Targeting oncogenic mutations with cleaving endonucleases

Abstract

Provided herein are compositions and methods using chimeric nucleases comprising an I-TevI ​​nuclease domain and a Cas domain for targeting oncogenes.

Description

[Technical Field]

[0001] cross reference [1] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 166,763, filed March 26, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] [2] Cancer is a leading cause of death worldwide. In 2018, there were 18.1 million new cancer cases and 9.5 million cancer-related deaths worldwide. By 2040, the number of new cancer cases and cancer-related deaths per year is expected to increase to 29.5 million and 16.4 million, respectively. Proto-oncogenes are genes that can cause cancer. Once mutated, proto-oncogenes become oncogenes. In tumor cells, proto-oncogenes are often mutated or expressed at elevated levels, which can contribute to the uncontrolled cell proliferation that characterizes cancer. While many current therapies target mutated proteins expressed from oncogenes, no treatments target the oncogenes themselves. Therefore, it is important to develop new technologies to disrupt oncogenes. Summary of the Invention

[0003] [3] Described herein are chimeric nucleases comprising an I-TevI ​​domain (1), a Cas domain, and a guide RNA that targets the chimeric nuclease to an oncogenic gene mutation. Such chimeric nucleases advantageously enable precise targeting and editing of a cell's genome, restoring the non-oncogenic function of the oncogenic gene. Compared to the use of the Cas enzyme alone, the inclusion of the I-TevI ​​domain allows for more precise editing and replacement of oncogenic gene sequences in cancer cells.

[0004] [4] In one aspect, the present disclosure provides a composition comprising a chimeric nuclease, the chimeric nuclease comprising an I-TEVI nuclease domain, an RNA-guided nuclease Cas domain, and a guide RNA, wherein the guide RNA comprises a nucleic acid sequence that targets an oncogenic mutation in exon 19 of EGFR that is not a deletion.

[0005] [5] In some embodiments, the oncogenic mutation is a single nucleotide polymorphism. In some embodiments, the sequence comprising the oncogenic mutation is selected from the mutations set forth in any one of SEQ ID NOs: 1-683, or a combination thereof. In some embodiments, the sequence comprising the oncogenic mutation is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to a mutation set forth in any one of SEQ ID NOs: 1-683, or a combination thereof. In some embodiments, the oncogenic mutation comprises an EGFR L858R mutation or a mutation corresponding to an EGFR V769_D770insASV mutation. In some embodiments, the oncogenic mutation comprises a mutation corresponding to an EGFR L858R mutation. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 45, 130, or 141, or comprises a nucleotide sequence set forth in SEQ ID NO: 1045, 1130, 1141, or 1686. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 45, 130, 141, or comprises a nucleotide sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1045, 1130, 1141, or 1686. In some embodiments, the oncogenic mutation comprises a mutation corresponding to the EGFR V769_D770insASV mutation. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 683, or comprises the nucleotide sequence set forth in SEQ ID NO: 1683 or 1684. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 683, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1683 or 1684. In some embodiments, the oncogenic mutation is an oncogenic mutation in a gene selected from Muc4, PIK3CA, KRAS, or a combination thereof. In some embodiments, the oncogenic mutation comprises a mutation in Muc4.In some embodiments, the Muc4 mutation is an in-frame deletion of exon 2 or an in-frame deletion of exon 3. In some embodiments, the Muc4 mutation comprises a mutation corresponding to any one of positions P1542, P1680, T1711, V1721, P1826, A1830, S3560, A1833, D2253, V2281, P3088, T3119, T3183, V3817, A3902 of the human Muc4 protein, or a combination thereof. In some embodiments, the Muc4 mutation is selected from mutations corresponding to any one of P1542L, P1680S, T1711I, V1721A, P1826H, A1830T, S3560S, A1833V, D2253H, V2281AM, P3088L, T3119T, T3183M, V3817A, A3902V of human Muc4 protein, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 676, 677, 678, 679, or 682, or comprises a nucleotide sequence set forth in SEQ ID NO: 1676, 1677, 1678, 1679, 1682, or 1685. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 676, 677, 678, 679, or 682, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1676, 1677, 1678, 1679, 1682, or 1685. In some embodiments, the oncogenic mutation comprises a mutation in PIK3CA. In some embodiments, the PIK3CA mutation includes a mutation corresponding to any one of positions H1047, E542, E545, N345, C1636, G1624, G1633, A3140, C3075, A1634, and A1173 of the human PIK3A protein, or a combination thereof.In some embodiments, the PIK3CA mutation is selected from mutations corresponding to any one of H1047R, H1047L, E542K, E545K, N345K, C1636A, G1624A, G1633A, A3140T, A3140G, C3075T, A1634C, A1173G of human PIK3A protein, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 33, 202, 204, 209, or 210, or comprises a nucleotide sequence set forth in SEQ ID NO: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, or 1210. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5, 6, 7, 8, 33, 202, 204, 209, or 210, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, or 1210. In some embodiments, the oncogenic mutation comprises a mutation in KRAS. In some embodiments, the KRAS mutation comprises a mutation selected from mutations corresponding to any one of positions A59, D119, D33, G21, G12, G13, Q61, A146, K117 of the human KRAS protein, or a combination thereof. In some embodiments, the KRAS mutation is selected from mutations corresponding to any one of A59T, A59E, A59T, D119N, D33E, G21C, G12C, G12D, G12V, G12R, G12A, G12S, G13D, G13C, G13V, G13R, Q61R, Q61V, Q61L, Q61K, Q61H, Q61A, Q61P, Q61E, A146T, A146V, K117N, K117R of the human KRAS protein, or a combination thereof.In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 37, 42, 51, 52, 62, 63, or 77, or comprises a nucleotide sequence set forth in SEQ ID NO: 1037, 1042, 1051, 1052, 1062, 1063, or 1077. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 37, 42, 51, 52, 62, 63, or 77, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1037, 1042, 1051, 1052, 1062, 1063, or 1077. In some embodiments, the guide RNA comprises one or more of an unnatural internucleoside linkage, a nucleic acid mimic, a modified sugar moiety, or a modified nucleobase. In some embodiments, the unnatural internucleoside linkage comprises one or more of a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'-alkylenephosphonate, a 5'-alkylenephosphonate, a chiral phosphonate, a phosphinate, a 3'-aminophosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, or a boranophosphate. In some embodiments, the nucleic acid mimic comprises one or more of a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), or a locked nucleic acid (LNA). In some embodiments, the modified sugar moiety comprises one or more of 2'-O-(2-methoxyethyl), 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O-methyl, or 2'-fluoro.In some embodiments, the modified nucleobase is 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivative of adenine, 6-methyl derivative of guanine, 2-propyl derivative of adenine, 2-propyl derivative of guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 5-halo, 5-bromo, 5-trifluoromethyl, 5-substituted uracil The present invention also includes one or more of the following: 1-aminopyridine, 2-aminopyridine, 5-substituted cytosine, 7-methylguanine, 7-methyladenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidine, phenoxazine cytidine, phenothiazine cytidine, substituted phenoxazine cytidine, carbazole cytidine, pyridoindole cytidine, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, 5-substituted pyrimidine, 6-azapyrimidine, N-2, N-6, or O-6 substituted purine, 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine. In some embodiments, the composition further comprises a linker operably linked to the I-TEVI nuclease domain and the RNA-guided nuclease Cas domain. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 701, 702, 703, or 704. In some embodiments, the linker comprises a mutation corresponding to any one of positions T95, S101, A119, K120, K135, P126, D127, N140, T147, Q158, A161, VI17, S165, or a combination thereof. In some embodiments, the linker comprises a mutation selected from the mutations corresponding to any one of T95S, S101Y, A119D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, V117F, S165G, or a combination thereof.In some embodiments, the linker comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 701, 702, 703, or 704. In some embodiments, the linker comprises a mutation corresponding to any one of positions T95, S101, A119, K120, K135, P126, D127, N140, T147, Q158, A161, V117, S165, or a combination thereof. In some embodiments, the linker comprises a mutation selected from the mutations corresponding to any one of T95S, S101Y, A119D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, V117F, S165G, or a combination thereof.

[0006] In some embodiments, the RNA-guided nuclease Cas domain is an RNA-guided nuclease Cas9 domain, ie, any one or combination of an RNA-guided nuclease Staphylococcus aureus Cas9 domain, an RNA-guided nuclease Streptococcus pyogenes Cas9 domain, an RNA-guided nuclease Neisseria meningitidis Cas9 domain, an RNA-guided nuclease Campylobacter jejuni Cas9 domain, an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain, an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain, an RNA-guided nuclease Clostridium cellulolyticum Cas9 domain, or an RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain. In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Staphylococcus aureus Cas9 domain. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 710.

[0007] Part of the form of Staphylococcus aureus, RNA-inducible Staphylococcus aureus Cas9 is denoted by, D10, H557, N580, H840, D1135, R1335, T1337, T267, L325, V327, D333, A336, I341, E345, D348, K352, S360, T368, N369, N371, S372, E373, K386, N393, H408, N410, I414, A415, T438, Y467, N471, D485, M489, E506, R409, T510, N515, Y 518, A539, ​​F550, N551, S596, T602, A611, I617, T620, G654, N667, R685, K695, I706, K722, A723, K724, M731, F732, K735, S739, P741, E742, E746, Q747, I754, T755, H757, K760, H761, P778, E781, I783, N784, D785, T786L, L787, Y788, K792, D 794, T798, L799, V801, N803, L804, N805, G806, D813, K814, L818, I819, S822, E824, L841, G847, D848, Y857, V875, I876, N884, A888, L890, D894, D895, P897, V903, G920, F924, N929, E936, N937, V941, N942, S943, C945, E947, K951, L952, S9 56, N957, Q958, A959, N974, G975, V983, N984, N985, D986, I991, V993, M995, I996, T999, Y1000, R1001, E1002, L1004, E1005, N1006, M1007, D1009, K1010, R1011, P1012, P1013, I1015, I1016, A1020, S1021, Q1024, K1027, E1039, H1045, I0148,In some embodiments, the RNA-guided nuclease Staphylococcus aureus comprises a mutation corresponding to any one of positions K1050 and K1060, or a combination thereof. The Cas9 domains are D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, T267A, L325F, V327I, D333G, A336S, I341L, E345D, D348N, K352E, S360A, T368A, N369E, N371E, S372P, E373K, K386T, N393R, H408N, N410S, I414M, A415T, T438S, Y467F, N471K, D485E, M489F, E506K, R409K, and T51 0E, N515K, Y518F, A539P, F550Y, N551H, S596A, T602I, A611S, I617V, T620K, G654E, N667D, R685K, K695Q, I706V, K722T, A723T, K724N, M731 T, F732V, K735Q, S739N, P741L, E742G, E746D, Q747D, I754D, T755I, H757R, K760Q, H761S, P778I, E781K, I783V, N784D, D785E, T786L, L787V Y788H, K792E, D794T, T798R, L799I, V801I, N803S, L804I, N805K, G806N, D813G, K814E, L818I, I819F, S822P, E824G, L841T, G847S, D848N, Y 857H, V875I, I876V, N884K, A888V, L890R, D894G, D895H, P897L, V903I , G920D, F924L, N929Y, E936D, N937G, V941I, N942D, S943L, C945A, E94 7K, K951R, L952Q, S956N, N957E, Q958K, A959S, N974D, G975K, V983A, N984S, N985D, D986G, I991V, V993L, M995F, I996V, T999N, Y1000K, R10 01E, E1002D, L1004I, E1005K, N1006M, M1007N, D1009L, K1010S, R1011 T, P1012S, P1013F, I1015L, I1016R, A1020G, S1021K, Q1024K, K1027S,In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 710.

