (e.g., human subject).

139. A method of cleaving a target site in a target nucleic acid (e.g., DNA) molecule (e.g., a double stranded target nucleic acid sequence (e.g., dsDNA, (e.g., genomic dsDNA))), the method comprising contacting the cell with the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceuticalAttorney Docket No.62801.37WO01 composition of any one of claims 69 or 130, or system of any one of claims 72-111, to thereby cleave the target site in the target nucleic acid (e.g., DNA) molecule.

140. A method of modifying a target site in a target nucleic acid (e.g., DNA) molecule (e.g., a double stranded target nucleic acid sequence (e.g., dsDNA, (e.g., genomic dsDNA))), the method comprising contacting the cell with the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72-111, to thereby modify the target site in the target nucleic acid (e.g., DNA) molecule.

141. A method of modifying a target site in genomic dsDNA in a cell, the method comprising, contacting the nuclease of any one of claims 1-26, the conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72-111, to thereby modify the target site in the genomic DNA of the cell.

142. A method of modifying a target site in a dsDNA molecule (e.g., genomic dsDNA (e.g., in a cell)), the method comprising: contacting a dsDNA molecule with (a) the fusion protein of any one of claims 41-44 (or a nucleic acid molecule (e.g., a DNA, RNA, nucleic acid molecule) encoding the fusion protein of any one of claims 41-44), and (b) a template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5' to 3') a crRNA, a heterologous object sequence, and a 3ˈ target homology domain, to thereby modify the target site in the dsDNA molecule (or a nucleic acid molecule (e.g., a DNA nucleic acid molecule) encoding the template RNA).

143. The method of claim 142, wherein the template RNA (e.g., a single template RNA, a plurality of different template RNAs) that comprises (e.g., from 5ˈ to 3ˈ) a crRNA, a tracrRNA, a heterologous object sequence, and a 3ˈ target homology domain, to thereby modify the target site in the dsDNA molecule (or a nucleic acid molecule (e.g., a DNA nucleic acid molecule) encoding the template RNA).Attorney Docket No.62801.37WO01 144. A method of modifying a target site in a dsDNA molecule (e.g., genomic dsDNA (e.g., in a cell)), the method comprising: contacting a dsDNA molecule with (a) the fusion protein of any one of claims 45-49 (or a nucleic acid molecule (e.g., a DNA, RNA, nucleic acid molecule) encoding the fusion protein of any one of claims 45-49), and (b) a gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA); or a nucleic acid molecule (e.g., a DNA molecule) encoding the gRNA (e.g., a crRNA, a tracrRNA and a crRNA, a sgRNA), to thereby modify the target site in the dsDNA molecule (or a nucleic acid molecule (e.g., a DNA nucleic acid molecule) encoding the gRNA).

145. The method of any one of claims 132-144, wherein the nucleic acid molecule is in a cell (e.g., a eukaryotic cell).

146. The method of any one of claims 132-145, wherein the cell is a eukaryotic cell.

147. The method of any one of claims 132-146, wherein the cell is a human cell.

148. The method of any one of claims 132-147, wherein the cell is a plant cell.

149. The method of any one of claims 132-148, wherein the cell is in vitro, ex vivo, or in vivo.

150. The method of any one of claims 132-149, wherein the cell is in a subject (e.g., a human subject).

151. The method of any one of claims 132-145, wherein the cell is in a plant.

152. The method of any one of claims 132-151, wherein the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the genomic dsDNA in the cell.

153. The method of any one of claims 132-152, wherein the modification comprises an insertion, a deletion, or a substitution of one or more nucleotides into / from the target site of the target nucleic acid molecule.

154. The method of any one of claims 132-153, wherein the insertion comprises the insertion of from about 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site.

155. The method of any one of claims 128-150, wherein the deletion comprises the deletion of from about 1-500, 1-400, 1-300, 1-200, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1- 10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 nucleotides at the target site.Attorney Docket No.62801.37WO01 156. A method of treating or preventing a disease in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject the nuclease of any one of claims 1- 26, the conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116- 118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72- 111, thereby treating or preventing the disease in the subject.

157. The method of claim 156, wherein the disease is associated with a genetic defect.

158. The method of claim 156 or 157, wherein the gRNA of the system is capable of targeting the endonuclease to the site of the genetic defect.

159. The method of any one of claims 156-158, wherein the genetic defect comprises a duplication of a gene, deletion of a gene, or a mutation of a gene.

160. The method of any one of claims 156-159, wherein the administration results in the correction of the genetic defect.

161. The method of any one of claims 156-160, wherein the subject is a human subject.

162. A nuclease of any one of claims 1-26, conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72-111 for use in a method of treating or preventing a disease in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject the nuclease, conjugate, fusion protein, nucleic acid molecule, vector, carrier, cell, pharmaceutical, or system, thereby treating or preventing the disease in the subject.

163. Use of a nuclease of any one of claims 1-26, conjugate of any one of claims 27-32, fusion protein of any one of claims 33-49, nucleic acid molecule of any one of claims 50-55 or 112-115, vector of any one of claims 56-59 or 116-118, carrier of any one of claims 60-65 or 119-125, cell of any one of claims 66-68 or 127-129, pharmaceutical composition of any one of claims 69 or 130, or system of any one of claims 72-111 in the manufacture of a medicament for use in a method of treating or preventing a disease in a subject (e.g., a human subject) in need thereof, the method comprising administering to the subject the nuclease, conjugate, fusion protein, nucleicAttorney Docket No.62801.37WO01 acid molecule, vector, carrier, cell, pharmaceutical, or system, thereby treating or preventing the disease in the subject.

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