[0008] Part of the form of Staphylococcus aureus, RNA-inducible Staphylococcus aureus Cas9 is denoted by, D10, H557, N580, H840, D1135, R1335, T1337, T267, L325, V327, D333, A336, I341, E345, D348, K352, S360, T368, N369, N371, S372, E373, K386, N393, H408, N410, I414, A415, T438, Y467, N471, D485, M489, E506, R409, T510, N515, Y 518, A539, ​​F550, N551, S596, T602, A611, I617, T620, G654, N667, R685, K695, I706, K722, A723, K724, M731, F732, K735, S739, P741, E742, E746, Q747, I754, T755, H757, K760, H761, P778, E781, I783, N784, D785, T786L, L787, Y788, K792, D 794, T798, L799, V801, N803, L804, N805, G806, D813, K814, L818, I819, S822, E824, L841, G847, D848, Y857, V875, I876, N884, A888, L890, D894, D895, P897, V903, G920, F924, N929, E936, N937, V941, N942, S943, C945, E947, K951, L952, S9 56, N957, Q958, A959, N974, G975, V983, N984, N985, D986, I991, V993, M995, I996, T999, Y1000, R1001, E1002, L1004, E1005, N1006, M1007, D1009, K1010, R1011, P1012, P1013, I1015, I1016, A1020, S1021, Q1024, K1027, E1039, H1045, I0148,In some embodiments, the RNA-guided nuclease Staphylococcus aureus comprises a mutation corresponding to any one of positions K1050 and K1060, or a combination thereof. The Cas9 domains are D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, T267A, L325F, V327I, D333G, A336S, I341L, E345D, D348N, K352E, S360A, T368A, N369E, N371E, S372P, E373K, K386T, N393R, H408N, N410S, I414M, A415T, T438S, Y467F, N471K, D485E, M489F, E506K, R409K, and T51 0E, N515K, Y518F, A539P, F550Y, N551H, S596A, T602I, A611S, I617V, T620K, G654E, N667D, R685K, K695Q, I706V, K722T, A723T, K724N, M731 T, F732V, K735Q, S739N, P741L, E742G, E746D, Q747D, I754D, T755I, H757R, K760Q, H761S, P778I, E781K, I783V, N784D, D785E, T786L, L787V Y788H, K792E, D794T, T798R, L799I, V801I, N803S, L804I, N805K, G806N, D813G, K814E, L818I, I819F, S822P, E824G, L841T, G847S, D848N, Y 857H, V875I, I876V, N884K, A888V, L890R, D894G, D895H, P897L, V903I , G920D, F924L, N929Y, E936D, N937G, V941I, N942D, S943L, C945A, E94 7K, K951R, L952Q, S956N, N957E, Q958K, A959S, N974D, G975K, V983A, N984S, N985D, D986G, I991V, V993L, M995F, I996V, T999N, Y1000K, R10 01E, E1002D, L1004I, E1005K, N1006M, M1007N, D1009L, K1010S, R1011 T, P1012S, P1013F, I1015L, I1016R, A1020G, S1021K, Q1024K, K1027S,In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation selected from the group consisting of E1039K, H1045K, I0148M, and K1050M, or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation corresponding to a D10E mutation.

[0009] One type of Streptococcus pyogenes, RNA-inducible Cas9 Cas9, and RNA-inducible Cas9 Streptococcus pyogenes. Part 1 of the application form, RNA-inducible Streptococcus pyogenes Cas9 ドメインは, registration number 711, record of the されるアミノ acid preparation system. One piece of Streptococcus pyogenes, RNA-inducible Streptococcus pyogenes Cas9ドメインは, D10 bit, S29 bit, F32 bit, D39 bit, R40 bit, H41 bit, S42 bit, I48 bit, C80 bit, S87 bit, K112 bit, H113 bit, K132 bit, K141 bit, D147 bit, L158 bit, E17 1 bit, P176 bit, I186 bit, V189 bit, Q190 bit, Q194 bit, N199 bit, I201 bit, N202 bit, A203 bit, S204 bit, R205 bit, A210 bit, Q228 bit, L229 bit, G231 bit, S245 bit, T249, S254, D261, T270, N295, T300, D304, V308, N309, I312, T333, A337, E345, F352, Q354, S355, K356, G366, A367, E396, L398, I414, D428, F429, D435, K468, S469, E470, T472, E480, A486, S490, F498, K500, N501, N504, K528, V530, E532, G533, A538, T555, K570, F575, D605, E611, R629, E634, T638, R655, R664, R671, K705, E706, Q709, K710, S714, G7115, G717, H721, H723, A725, N726, V743, L747, V748 , K772, K775, N776, I788, G792, K797, Y799, T804, N808, L811, R820, N831, R832, ​​V842, L847, N869, E874, N881, Q885, N888, T893, L911, Y945, D946, L949, E952, A1023, Y1036, G1067, G1077, R1078, N1093,R1114th, N1115th, D1117th, A1121st, D1125th, P1128th, K1129th, V1146th, S1154th, S1159th, L1164th, S1172nd, N1177th, P1178th, I1 179th, D1180th, K1211th, M1213th, G1218th, N1234th, E1243rd, K1244th, E1253rd, E1260th, K1263rd, H1264th, E1271st, Q1272, E1275th , V1290, L1291, S1292, A1293, N1295, H1297, R1298, D1299, K1300, R1303, E1307, N1308, I1309, I1310, H1311, L1312, L1315, T1316, N1317, Y1326, D1328, V1342, A1345, I1360, S1363, or a combination thereof. The Cas9 domains are D10E, D10A, S29T, F32M, D39N, R40K, H41Q, S42T, I48L, C80R, S87A, K112D, H113N, K132N, K141E, D147E, L158V, E171Q, P176S, I186K, V189L, Q190H, Q194E, N 199R, I201L, N202E, A203E, S204I, R205K, A210G, Q228A, L229F, G231N, S245A, T249 M, S254A, D261N, T270S, N295K, T300I, D304G, V308A, N309D, I312V, T333A, A337V, E 345K, F352S, Q354K, S355T, K356T, G366K, A367T, E396D, L398F, I414V, D428A, F429 Y, D435E, K468Q, S469R, E470N, T472A, E480D, A486T, S490L, F498V, K500E, N501H, N 504T, K528R, V530I, E532D, G533E, A538E, T555A, K570Q, F575C, D605E, E611D, R629 K, E634K, T638K, R655H, R664K, R671K, K705V, E706D, Q709K, K710A, S714F, G7115E,G717K, H721K, H723Q, A725S, N726A, V743I, L747I, V748I, K772Q, K775R, N776R, I788M, G792R, K797 E, Y799H, T804A, N808D, L811R, R820K, N831D, R832H, V842I, L847I, N869D, E874A, N881S, Q885R, N8 88K, T893S, L911A, Y945H, D946G, L949P, E952A, A1023G, Y1036R, G1067E, G1077E, R1078K, N1093T, R1114G, N1115E, D1117A, A1121P, D1125G, P1128T, K1129T, V1146I, S1154T, S1159P, L1164V, S1172N , N1177D, P1178S, I1179V, D1180S, K1211R, M1213L, G1218T, N1234H, E1243D, K1244T, E1253K, E126 0D, K1263Q, H1264Y, E1271D, Q1272W, E1275H, V1290L, L1291R, S1292A, A1293T, N1295E, H1297N, R12 98T, D1299H, K1300L, R1303S, E1307D, N1308S, I1309M, I1310L, H1311N, L1312A, L1315F, T1316S, N1317R, Y1326F, D1328N, V1342I, A1345S, I1360L, S1363N, or a combination thereof. In some embodiments, the RNA-guided nuclease Streptococcus pyogenes Cas9 domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 711.

[0010] 1st Staphylococcus pyogenes, RNA-inducible Staphylococcus pyogenes Cas9ドメインは, D10 bit, S29 bit, F32 bit, D39 bit, R40 bit, H41 bit, S42 bit, I48 bit, C80 bit, S87 bit , K112 bit, H113 bit, K132 bit, K141 bit, D147 bit, L158 bit, E171 bit, P176 bit, I186 bit, V189 bit , Q190 bit, Q194 bit, N199 bit, I201 bit, N202 bit, A203 bit, S204 bit, R205 bit, A210 bit, Q228 bit, L229 bit, G231 bit, S245 bit, T249 bit, S254 bit, D261 bit, T270 bit, N295 bit, T300 bit, D304 bit, V308, N309, I312, T333, A337, E345, F352, Q354, S355, K356, G366, A367, E396, L398, I414, D428, F429, D435, K468, S469, E470, T472, E480, A486, S490, F498, K500, N501, N504, K528, V530, E532, G533, A538, T555, K570, F575, D605, E611, R62 9, E634, T638, R655, R664, R671, K705, E706, Q709, K710, S714, G7115, G717, H721, H723, A725, N726, V743, L747, V748, K772, K775, N776, I788, G792, K797, Y799, T804, N808, L811, R820, N831, R832, ​​V842, L847, N869, E874, N881, Q885, N888, T893, L911, Y945, D946, L949, E952, A1023, Y1036, G1067, G1077, R1078, N1093, R1114, N1115, D1117, A1121, D1125, P1128, K1129, V1146, S1154, S1159, L1164, S1172, N1177, P1178, I1179, D1180, K1211, M1213, G1218, N1234, E1243, K1244, E1253,In some embodiments, the RNA-guided nuclease Streptococcus pyogenes comprises a mutation corresponding to any one of positions E1260, K1263, H1264, E1271, Q1272, E1275, V1290, L1291, S1292, A1293, N1295, H1297, R1298, D1299, K1300, R1303, E1307, N1308, I1309, I1310, H1311, L1312, L1315, T1316, N1317, Y1326, D1328, V1342, A1345, I1360, S1363, or a combination thereof. The Cas9 domains are D10E, D10A, S29T, F32M, D39N, R40K, H41Q, S42T, I48L, C80R, S87A, K112D, H113N, K132N, K141E, D147E, L158V, E171Q, P176S, I186K, V189L, Q190H, Q194E, N199R, I201L, N202E, A203E, S204I, R205K, and A210. G, Q228A, L229F, G231N, S245A, T249M, S254A, D261N, T270S, N295K, T300I, D304G, V308A, N309D, I312V, T3 33A, A337V, E345K, F352S, Q354K, S355T, K356T, G366K, A367T, E396D, L398F, I414V, D428A, F429Y, D435E, K 468Q, S469R, E470N, T472A, E480D, A486T, S490L, F498V, K500E, N501H, N504T, K528R, V530I, E532D, G533E , A538E, T555A, K570Q, F575C, D605E, E611D, R629K, E634K, T638K, R655H, R664K, R671K, K705V, E706D, Q709 K, K710A, S714F, G7115E, G717K, H721K, H723Q, A725S, N726A, V743I, L747I, V748I, K772Q, K775R, N776R, I 788M, G792R, K797E, Y799H, T804A, N808D, L811R, R820K, N831D, R832H, V842I, L847I, N869D, E874A, N881S,Q885R, N888K, T893S, L911A, Y945H, D946G, L949P, E952A, A1023G, Y1036R, G1067E, G1077E, R1078K, N1093T, R1114G, N1115E, D1117A, A1121P, D1125G, P1128T, K1129T, V1146I, S1154T, S1159P, L1164V, S1172N, N1177D, P1178S, I1179V, D1180S, K1211R, M1213L, G1218T, N1234H, E1243D, K1244T, E1253K, The mutations include those selected from the group consisting of E1260D, K1263Q, H1264Y, E1271D, Q1272W, E1275H, V1290L, L1291R, S1292A, A1293T, N1295E, H1297N, R1298T, D1299H, K1300L, R1303S, E1307D, N1308S, I1309M, I1310L, H1311N, L1312A, L1315F, T1316S, N1317R, Y1326F, D1328N, V1342I, A1345S, I1360L, and S1363N, or a combination thereof.

[0011] In some embodiments, the RNA-guided nuclease Cas9 domain is a RNA-guided nuclease Neisseria meningitidis Cas9 domain. In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 712.Neisseria meningitidis Cas9ドメインは, I9 bit, D16 bit, D30 bit, E31 bit, A94 bit, I103 bit, P124 bit, N164 bit, I213 bit , G229 bit, T241 bit, S376 bit, E393 bit, G454 bit, K471 bit, G490 bit, D660 bit, C665 bit, K7 64-bit, T770-bit, P803-bit, A841-bit, H842-bit, K843-bit, D844-bit, L846-bit, R847-bit, K854-bit, H855-bit, N856-bit, K858-bit, K862-bit, W865-bit, E868-bit, I869-bit, A872-bit, D873-bit, N87 6, Y880, G883, I886, E887, E890, R895, A898, Y899, G900, G901, N902, A903, K904, Q905, D908, N912, K917, G919, L921, V927, K929, T930, E932, S933, L936, L937, N938, K939, K940, Y943, T944, G949, D950, C958, K965, N966, Q967, F969 , A975, E980, N981, I986, D987, C988, K989, G990, Y991, R992, I993, D994, Y997, T998, C1000, S1002, H1004, K1005, Y1006, A1010, F1011, Q1012, K1013, D1014, E1015, K1018, V1019, E1020, F1021, A1022, Y1024, I1025, N1026, C1027, D1028 Position, S1029, S1030, N1031, R1033, F1034, Y1035, L1036, A1037, W1038, K1041, G1042, K1044, E1045, Q1046, Q1047, F1048, R1 049 bit, I1050 bit, S1051 bit, T1052 bit, Q1053 bit, N1054 bit, L1055 bit, V1056 bit, L10 57 bits, I1058 bits, Y1061 bits, V1063 bits, and N1064 bits are all the same.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain is selected from the group consisting of I9M, D16E, D30E, E31K, A94D, I103V, P124C, N164D, I213N, G229D, T241A, S376T, E393K, G454C, K471E, G490C, D660E, C665R, K764E, T770A, P803S, A841Q, H842G, K843H, D844E, L846V, R847K, K854R, H855L, N856D, K858G, K862L, W865P, E868Q, I869L, A872K, D873G, N876K, Y880R, G883E, I886P, E887K, E890E, R895Q, A898T, Y899H, G900K, G901D, N902D, A903P, K904T, Q905K, D908A, N912E, K917Y, G919T, L921Q, V9 27I, K929Q, T930V, E932K, S933T, L936W, L937V, N938R, K939N, K940H, Y943N, T944G, G949A, D950T, C958E, K965G, N966G, Q967K, F969Y, A975 S, E980K, N981G, I986R, D987A, C988V, K989V, G990A, Y991F, R992K, I993D, D994E, Y997F, T998E, C1000R, S1002I, H1004Y, K1005A, Y1006N, A 1010K, F1011L, Q1012T, K1013A, D1014K, E1015K, K1018N, V1019E, E10 20F, F1021L, A1022G, Y1024F, I1025V, N1026S, C1027L, D1028N, S1029 The mutations include those selected from the group consisting of mutations corresponding to any one of R, S1030A, N1031T, R1033A, F1034I, Y1035D, L1036I, A1037R, W1038T, K1041T, G1042D, K1044T, E1045K, Q1046G, Q1047E, F1048Q, R1049S, I1050V, S1051G, T1052V, Q1053K, N1054T, L1055A, V1056L, L1057S, I1058F, Y1061N, V1063I, and N1064D, or a combination thereof.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:712.

[0012] Neisseria meningitidis Cas9ドメインは, I9 bit, D16 bit, D30 bit, E31 bit, A94 bit, I103 bit, P124 bit, N164 bit, I213 bit , G229 bit, T241 bit, S376 bit, E393 bit, G454 bit, K471 bit, G490 bit, D660 bit, C665 bit, K76 4-bit, T770-bit, P803-bit, A841-bit, H842-bit, K843-bit, D844-bit, L846-bit, R847-bit, K854-bit, H855-bit, N856-bit, K858-bit, K862-bit, W865-bit, E868-bit, I869-bit, A872-bit, D873-bit, N876 , Y880, G883, I886, E887, E890, R895, A898, Y899, G900, G901, N902, A903, K904, Q905, D908, N912, K917, G919, L921, V927, K929, T930, E932, S933, L936, L937, N938, K939, K940, Y943, T944, G949, D950, C958, K965, N966, Q967, F969, A975, E980, N981, I986, D987, C988, K989, G990, Y991, R992, I993, D994, Y997, T998, C1000, S1002, H1004, K1005, Y1006, A1010, F1011, Q1012, K1013, D1014, E1015, K1018, V1019, E1020, F1021, A1022, Y1024, I1025, N1026, C1027, D1028 , S1029, S1030, N1031, R1033, F1034, Y1035, L1036, A1037, W1038, K1041, G1042, K1044, E1045, Q1046, Q1047, F1048, R1 049 bit, I1050 bit, S1051 bit, T1052 bit, Q1053 bit, N1054 bit, L1055 bit, V1056 bit, L10 57 bits, I1058 bits, Y1061 bits, V1063 bits, and N1064 bits are all the same.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain is selected from the group consisting of I9M, D16E, D30E, E31K, A94D, I103V, P124C, N164D, I213N, G229D, T241A, S376T, E393K, G454C, K471E, G490C, D660E, C665R, K764E, T770A, P803S, A841Q, H842G, K843H, D844E, L846V, R847K, K854R, H855L, N856D, K858G, K862L, W865P, E868Q, I869L, A872K, D873G, N876K, Y880R, G883E, I886P, E887K, E890E, R895Q, A898T, Y899H, G900K, G901D, N902D, A903P, K904T, Q905K, D908A, N912E, K917Y, G919T, L921Q, V9 27I, K929Q, T930V, E932K, S933T, L936W, L937V, N938R, K939N, K940H, Y943N, T944G, G949A, D950T, C958E, K965G, N966G, Q967K, F969Y, A975 S, E980K, N981G, I986R, D987A, C988V, K989V, G990A, Y991F, R992K, I993D, D994E, Y997F, T998E, C1000R, S1002I, H1004Y, K1005A, Y1006N, A 1010K, F1011L, Q1012T, K1013A, D1014K, E1015K, K1018N, V1019E, E10 20F, F1021L, A1022G, Y1024F, I1025V, N1026S, C1027L, D1028N, S1029 The mutations include those selected from the group consisting of mutations corresponding to any one of R, S1030A, N1031T, R1033A, F1034I, Y1035D, L1036I, A1037R, W1038T, K1041T, G1042D, K1044T, E1045K, Q1046G, Q1047E, F1048Q, R1049S, I1050V, S1051G, T1052V, Q1053K, N1054T, L1055A, V1056L, L1057S, I1058F, Y1061N, V1063I, and N1064D, or a combination thereof.

[0013] In some embodiments, the RNA-guided nuclease Cas9 domain is the RNA-guided nuclease Campylobacter jejuni Cas9 domain. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 713. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain is at positions L5, A6, D8, I9, S12, S13, F18, S19, L24, K25, I31, T40, E42, L50, L58, A59, R61, L58, L65, H67, N74, K77, L98, I99, P101, N110, L113, A119, A126, R128, I134, K140, A144, K147, Q151, L156, V184, S190, F199, D202, G203, R212, F214, K221, E223, Y232, A235, V243, S247, D251, P256, L261, T269, N276, N277, L285, T287, L291, K300, T305, Q308, L312, G314, Y335, K336, I339, H345, D351, N353, E354, I362, K370, D383, S384, K391, I396, L403, T405, K413, N419, L421, D430, K432, A437, L453, K457, V462, A465, K472, N477, A492, E495, L525, K526, L527, K531, E532, E542, Q550, E556, H559, Y561, S564, M572, V577, Q581, N587, N596, K600, Q602, K603, Q616, K617, N623, Y624, K633, D634, Y642, N649, D656, L660, D662, K667, V677, E680, K682, L686, H692, T693, V712, I714, V722, K723, S736, L739, K742, L747,In some embodiments, the nucleic acid sequence includes a mutation corresponding to any one of positions N751, F756, R763, Q764, E772, K777, A786, E790, F792, Q800, S801, G804, L812, E813, V833, I835, T841, Y845, A855, L856, A863, V864, D879, E883, D900, Q902, K927, F928, V971, T972, or a combination thereof. The Cas9 domains are L5I, A6G, D8N, D8E, I9L, S12A, S13N, F18L, S19R, L24I, K25I, I31V, T40N, E42N, L50E, L58V, A59K, R61K, L58V, L65M, H67A, N74K, K77N, L98T, I99Q, P101I, N110S, L113I, A119S, A126V, R128H, I134S, K140N, A144T, and K14 7E, Q151K, L156M, V184I, S190D, F199L, D202Q, G203E, R212K, F214L, K221K, E223K, Y232F, A235P, V243I, S247I, D251N, P256A, L261S, T269G, N276K, N277S, L285V, T287E, L291I, K300D, T305S, Q308K, L312I, G314N, Y335L, K336 N, I339K, H345T, D351I, N353D, E354S, I362T, K370E, D383E, S384K, K391N, I396L, L403Q, T405I, K413R, N419E, L 421C, D430E, K432S, A437L, L453I, K457C, V462L, A465D, K472S, N477H, A492K, E495I, L525Q, K526I, L527V, K531E , E532D, E542L, Q550D, E556V, H559Y, Y561R, S564N, M572S, V577T, Q581L, N587G, N596E, K600L, Q602A, K603E, Q6 16R, K617F, N623F, Y624F, K633T, D634E, Y642W, N649S, D656S, L660I, D662E, K667A, V677Q, E680V, ​​K682S, L686I,H692N, T693F, V712I, I714V, V722I, K723F, S736K, L739F, K742N, L747S, N751L, F7 56L, R763K, Q764E, E772N, K777H, A786T, E790L, F792P, Q800N, S801T, G804D, L812V , E813K, V833S, I835L, T841K, Y845H, A855S, L856T, A863T, V864P, D879N, E883N, D900G, Q902K, K927N, F928Y, V971L, T972S, or a combination thereof. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 713.

[0014] In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain has amino acid residues at positions L5, A6, D8, I9, S12, S13, F18, S19, L24, K25, I31, T40, E42, L50, L58, A59, R61, L58, L65, H67, N74, K77, L98, I99, P101, N110, L113, A119, A126, R128, I134, K140, A144, K147, Q151, L156, V184, S190, F199, D202, G203, R212, F214, K221, E223, Y232, A235, V243, S247, D251, P256, L261, T269, N276, N277, L285, T287, L291, K300, T305, Q308, L312, G314, Y335, K336, I339, H345, D351, N353, E354, I362, K370, D383, S384, K391, I396, L403, T405, K413, N419, L421, D430, K432, A437, L453, K457, V462, A465, K472, N477, A492, E495, L525, K526, L527, K531, E532, E542, Q550, E556, H559, Y561, S564, M572, V577, Q581, N587, N596, K600, Q602, K603, Q616, K617, N623, Y624, K633, D634, Y642, N649, D656, L660, D662, K667, V677, E680, K682, L686, H692, T693, V712, I714, V722, K723, S736, L739, K742, L747, N751, F756, R763, Q764, E772, K777, A786, E790, F792, Q800, S801, G804, L812, E813, V833, I835, T841, Y845, A855, L856, A863, V864, D879, E883, D900.In some embodiments, the nucleic acid sequence of the present invention comprises a mutation corresponding to any one of positions Q902, K927, F928, V971, T972, or a combination thereof. The Cas9 domains are L5I, A6G, D8N, D8E, I9L, S12A, S13N, F18L, S19R, L24I, K25I, I31V, T40N, E42N, L50E, L58V, A59K, R61K, L58V, L65M, H67A, N74K, K77N, L98T, I99Q, P101I, N110S, L113I, A119S, A126V, R128H, I134S, K140N, A144T, K147E, Q151K, L156M, V184I, S190D, F199L, and D202. Q, G203E, R212K, F214L, K221K, E223K, Y232F, A235P, V243I, S247I, D251N, P256A, L261S, T269G, N276K, N277S, L285V, T287E, L291I, K3 00D, T305S, Q308K, L312I, G314N, Y335L, K336N, I339K, H345T, D351I, N353D, E354S, I362T, K370E, D383E, S384K, K391N, I396L, L403Q, T 405I, K413R, N419E, L421C, D430E, K432S, A437L, L453I, K457C, V462L, A465D, K472S, N477H, A492K, E495I, L525Q, K526I, L527V, K531E , E532D, E542L, Q550D, E556V, H559Y, Y561R, S564N, M572S, V577T, Q581L, N587G, N596E, K600L, Q602A, K603E, Q616R, K617F, N623F, Y62 4F, K633T, D634E, Y642W, N649S, D656S, L660I, D662E, K667A, V677Q, E680V, ​​K682S, L686I, H692N, T693F, V712I, I714V, V722I, K723F, S 736K, L739F, K742N, L747S, N751L, F756L, R763K, Q764E, E772N, K777H, A786T, E790L, F792P, Q800N, S801T, G804D, L812V, E813K, V833S,The mutations include those selected from the group consisting of I835L, T841K, Y845H, A855S, L856T, A863T, V864P, D879N, E883N, D900G, Q902K, K927N, F928Y, V971L, and T972S, or a combination thereof.

[0015] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 714. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 714. The Cas9 domain contains the following positions: D11, E85, A88, T92, E96, Y100, T109, D110, D113, E115, R116, D125, I127, K128, E132, S147, I185, A187, K228, Y229, T232, M255, S271, N273, A294, A327, E355, K357, N379 Place, T380th, S382nd, A385th, D439th, R440th, S464th, H469th, Y519th, I528th, N569th, I581st, A607th, K632nd, D633rd, H6 35th place, E636th place, A647th place, D648th place, T703rd place, P705th place, K712th place, S713th place, A724th place, V750th place, D882nd, S951st, D977th, E979th, S1014th, H1027th, I1030th, E1081st, D1082nd, D1086th, K1088th, S1089th, N1090th, R1092nd, T1093rd, I1094th, C1095th, A113 8th place, Y1139th, D1141st, T1142nd, F1158th, A1168th, E1190th, E1198th, H1202nd, I1204th, R1205th, I1210th, K1224th, S1 The mutations include any one of positions 232, M1240, V1241, I1242, P1243, G1424, K1248, Q1254, N1257, S1258, T1262, K1263, Y1264, D1266, A1270, K1277, D1284, L1288, V1302, N1316, T1346, and I1374, or a combination thereof.In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain is selected from the group consisting of D11E, D11A, E85D, A88T, T92A, E96D, Y100Q, T109D, D110N, D113N, E115D, R116S, D125E, I127D, K128A, E132K, S147T, I185L, A187T, K228N, Y229N, T232K, M255T, S271T, N273E, A294S, A327V, E355K, K357Q, N3 79G, T380I, S382T, A385N, D439E, R440E, S464A, H469R, Y519F, I528V, N569D, I581V, A607S, K632R, D633E, H6 35Q, E636Q, A647K, D648Q, T703A, P705S, K712E, S713A, A724T, V750I, D882G, S951R, D977E, E979K, S1014P, H1 027R, I1030V, E1081G, D1082E, D1086N, K1088R, S1089T, N1090D, R1092E, T1093K, I1094V, C1095R, A1138V, Y 1139L, D1141E, T1142P, F1158L, A1168T, E1190K, E1198K, H1202Q, I1204V, R1205Q, I1210M, K1224R, S1232T, M 1240I, V1241M, I1242L, P1243S, G1424A, K1248A, Q1254H, N1257G, S1258N, T1262A, K1263E, Y1264H, D1266K, A1270E, K1277E, D1284N, L1288V, V1302A, N1316D, T1346N, I1374L, or a combination thereof. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:714.

[0016] In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises a mutation corresponding to any one of positions D11, E85, A88, T92, E96, Y100, T109, D110, D113, E115, R116, D125, I127, K128, E132, S147, I185, A187, K228, Y229, T232, M255, S271, N273, A294, A327, E355, K357, N379, T380, S382, A385, D439, R440, S464, H469, Y519, I528, N569, I581, A607, K632, D633, H635, E636, A647, D648, T703, P705, K712, S713, A724, V750, D882, S951, D977, E979, S1014, H1027, I1030, E1081, D1082, D1086, K1088, S1089, N1090, R1092, T1093, I1094, C1095, A1138, Y1139, D1141, T1142, F1158, A1168, E1190, E1198, H1202, I1204, R1205, I1210, K1224, S1232, M1240, V1241, I1242, P1243, G1424, K1248, Q1254, N1257, S1258, T1262, K1263, Y1264, D1266, A1270, K1277, D1284, L1288, V1302, N1316, T1346, I1374 or a combination thereof.In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain is selected from the group consisting of D11E, D11A, E85D, A88T, T92A, E96D, Y100Q, T109D, D110N, D113N, E115D, R116S, D125E, I127D, K128A, E132K, S147T, I185L, A187T, K228N, Y229N, T232K, M255T, S271T, N273E, A294S, A327V, E355K, K357Q, N3 79G, T380I, S382T, A385N, D439E, R440E, S464A, H469R, Y519F, I528V, N569D, I581V, A607S, K632R, D633E, H6 35Q, E636Q, A647K, D648Q, T703A, P705S, K712E, S713A, A724T, V750I, D882G, S951R, D977E, E979K, S1014P, H1 027R, I1030V, E1081G, D1082E, D1086N, K1088R, S1089T, N1090D, R1092E, T1093K, I1094V, C1095R, A1138V, Y 1139L, D1141E, T1142P, F1158L, A1168T, E1190K, E1198K, H1202Q, I1204V, R1205Q, I1210M, K1224R, S1232T, M The mutations include those selected from the group consisting of 1240I, V1241M, I1242L, P1243S, G1424A, K1248A, Q1254H, N1257G, S1258N, T1262A, K1263E, Y1264H, D1266K, A1270E, K1277E, D1284N, L1288V, V1302A, N1316D, T1346N, and I1374L, or a combination thereof.

[0017] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Clostridium cellulolyticum Cas9 domain. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 715. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is T4, D10, V9, D20, K21, I27, C33, K36, A47, A49, S64, Q65, E102, L103, T1 22nd place, I124th place, K131st place, D137th place, R163rd place, G166th place, I169th place, F170th place, V183rd place, D184th place, I187th place, E193rd place, K200th place, K208th place, L209th, D221st, N224th, E227th, F228th, S234th, V242nd, K244th, L252nd, T256th, C258th, S261st, V413th, M415 Place, K416th, R417th, K424th, Y426th, K427th, S429th, D430th, A468th, T470th, A472nd, A478th, Q481st, K482nd, L48 5th place, A497th place, L535th place, W540th place, R541st, E544th, G554th, P556th, I570th, Y574th, M580th, Y584th, M585th, T592nd, D 593rd place, V606th place, W607th place, I647th place, N650th place, S693rd place, L697th place, E702nd place, S704th place, A713th place, V714th place, I715th place, D776th place, L847th place , G850, G853, A854, R860, I900, H904, M905, I906, E921, Q923, S929, T930, H931, Q939, N994, I997, N1000, K1001, S1002, I1003, K1005, P1008, or a combination thereof.In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is selected from the group consisting of T4S, D10E, V9I, D20N, K21E, I27E, C33I, K36V, A47S, A49P, S64R, Q65H, E102L, L103V, T122V, I124F, K131Q, D137E, R163Q, G166S, I169L, F170L, V183G, D184G, I187T, E193S, K200Q, K208A, L 209Y, D221K, N224Q, E227S, F228S, S234T, V242I, K244N, L252K, T256K, C258T, S261F, V413K, M415L , K416R, R417N, K424Q, Y426I, K427P, S429H, D430Q, A468S, T470S, A472V, A478G, Q481K, K482R, L485 S, A497M, L535H, W540Y, R541K, E544Q, G554F, P556S, I570V, Y574I, M580F, Y584N, M585N, T592A, D5 93A, V606W, W607F, I647R, N650H, S693K, L697F, E702Q, S704N, A713V, V714I, I715V, D776E, L847A, G In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:715.

[0018] In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises a mutation corresponding to any one of positions T4, D10, V9, D20, K21, I27, C33, K36, A47, A49, S64, Q65, E102, L103, T122, I124, K131, D137, R163, G166, I169, F170, V183, D184, I187, E193, K200, K208, L209, D221, N224, E227, F228, S234, V242, K244, L252, T256, C258, S261, V413, M415, K416, R417, K424, Y426, K427, S429, D430, A468, T470, A472, A478, Q481, K482, L485, A497, L535, W540, R541, E544, G554, P556, I570, Y574, M580, Y584, M585, T592, D593, V606, W607, I647, N650, S693, L697, E702, S704, A713, V714, I715, D776, L847, G850, G853, A854, R860, I900, H904, M905, I906, E921, Q923, S929, T930, H931, Q939, N994, I997, N1000, K1001, S1002, I1003, K1005, P1008 or a combination thereof.In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is selected from the group consisting of T4S, D10E, V9I, D20N, K21E, I27E, C33I, K36V, A47S, A49P, S64R, Q65H, E102L, L103V, T122V, I124F, K131Q, D137E, R163Q, G166S, I169L, F170L, V183G, D184G, I187T, E193S, K200Q, K208A, L2 09Y, D221K, N224Q, E227S, F228S, S234T, V242I, K244N, L252K, T256K, C258T, S261F, V413K, M415L, K 416R, R417N, K424Q, Y426I, K427P, S429H, D430Q, A468S, T470S, A472V, A478G, Q481K, K482R, L485S, A 497M, L535H, W540Y, R541K, E544Q, G554F, P556S, I570V, Y574I, M580F, Y584N, M585N, T592A, D593A, V606W, W607F, I647R, N650H, S693K, L697F, E702Q, S704N, A713V, V714I, I715V, D776E, L847A, G850P, The mutations include those selected from the group consisting of G853A, A854P, R860K, I900V, H904D, M905V, I906L, E921Y, Q923E, S929D, T930E, H931Y, Q939P, N994Q, I997P, N1000R, K1001M, S1002N, I1003K, K1005H, and P1008K, or a combination thereof.

[0019] In some embodiments, the RNA-guided nuclease Cas9 domain is a RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO:716.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions K2, D8, I14, D35, K41, F74, V75, K91, I117, R128, T136, Q151, S152, S156, A161, V164, S171, E178, D179, V185, R192, K195, A199, Y204, I207, V208, A212, H215, S219, F227, T260, V261, V271, G274, I276, A278, L279, D282, I287, K289, H293, F299, V302, N307, R313, L317, L318, V331, G337, K341, S348, A354, A355, K356, R359, M372, T377, R380, E395, D399, E404, S416, T441, R445, N464, E504, S508, M515, Q516, E520, G521, V534, L545, K559, T578, K603, T612, L619, S621, N656, N660, L673, D685, I699, N708, N717, R737, V738, S752, D756, Q771, N777, N792, E793, I811, I824, K839, Q845, K848, T849, L895, I902, T908, V929, I943, I946, M948, F990, T995, V1000, Q1014, D1017, S1019, N1020, G1021, S1024, N1030, N1031, R1035, S1036, I1037, V1067, S1071, A1075, I1079 or a combination thereof.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain is selected from the group consisting of K2R, D8E, D8A, I14V, D35E, K41Q, F74V, V75I, K91E, I117V, R128K, T136S, Q151R, S152A, S156G, A161G, V164I, S171A, E178G, D179E, V185I, R192H, K195R, A199S, Y204F, I207M, V208S, A212K, H215N, S219T, F227V, T260I, V261A, V271I , G274S, I276A, A278G, L279P, D282E, I287L, K289E, H293Q, F299Y, V302I, N307R, R313Y, L317I, L318V, V331I, G337D, K34 1Q, S348K, A354K, A355S, K356S, R359L, M372L, T377A, R380H, E395P, D399N, E404N, S416T, T441S, R445K, N464T, E504D, S5 08T, M515T, Q516K, E520D, G521E, V534M, L545H, K559R, T578V, K603R, T612I, L619V, S621T, N656M, N660S, L673F, D685E, I699V, N708E, N717D, ​​R737K, V738I, S752A, D756E, Q771R, N777H, N792D, E793Q, I811V, I824V, K839T, Q845K, K848A, T849S , L895P, I902V, T908K, V929V, I943V, I946M, M948I, F990L, T995I, V1000G, Q1014K, D1017H, S1019G, N1020T, G1021A, S1024E, N1030C, N1031S, R1035S, S1036G, I1037V, V1067L, S1071A, A1075T, I1079V, or a combination thereof. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:716.

[0020] In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions K2, D8, I14, D35, K41, F74, V75, K91, I117, R128, T136, Q151, S152, S156, A161, V164, S171, E178, D179, V185, R192, K195, A199, Y204, I207, V208, A212, H215, S219, F227, T260, V261, V271, G274, I276, A278, L279, D282, I287, K289, H293, F299, V302, N307, R313, L317, L318, V331, G337, K341, S348, A354, A355, K356, R359, M372, T377, R380, E395, D399, E404, S416, T441, R445, N464, E504, S508, M515, Q516, E520, G521, V534, L545, K559, T578, K603, T612, L619, S621, N656, N660, L673, D685, I699, N708, N717, R737, V738, S752, D756, Q771, N777, N792, E793, I811, I824, K839, Q845, K848, T849, L895, I902, T908, V929, I943, I946, M948, F990, T995, V1000, Q1014, D1017, S1019, N1020, G1021, S1024, N1030, N1031, R1035, S1036, I1037, V1067, S1071, A1075, I1079 or a combination thereof.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain is selected from the group consisting of K2R, D8E, D8A, I14V, D35E, K41Q, F74V, V75I, K91E, I117V, R128K, T136S, Q151R, S152A, S156G, A161G, V164I, S171A, E178G, D179E, V185I, R192H, K195R, A199S, Y204F, I207M, V208S, A212K, H215N, S219T, F227V, T260I, V261A, V271I , G274S, I276A, A278G, L279P, D282E, I287L, K289E, H293Q, F299Y, V302I, N307R, R313Y, L317I, L318V, V331I, G337D, K34 1Q, S348K, A354K, A355S, K356S, R359L, M372L, T377A, R380H, E395P, D399N, E404N, S416T, T441S, R445K, N464T, E504D, S5 08T, M515T, Q516K, E520D, G521E, V534M, L545H, K559R, T578V, K603R, T612I, L619V, S621T, N656M, N660S, L673F, D685E, I699V, N708E, N717D, ​​R737K, V738I, S752A, D756E, Q771R, N777H, N792D, E793Q, I811V, I824V, K839T, Q845K, K848A, T849S , L895P, I902V, T908K, V929V, I943V, I946M, M948I, F990L, T995I, V1000G, Q1014K, D1017H, S1019G, N1020T, G1021A, S1024E, N1030C, N1031S, R1035S, S1036G, I1037V, V1067L, S1071A, A1075T, I1079V, or a combination thereof.

[0021] In some embodiments, the RNA-guided nuclease Cas domain is an RNA-guided nuclease Cas12 domain. In some embodiments, the RNA-guided nuclease Cas domain is an RNA-guided nuclease CasX domain. In some embodiments, the I-TEVI nuclease domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 700. In some embodiments, the I-TEVI nuclease domain comprises a mutation at any one of positions corresponding to T11, V16, N14, E25, K26, R27, E36, K37, G38, C39, S41, L45, F49, I60, and E81, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation selected from any one corresponding to T11V, V16I, N14G, E25D, K26R, R27A, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, E81I, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation corresponding to the K26R mutation. In some embodiments, the I-TEVI nuclease domain comprises the amino acid sequence set forth in SEQ ID NO: 700. In some embodiments, the I-TEVI nuclease domain comprises a mutation corresponding to any one of positions T11, V16, N14, E25, K26, R27, E36, K37, G38, C39, S41, L45, F49, I60, and E81, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation selected from mutations corresponding to any one of positions T11V, VI6I, N14G, E25D, K26R, R27A, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, and E81I, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation corresponding to the K26R mutation. In some embodiments, the chimeric nuclease further comprises a nuclear localization signal. In some embodiments, the nuclear localization signal comprises an SV40 nuclear localization signal. In some embodiments, the nuclear localization signal comprises a nucleoplasmin nuclear localization signal. In some embodiments, the composition further comprises a donor nucleic acid.In some embodiments, the donor nucleic acid restores non-oncogenic function of a gene containing an oncogenic mutation. In some embodiments, the donor nucleic acid comprises a non-oncogenic version of the oncogenic mutation. In some embodiments, the donor nucleic acid is DNA. In some embodiments, the donor nucleic acid comprises a blunt end and a 3' overhang of at least two nucleotides. In some embodiments, the donor nucleic acid comprises 5' and 3' homology adjacent to the non-oncogenic version of the oncogenic mutation. In some embodiments, the composition does not comprise a donor nucleic acid. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, the composition is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic or neutral lipid.

[0022] [6] In another aspect, the present disclosure provides a nucleic acid or multiple nucleic acids encoding the chimeric nuclease or guide RNA of the present disclosure. In some embodiments, the chimeric nuclease or guide RNA is operably linked to a eukaryotic promoter, enhancer, polyadenylation site, or a combination thereof. In some embodiments, the nucleic acid is an expression vector selected from a plasmid, a lentiviral vector, an adeno-associated viral vector, or an adenoviral vector. In some embodiments, the nucleic acid or multiple nucleic acids further comprise a donor nucleic acid portion.

[0023] [7] In another aspect, the present disclosure provides a method of targeting an oncogenic mutation in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0024] [8] In another aspect, the present disclosure provides a use of a composition of the present disclosure in a cell to target an oncogenic mutation in the cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0025]

[0009] In another aspect, the present disclosure provides a method of editing a genome in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual suffering from cancer.

[0026]

[10] In another aspect, the present disclosure provides a use of a composition of the present disclosure for editing the genome in a cell. In some embodiments, the cell is a cell of an individual suffering from cancer.

[11] In another aspect, the present disclosure provides a method for deleting at least some oncogenic mutations in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual suffering from cancer.

[0027]

[12] In another aspect, the present disclosure provides a use of a composition of the present disclosure for deleting at least some oncogenic mutations in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0028]

[13] In another aspect, the present disclosure provides a method of silencing or disrupting at least some oncogenic mutations in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0029]

[14] In another aspect, the present disclosure provides a use of a composition of the present disclosure for silencing or disrupting at least a portion of oncogenic mutations in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0030]

[15] In another aspect, the present disclosure provides a method of replacing at least a portion of oncogenic mutations in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0031]

[16] In another aspect, the present disclosure provides a use of a composition of the present disclosure for replacing at least a portion of an oncogenic mutation in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0032]

[17] In another aspect, the present disclosure provides a method of restoring a non-oncogenic function in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0033]

[18] In another aspect, the present disclosure provides a use of a composition of the present disclosure for restoring a non-oncogenic function in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0034]

[19] In another aspect, the present disclosure provides a method of treating cancer in an individual, comprising administering a composition of the present disclosure to an individual having cancer, thereby treating the cancer in the individual.

[20] In another aspect, the present disclosure provides a use of a composition of the present disclosure for treating cancer in an individual.

[0035]

[21] In another aspect, the present disclosure provides a composition comprising a chimeric nuclease, the chimeric nuclease comprising an I-TEVI nuclease domain, an RNA-guided nuclease Cas domain, and a guide RNA, wherein the guide RNA comprises a nucleic acid sequence that targets an oncogenic mutation, the oncogenic mutation being (i) an insertion of one or more nucleotides, or (ii) a substitution or deletion of 10 or fewer nucleotides.

[0036]

[22] In some embodiments, the oncogenic mutation is a single nucleotide polymorphism. In some embodiments, the sequence comprising the oncogenic mutation is selected from the mutations set forth in any one of SEQ ID NOs: 1-683 or a combination thereof. In some embodiments, the sequence comprising the oncogenic mutation is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to a mutation set forth in any one of SEQ ID NOs: 1-683 or a combination thereof. In some embodiments, the oncogenic mutation comprises an EGFR L858R mutation or a mutation corresponding to an EGFR V769_D770insASV mutation. In some embodiments, the oncogenic mutation comprises a mutation corresponding to an EGFR L858R mutation. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 45, 130, or 141, or comprises a nucleotide sequence set forth in SEQ ID NO: 1045, 1130, 1141, or 1686. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 45, 130, 141, or comprises a nucleotide sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1045, 1130, 1141, or 1686. In some embodiments, the oncogenic mutation comprises a mutation corresponding to the EGFR V769_D770insASV mutation. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 683, or comprises the nucleotide sequence set forth in SEQ ID NO: 1683 or 1684. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 683, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1683 or 1684.

[0037] In some embodiments, the oncogenic mutation is an oncogenic mutation in a gene selected from any one of Muc4, PIK3CA, and KRAS, or a combination thereof. In some embodiments, the oncogenic mutation comprises a Muc4 mutation. In some embodiments, the Muc4 mutation is an in-frame deletion of exon 2 or an in-frame deletion of exon 3. In some embodiments, the Muc4 mutation comprises a mutation corresponding to any one of positions P1542, P1680, T1711, V1721, P1826, A1830, S3560, A1833, D2253, V2281, P3088, T3119, T3183, V3817, and A3902 of the human Muc4 protein, or a combination thereof. In some embodiments, the Muc4 mutation is selected from mutations corresponding to any one of P1542L, P1680S, T1711I, V1721A, P1826H, A1830T, S3560S, A1833V, D2253H, V2281AM, P3088L, T3119T, T3183M, V3817A, A3902V of human Muc4 protein, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 676, 677, 678, 679, or 682, or comprises a nucleotide sequence set forth in SEQ ID NO: 1676, 1677, 1678, 1679, 1682, or 1685. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 676, 677, 678, 679, or 682, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1676, 1677, 1678, 1679, 1682, or 1685.

[0038] In some embodiments, the oncogenic mutation comprises a PIK3CA mutation. In some embodiments, the PIK3CA mutation comprises a mutation corresponding to any one of positions H1047, E542, E545, N345, C1636, G1624, G1633, A3140, C3075, A1634, A1173 of the human PIK3A protein, or a combination thereof. In some embodiments, the PIK3CA mutation is selected from a mutation corresponding to any one of positions H1047R, H1047L, E542K, E545K, N345K, C1636A, G1624A, G1633A, A3140T, A3140G, C3075T, A1634C, A1173G of the human PIK3A protein, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 33, 202, 204, 209, or 210, or comprises a nucleotide sequence set forth in SEQ ID NO: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, or 1210. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5, 6, 7, 8, 33, 202, 204, 209, or 210, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, or 1210.

[0039] In some embodiments, the oncogenic mutation comprises a KRAS mutation selected from mutations corresponding to any one of positions A59, D119, D33, G21, G12, G13, Q61, A146, K117 of the human KRAS protein, or a combination thereof. In some embodiments, the KRAS mutation is selected from mutations corresponding to any one of A59T, A59E, A59T, D119N, D33E, G21C, G12C, G12D, G12V, G12R, G12A, G12S, G13D, G13C, G13V, G13R, Q61R, Q61V, Q61L, Q61K, Q61H, Q61A, Q61P, Q61E, A146T, A146V, K117N, K117R of the human KRAS protein, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 37, 42, 51, 52, 62, 63, or 77, or comprises a nucleotide sequence set forth in SEQ ID NO: 1037, 1042, 1051, 1052, 1062, 1063, or 1077. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 37, 42, 51, 52, 62, 63, or 77, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1037, 1042, 1051, 1052, 1062, 1063, or 1077.

[0040] In some embodiments, the guide RNA comprises one or more of an unnatural internucleoside linkage, a nucleic acid mimic, a modified sugar moiety, or a modified nucleobase. In some embodiments, the unnatural internucleoside linkage comprises one or more of a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'-alkylenephosphonate, a 5'-alkylenephosphonate, a chiral phosphonate, a phosphinate, a 3'-aminophosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, or a boranophosphate. In some embodiments, the nucleic acid mimic comprises one or more of a peptide nucleic acid (PNA), a morpholino nucleic acid, a cyclohexenyl nucleic acid (CeNA), or a locked nucleic acid (LNA). In some embodiments, the modified sugar moiety comprises one or more of 2'-O-(2-methoxyethyl), 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O-methyl, or 2'-fluoro.In some embodiments, the modified nucleobase is 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivative of adenine, 6-methyl derivative of guanine, 2-propyl derivative of adenine, 2-propyl derivative of guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 5-halo, 5-bromo, 5-trifluoromethyl, 5-substituted uracil cytidine, 5-substituted cytosine, 7-methylguanine, 7-methyladenine, 2-F adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidine, phenoxazine cytidine, phenothiazine cytidine, substituted phenoxazine cytidine, carbazole cytidine, pyridoindole cytidine, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, 5-substituted pyrimidine, 6-azapyrimidine, N-2, N-6, or O-6 substituted purine, 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine.

[0041] In some embodiments, the composition further comprises a linker operably linked to the I-TEVI nuclease domain and the RNA-guided nuclease Cas domain. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 701, 702, 703, or 704. In some embodiments, the linker comprises a mutation corresponding to any one of positions T95, S101, A119, K120, K135, P126, D127, N140, T147, Q158, A161, V117, S165, or a combination thereof. In some embodiments, the linker comprises a mutation selected from mutations corresponding to any one of T95S, S101Y, A119D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, V117F, S165G, or a combination thereof. In some embodiments, the linker comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 701, 702, 703, or 704. In some embodiments, the linker comprises a mutation corresponding to any one of positions T95, S101, A119, K120, K135, P126, D127, N140, T147, Q158, A161, V117, S165, or a combination thereof. In some embodiments, the linker comprises a mutation selected from a mutation corresponding to any one of positions T95S, S101Y, A119D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, V117F, S165G, or a combination thereof.

[0042] In some embodiments, the RNA-guided nuclease Cas domain is an RNA-guided nuclease Cas9 domain. In some embodiments, the RNA-guided nuclease Cas9 domain is any one of an RNA-guided nuclease Staphylococcus aureus Cas9 domain, an RNA-guided nuclease Streptococcus pyogenes Cas9 domain, an RNA-guided nuclease Neisseria meningitidis Cas9 domain, an RNA-guided nuclease Campylobacter jejuni Cas9 domain, an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain, an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain, an RNA-guided nuclease Clostridium cellulolyticum Cas9 domain, an RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain, or a combination thereof. In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Staphylococcus aureus Cas9 domain. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 710. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 710. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 710 at positions D10, H557, N580, H840, D1135, R1335, T1337, T267, L325, V327, D333, A336, I341, E345, D348, K352, S360, T368, N369, N371, S372, E373, K386, N393, H40 8th, N410th, I414th, A415th, T438th, Y467th, N471st, D485th, M489th, E506th, R409th, T510th, N515th, Y518 A539th, F550th, N551st, S596th, T602nd, A611th, I617th, T620th, G654th, N667th, R685th, K695th, I706th,Contains a mutation corresponding to any one of K722, A723, K724, M731, F732, K735, S739, P741, E742, E746, Q747, I754, T755, H757, K760, H761, P778, E781, I783, N784, D785, T786L, L787, Y788, K792, D794, T798, L799, V801, N803, L804, N805, G806, D813, K814, L818, I819, S822, E824, L841, G847, D848, Y857, V875, I876, N884, A888, L890, D894, D895, P897, V903, G920, F924, N929, E936, N937, V941, N942, S943, C945, E947, K951, L952, S956, N957, Q958, A959, N974, G975, V983, N984, N985, D986, I991, V993, M995, I996, T999, Y1000, R1001, E1002, L1004, E1005, N1006, M1007, D1009, K1010, R1011, P1012, P1013, I1015, I1016, A1020, S1021, Q1024, K1027, E1039, H1045, I0148, K1050 or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain is D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, T267A, L325F, V327I, D333G, A336S, I341L, E345D, D348N, K352E, S360A, T368A, N369E, N371E, S372P, E373K, K386T, N393R, H408N, N410S, I414M, A415T, T438S, Y467F, N471K, D485E, M489F, E506K, R409K, T510E, N515K, Y518F, A539P, F550Y, N551H, S596A, T602I, A611S, I617V, T620K, G654E,N667D, R685K, K695Q, I706V, K722T, A723T, K724N, M731T, F732V, K735Q, S739N, P741L, E742G, E746D, Q 747D, I754D, T755I, H757R, K760Q, H761S, P778I, E781K, I783V, N784D, D785E, T786L, L787V, Y788H, K79 2E, D794T, T798R, L799I, V801I, N803S, L804I, N805K, G806N, D813G, K814E, L818I, I819F, S822P, E824G , L841T, G847S, D848N, Y857H, V875I, I876V, N884K, A888V, L890R, D894G, D895H, P897L, V903I, G920D, F 924L, N929Y, E936D, N937G, V941I, N942D, S943L, C945A, E947K, K951R, L952Q, S956N, N957E, Q958K, A95 9S, N974D, G975K, V983A, N984S, N985D, D986G, I991V, V993L, M995F, I996V, T999N, Y1000K, R1001E, E10 02D, L1004I, E1005K, N1006M, M1007N, D1009L, K1010S, R1011T, P1012S, P1013F, I1015L, I1016R, A1020G, S1021K, Q1024K, K1027S, E1039K, H1045K, I0148M, K1050M, or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 710.

[0043] Part of the form of Staphylococcus aureus, RNA-inducible Staphylococcus aureus Cas9 is denoted by, D10, H557, N580, H840, D1135, R1335, T1337, T267, L325, V327, D333, A336, I341, E345, D348, K352, S360, T368, N369, N371, S372, E373, K386, N393, H408, N410, I414, A415, T438, Y467, N471, D485, M489, E506, R409, T510, N515, Y 518, A539, ​​F550, N551, S596, T602, A611, I617, T620, G654, N667, R685, K695, I706, K722, A723, K724, M731, F732, K735, S739, P741, E742, E746, Q747, I754, T755, H757, K760, H761, P778, E781, I783, N784, D785, T786L, L787, Y788, K792, D 794, T798, L799, V801, N803, L804, N805, G806, D813, K814, L818, I819, S822, E824, L841, G847, D848, Y857, V875, I876, N884, A888, L890, D894, D895, P897, V903, G920, F924, N929, E936, N937, V941, N942, S943, C945, E947, K951, L952, S9 56, N957, Q958, A959, N974, G975, V983, N984, N985, D986, I991, V993, M995, I996, T999, Y1000, R1001, E1002, L1004, E1005, N1006, M1007, D1009, K1010, R1011, P1012, P1013, I1015, I1016, A1020, S1021, Q1024, K1027, E1039, H1045, I0148,In some embodiments, the RNA-guided nuclease Staphylococcus aureus comprises a mutation corresponding to any one of positions K1050 and K1060, or a combination thereof. The Cas9 domains are D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, T267A, L325F, V327I, D333G, A336S, I341L, E345D, D348N, K352E, S360A, T368A, N369E, N371E, S372P, E373K, K386T, N393R, H408N, N410S, I414M, A415T, T438S, Y467F, N471K, D485E, M489F, E506K, R409K, and T51 0E, N515K, Y518F, A539P, F550Y, N551H, S596A, T602I, A611S, I617V, T620K, G654E, N667D, R685K, K695Q, I706V, K722T, A723T, K724N, M731 T, F732V, K735Q, S739N, P741L, E742G, E746D, Q747D, I754D, T755I, H757R, K760Q, H761S, P778I, E781K, I783V, N784D, D785E, T786L, L787V Y788H, K792E, D794T, T798R, L799I, V801I, N803S, L804I, N805K, G806N, D813G, K814E, L818I, I819F, S822P, E824G, L841T, G847S, D848N, Y 857H, V875I, I876V, N884K, A888V, L890R, D894G, D895H, P897L, V903I , G920D, F924L, N929Y, E936D, N937G, V941I, N942D, S943L, C945A, E94 7K, K951R, L952Q, S956N, N957E, Q958K, A959S, N974D, G975K, V983A, N984S, N985D, D986G, I991V, V993L, M995F, I996V, T999N, Y1000K, R10 01E, E1002D, L1004I, E1005K, N1006M, M1007N, D1009L, K1010S, R1011 T, P1012S, P1013F, I1015L, I1016R, A1020G, S1021K, Q1024K, K1027S,In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation selected from the group consisting of E1039K, H1045K, I0148M, and K1050M, or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation corresponding to a D10E mutation. One type of Streptococcus pyogenes, RNA-inducible Cas9 Cas9, and RNA-inducible Cas9 Streptococcus pyogenes. Part 1 of the application form, RNA-inducible Streptococcus pyogenes Cas9 ドメインは, registration number 711, record of the されるアミノ acid preparation system. One piece of Streptococcus pyogenes, RNA-inducible Streptococcus pyogenes Cas9ドメインは, D10 bit, S29 bit, F32 bit, D39 bit, R40 bit, H41 bit, S42 bit, I48 bit, C80 bit, S87 bit, K112 bit, H113 bit, K132, K141 bit, D147 bit, L158 bit, E171 bit, P176 bit, I186 bit, V189 bit, Q190 bit, Q194 bit, N199 bit, I201 bit, N202 bit, A203 bit, S204 bit, R205 bit, A210 bit, Q228 bit, L229 bit, G231 bit, S245 bit, T2 49, S254, D261, T270, N295, T300, D304, V308, N309, I312, T333, A337, E345, F352, Q354, S355, K356, G366, A367, E396, L398, I414, D428, F429, D435, K468, S469, E470, T472, E480, A486, S490, F498, K500 bit, N501, N504, K528, V530, E532, G533, A538, T555, K570, F575, D605, E611, R629, E634, T638, R655, R664, R671, K705, E706, Q709, K710, S714, G7115, G717, H721, H723, A725, N726, V743, L747, V748, K772 , K775, N776, I788, G792, K797, Y799, T804, N808, L811, R820, N831, R832, ​​V842, L847, N869, E874, N881, Q885, N888, T893, L911, Y945, D946, L949, E952, A1023, Y1036, G1067, G1077, R1078, N1093, R1114,N1115th, D1117th, A1121st, D1125th, P1128th, K1129th, V1146th, S1154th, S1159th, L1164th, S1172nd, N1177th, P1178th, I1179th, D 1180th, K1211, M1213, G1218, N1234, E1243, K1244, E1253, E1260, K1263, H1264, E1271, Q1272, E1275, V1 In some embodiments, the RNA-guided nuclease comprises a mutation corresponding to any one of positions 290, L1291, S1292, A1293, N1295, H1297, R1298, D1299, K1300, R1303, E1307, N1308, I1309, I1310, H1311, L1312, L1315, T1316, N1317, Y1326, D1328, V1342, A1345, I1360, S1363, or a combination thereof. The Cas9 domains are D10E, D10A, S29T, F32M, D39N, R40K, H41Q, S42T, I48L, C80R, S87A, K112D, H113N, K132N, K141E, D147E, L158V, E171Q, P176S, I186K, V189L, Q190H, Q194E, and N1 99R, I201L, N202E, A203E, S204I, R205K, A210G, Q228A, L229F, G231N, S245A, T249M, S254A, D261N, T270S, N295K, T300I, D304G, V308A, N309D, I312V, T333A, A337V, E345 K, F352S, Q354K, S355T, K356T, G366K, A367T, E396D, L398F, I414V, D428A, F429Y, D 435E, K468Q, S469R, E470N, T472A, E480D, A486T, S490L, F498V, K500E, N501H, N504T , K528R, V530I, E532D, G533E, A538E, T555A, K570Q, F575C, D605E, E611D, R629K, E63 4K, T638K, R655H, R664K, R671K, K705V, E706D, Q709K, K710A, S714F, G7115E, G717K,H721K, H723Q, A725S, N726A, V743I, L747I, V748I, K772Q, K775R, N776R, I788M, G792R, K797E, Y79 9H, T804A, N808D, L811R, R820K, N831D, R832H, V842I, L847I, N869D, E874A, N881S, Q885R, N888K, T 893S, L911A, Y945H, D946G, L949P, E952A, A1023G, Y1036R, G1067E, G1077E, R1078K, N1093T, R111 4G, N1115E, D1117A, A1121P, D1125G, P1128T, K1129T, V1146I, S1154T, S1159P, L1164V, S1172N, N1 177D, P1178S, I1179V, D1180S, K1211R, M1213L, G1218T, N1234H, E1243D, K1244T, E1253K, E1260D , K1263Q, H1264Y, E1271D, Q1272W, E1275H, V1290L, L1291R, S1292A, A1293T, N1295E, H1297N, R129 8T, D1299H, K1300L, R1303S, E1307D, N1308S, I1309M, I1310L, H1311N, L1312A, L1315F, T1316S, N1317R, Y1326F, D1328N, V1342I, A1345S, I1360L, S1363N, or a combination thereof. In some embodiments, the RNA-guided nuclease Streptococcus pyogenes Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 711.

[0044] 1st Staphylococcus pyogenes, RNA-inducible Staphylococcus pyogenes Cas9ドメインは, D10 bit, S29 bit, F32 bit, D39 bit, R40 bit, H41 bit, S42 bit, I48 bit, C80 bit, S87 bit , K112 bit, H113 bit, K132 bit, K141 bit, D147 bit, L158 bit, E171 bit, P176 bit, I186 bit, V189 bit , Q190 bit, Q194 bit, N199 bit, I201 bit, N202 bit, A203 bit, S204 bit, R205 bit, A210 bit, Q228 bit, L229 bit, G231 bit, S245 bit, T249 bit, S254 bit, D261 bit, T270 bit, N295 bit, T300 bit, D304 bit, V308, N309, I312, T333, A337, E345, F352, Q354, S355, K356, G366, A367, E396, L398, I414, D428, F429, D435, K468, S469, E470, T472, E480, A486, S490, F498, K500, N501, N504, K528, V530, E532, G533, A538, T555, K570, F575, D605, E611, R62 9, E634, T638, R655, R664, R671, K705, E706, Q709, K710, S714, G7115, G717, H721, H723, A725, N726, V743, L747, V748, K772, K775, N776, I788, G792, K797, Y799, T804, N808, L811, R820, N831, R832, ​​V842, L847, N869, E874, N881, Q885, N888, T893, L911, Y945, D946, L949, E952, A1023, Y1036, G1067, G1077, R1078, N1093, R1114, N1115, D1117, A1121, D1125, P1128, K1129, V1146, S1154, S1159, L1164, S1172, N1177, P1178, I1179, D1180, K1211, M1213, G1218, N1234, E1243, K1244, E1253,In some embodiments, the RNA-guided nuclease Streptococcus pyogenes comprises a mutation corresponding to any one of positions E1260, K1263, H1264, E1271, Q1272, E1275, V1290, L1291, S1292, A1293, N1295, H1297, R1298, D1299, K1300, R1303, E1307, N1308, I1309, I1310, H1311, L1312, L1315, T1316, N1317, Y1326, D1328, V1342, A1345, I1360, S1363, or a combination thereof. The Cas9 domains are D10E, D10A, S29T, F32M, D39N, R40K, H41Q, S42T, I48L, C80R, S87A, K112D, H113N, K132N, K141E, D147E, L158V, E171Q, P176S, I186K, V189L, Q190H, Q194E, N199R, I201L, N202E, A203E, S204I, R205K, and A210. G, Q228A, L229F, G231N, S245A, T249M, S254A, D261N, T270S, N295K, T300I, D304G, V308A, N309D, I312V, T3 33A, A337V, E345K, F352S, Q354K, S355T, K356T, G366K, A367T, E396D, L398F, I414V, D428A, F429Y, D435E, K 468Q, S469R, E470N, T472A, E480D, A486T, S490L, F498V, K500E, N501H, N504T, K528R, V530I, E532D, G533E , A538E, T555A, K570Q, F575C, D605E, E611D, R629K, E634K, T638K, R655H, R664K, R671K, K705V, E706D, Q709 K, K710A, S714F, G7115E, G717K, H721K, H723Q, A725S, N726A, V743I, L747I, V748I, K772Q, K775R, N776R, I 788M, G792R, K797E, Y799H, T804A, N808D, L811R, R820K, N831D, R832H, V842I, L847I, N869D, E874A, N881S,Q885R, N888K, T893S, L911A, Y945H, D946G, L949P, E952A, A1023G, Y1036R, G1067E, G1077E, R1078K, N1093T, R1114G, N1115E, D1117A, A1121P, D1125G, P1128T, K1129T, V1146I, S1154T, S1159P, L1164V, S1172N, N1177D, P1178S, I1179V, D1180S, K1211R, M1213L, G1218T, N1234H, E1243D, K1244T, E1253K, The mutations include those selected from the group consisting of E1260D, K1263Q, H1264Y, E1271D, Q1272W, E1275H, V1290L, L1291R, S1292A, A1293T, N1295E, H1297N, R1298T, D1299H, K1300L, R1303S, E1307D, N1308S, I1309M, I1310L, H1311N, L1312A, L1315F, T1316S, N1317R, Y1326F, D1328N, V1342I, A1345S, I1360L, and S1363N, or a combination thereof.

[0045] In some embodiments, the RNA-guided nuclease Cas9 domain is a RNA-guided nuclease Neisseria meningitidis Cas9 domain. In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 712.Neisseria meningitidis Cas9ドメインは, I9 bit, D16 bit, D30 bit, E31 bit, A94 bit, I103 bit, P124 bit, N164 bit, I213 bit , G229 bit, T241 bit, S376 bit, E393 bit, G454 bit, K471 bit, G490 bit, D660 bit, C665 bit, K76 4-bit, T770-bit, P803-bit, A841-bit, H842-bit, K843-bit, D844-bit, L846-bit, R847-bit, K854-bit, H855-bit, N856-bit, K858-bit, K862-bit, W865-bit, E868-bit, I869-bit, A872-bit, D873-bit, N876 , Y880, G883, I886, E887, E890, R895, A898, Y899, G900, G901, N902, A903, K904, Q905, D908, N912, K917, G919, L921, V927, K929, T930, E932, S933, L936, L937, N938, K939, K940, Y943, T944, G949, D950, C958, K965, N966, Q967, F969, A975, E980, N981, I986, D987, C988, K989, G990, Y991, R992, I993, D994, Y997, T998, C1000, S1002, H1004, K1005, Y1006, A1010, F1011, Q1012, K1013, D1014, E1015, K1018, V1019, E1020, F1021, A1022, Y1024, I1025, N1026, C1027, D1028 , S1029, S1030, N1031, R1033, F1034, Y1035, L1036, A1037, W1038, K1041, G1042, K1044, E1045, Q1046, Q1047, F1048, R1 049 bit, I1050 bit, S1051 bit, T1052 bit, Q1053 bit, N1054 bit, L1055 bit, V1056 bit, L10 57 bits, I1058 bits, Y1061 bits, V1063 bits, and N1064 bits are all the same.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain is selected from the group consisting of I9M, D16E, D30E, E31K, A94D, I103V, P124C, N164D, I213N, G229D, T241A, S376T, E393K, G454C, K471E, G490C, D660E, C665R, K764E, T770A, P803S, A841Q, H842G, K843H, D844E, L846V, R847K, K854R, H855L, N856D, K858G, K862L, W865P, E868Q, I869L, A872K, D873G, N876K, Y880R, G883E, I886P, E887K, E890E, R895Q, A898T, Y899H, G900K, G901D, N902D, A903P, K904T, Q905K, D908A, N912E, K917Y, G919T, L921Q, V9 27I, K929Q, T930V, E932K, S933T, L936W, L937V, N938R, K939N, K940H, Y943N, T944G, G949A, D950T, C958E, K965G, N966G, Q967K, F969Y, A975 S, E980K, N981G, I986R, D987A, C988V, K989V, G990A, Y991F, R992K, I993D, D994E, Y997F, T998E, C1000R, S1002I, H1004Y, K1005A, Y1006N, A 1010K, F1011L, Q1012T, K1013A, D1014K, E1015K, K1018N, V1019E, E10 20F, F1021L, A1022G, Y1024F, I1025V, N1026S, C1027L, D1028N, S1029 The mutations include those selected from the group consisting of mutations corresponding to any one of R, S1030A, N1031T, R1033A, F1034I, Y1035D, L1036I, A1037R, W1038T, K1041T, G1042D, K1044T, E1045K, Q1046G, Q1047E, F1048Q, R1049S, I1050V, S1051G, T1052V, Q1053K, N1054T, L1055A, V1056L, L1057S, I1058F, Y1061N, V1063I, and N1064D, or a combination thereof.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:712.

[0046] Neisseria meningitidis Cas9ドメインは, I9 bit, D16 bit, D30 bit, E31 bit, A94 bit, I103 bit, P124 bit, N164 bit, I213 bit , G229 bit, T241 bit, S376 bit, E393 bit, G454 bit, K471 bit, G490 bit, D660 bit, C665 bit, K76 4-bit, T770-bit, P803-bit, A841-bit, H842-bit, K843-bit, D844-bit, L846-bit, R847-bit, K854-bit, H855-bit, N856-bit, K858-bit, K862-bit, W865-bit, E868-bit, I869-bit, A872-bit, D873-bit, N876 , Y880, G883, I886, E887, E890, R895, A898, Y899, G900, G901, N902, A903, K904, Q905, D908, N912, K917, G919, L921, V927, K929, T930, E932, S933, L936, L937, N938, K939, K940, Y943, T944, G949, D950, C958, K965, N966, Q967, F969, A975, E980, N981, I986, D987, C988, K989, G990, Y991, R992, I993, D994, Y997, T998, C1000, S1002, H1004, K1005, Y1006, A1010, F1011, Q1012, K1013, D1014, E1015, K1018, V1019, E1020, F1021, A1022, Y1024, I1025, N1026, C1027, D1028 , S1029, S1030, N1031, R1033, F1034, Y1035, L1036, A1037, W1038, K1041, G1042, K1044, E1045, Q1046, Q1047, F1048, R1 049 bit, I1050 bit, S1051 bit, T1052 bit, Q1053 bit, N1054 bit, L1055 bit, V1056 bit, L10 57 bits, I1058 bits, Y1061 bits, V1063 bits, and N1064 bits are all the same.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain is selected from the group consisting of I9M, D16E, D30E, E31K, A94D, I103V, P124C, N164D, I213N, G229D, T241A, S376T, E393K, G454C, K471E, G490C, D660E, C665R, K764E, T770A, P803S, A841Q, H842G, K843H, D844E, L846V, R847K, K854R, H855L, N856D, K858G, K862L, W865P, E868Q, I869L, A872K, D873G, N876K, Y880R, G883E, I886P, E887K, E890E, R895Q, A898T, Y899H, G900K, G901D, N902D, A903P, K904T, Q905K, D908A, N912E, K917Y, G919T, L921Q, V9 27I, K929Q, T930V, E932K, S933T, L936W, L937V, N938R, K939N, K940H, Y943N, T944G, G949A, D950T, C958E, K965G, N966G, Q967K, F969Y, A975 S, E980K, N981G, I986R, D987A, C988V, K989V, G990A, Y991F, R992K, I993D, D994E, Y997F, T998E, C1000R, S1002I, H1004Y, K1005A, Y1006N, A 1010K, F1011L, Q1012T, K1013A, D1014K, E1015K, K1018N, V1019E, E10 20F, F1021L, A1022G, Y1024F, I1025V, N1026S, C1027L, D1028N, S1029 The mutations include those selected from the group consisting of mutations corresponding to any one of R, S1030A, N1031T, R1033A, F1034I, Y1035D, L1036I, A1037R, W1038T, K1041T, G1042D, K1044T, E1045K, Q1046G, Q1047E, F1048Q, R1049S, I1050V, S1051G, T1052V, Q1053K, N1054T, L1055A, V1056L, L1057S, I1058F, Y1061N, V1063I, and N1064D, or a combination thereof.

[0047] In some embodiments, the RNA-guided nuclease Cas9 domain is the RNA-guided nuclease Campylobacter jejuni Cas9 domain. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 713. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain is at positions L5, A6, D8, I9, S12, S13, F18, S19, L24, K25, I31, T40, E42, L50, L58, A59, R61, L58, L65, H67, N74, K77, L98, I99, P101, N110, L113, A119, A126, R128, I134, K140, A144, K147, Q151, L156, V184, S190, F199, D202, G203, R212, F214, K221, E223, Y232, A235, V243, S247, D251, P256, L261, T269, N276, N277, L285, T287, L291, K300, T305, Q308, L312, G314, Y335, K336, I339, H345, D351, N353, E354, I362, K370, D383, S384, K391, I396, L403, T405, K413, N419, L421, D430, K432, A437, L453, K457, V462, A465, K472, N477, A492, E495, L525, K526, L527, K531, E532, E542, Q550, E556, H559, Y561, S564, M572, V577, Q581, N587, N596, K600, Q602, K603, Q616, K617, N623, Y624, K633, D634, Y642, N649, D656, L660, D662, K667, V677, E680, K682, L686, H692, T693, V712, I714, V722, K723, S736, L739, K742, L747,In some embodiments, the nucleic acid sequence includes a mutation corresponding to any one of positions N751, F756, R763, Q764, E772, K777, A786, E790, F792, Q800, S801, G804, L812, E813, V833, I835, T841, Y845, A855, L856, A863, V864, D879, E883, D900, Q902, K927, F928, V971, T972, or a combination thereof. The Cas9 domains are L5I, A6G, D8N, D8E, I9L, S12A, S13N, F18L, S19R, L24I, K25I, I31V, T40N, E42N, L50E, L58V, A59K, R61K, L58V, L65M, H67A, N74K, K77N, L98T, I99Q, P101I, N110S, L113I, A119S, A126V, R128H, I134S, K140N, A144T, and K14 7E, Q151K, L156M, V184I, S190D, F199L, D202Q, G203E, R212K, F214L, K221K, E223K, Y232F, A235P, V243I, S247I, D251N, P256A, L261S, T269G, N276K, N277S, L285V, T287E, L291I, K300D, T305S, Q308K, L312I, G314N, Y335L, K336 N, I339K, H345T, D351I, N353D, E354S, I362T, K370E, D383E, S384K, K391N, I396L, L403Q, T405I, K413R, N419E, L 421C, D430E, K432S, A437L, L453I, K457C, V462L, A465D, K472S, N477H, A492K, E495I, L525Q, K526I, L527V, K531E , E532D, E542L, Q550D, E556V, H559Y, Y561R, S564N, M572S, V577T, Q581L, N587G, N596E, K600L, Q602A, K603E, Q6 16R, K617F, N623F, Y624F, K633T, D634E, Y642W, N649S, D656S, L660I, D662E, K667A, V677Q, E680V, ​​K682S, L686I,H692N, T693F, V712I, I714V, V722I, K723F, S736K, L739F, K742N, L747S, N751L, F7 56L, R763K, Q764E, E772N, K777H, A786T, E790L, F792P, Q800N, S801T, G804D, L812V , E813K, V833S, I835L, T841K, Y845H, A855S, L856T, A863T, V864P, D879N, E883N, D900G, Q902K, K927N, F928Y, V971L, T972S, or a combination thereof. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 713.

[0048] In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain has amino acid residues at positions L5, A6, D8, I9, S12, S13, F18, S19, L24, K25, I31, T40, E42, L50, L58, A59, R61, L58, L65, H67, N74, K77, L98, I99, P101, N110, L113, A119, A126, R128, I134, K140, A144, K147, Q151, L156, V184, S190, F199, D202, G203, R212, F214, K221, E223, Y232, A235, V243, S247, D251, P256, L261, T269, N276, N277, L285, T287, L291, K300, T305, Q308, L312, G314, Y335, K336, I339, H345, D351, N353, E354, I362, K370, D383, S384, K391, I396, L403, T405, K413, N419, L421, D430, K432, A437, L453, K457, V462, A465, K472, N477, A492, E495, L525, K526, L527, K531, E532, E542, Q550, E556, H559, Y561, S564, M572, V577, Q581, N587, N596, K600, Q602, K603, Q616, K617, N623, Y624, K633, D634, Y642, N649, D656, L660, D662, K667, V677, E680, K682, L686, H692, T693, V712, I714, V722, K723, S736, L739, K742, L747, N751, F756, R763, Q764, E772, K777, A786, E790, F792, Q800, S801, G804, L812, E813, V833, I835, T841, Y845, A855, L856, A863, V864, D879, E883, D900.In some embodiments, the nucleic acid sequence of the present invention comprises a mutation corresponding to any one of positions Q902, K927, F928, V971, T972, or a combination thereof. The Cas9 domains are L5I, A6G, D8N, D8E, I9L, S12A, S13N, F18L, S19R, L24I, K25I, I31V, T40N, E42N, L50E, L58V, A59K, R61K, L58V, L65M, H67A, N74K, K77N, L98T, I99Q, P101I, N110S, L113I, A119S, A126V, R128H, I134S, K140N, A144T, K147E, Q151K, L156M, V184I, S190D, F199L, and D202. Q, G203E, R212K, F214L, K221K, E223K, Y232F, A235P, V243I, S247I, D251N, P256A, L261S, T269G, N276K, N277S, L285V, T287E, L291I, K3 00D, T305S, Q308K, L312I, G314N, Y335L, K336N, I339K, H345T, D351I, N353D, E354S, I362T, K370E, D383E, S384K, K391N, I396L, L403Q, T 405I, K413R, N419E, L421C, D430E, K432S, A437L, L453I, K457C, V462L, A465D, K472S, N477H, A492K, E495I, L525Q, K526I, L527V, K531E , E532D, E542L, Q550D, E556V, H559Y, Y561R, S564N, M572S, V577T, Q581L, N587G, N596E, K600L, Q602A, K603E, Q616R, K617F, N623F, Y62 4F, K633T, D634E, Y642W, N649S, D656S, L660I, D662E, K667A, V677Q, E680V, ​​K682S, L686I, H692N, T693F, V712I, I714V, V722I, K723F, S 736K, L739F, K742N, L747S, N751L, F756L, R763K, Q764E, E772N, K777H, A786T, E790L, F792P, Q800N, S801T, G804D, L812V, E813K, V833S,The mutations include those selected from the group consisting of I835L, T841K, Y845H, A855S, L856T, A863T, V864P, D879N, E883N, D900G, Q902K, K927N, F928Y, V971L, and T972S, or a combination thereof.

[0049] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 714. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 714. The Cas9 domain contains the following positions: D11, E85, A88, T92, E96, Y100, T109, D110, D113, E115, R116, D125, I127, K128, E132, S147, I185, A187, K228, Y229, T232, M255, S271, N273, A294, A327, E355, K357, N379 Place, T380th, S382nd, A385th, D439th, R440th, S464th, H469th, Y519th, I528th, N569th, I581st, A607th, K632nd, D633rd, H6 35th place, E636th place, A647th place, D648th place, T703rd place, P705th place, K712th place, S713th place, A724th place, V750th place, D882nd, S951st, D977th, E979th, S1014th, H1027th, I1030th, E1081st, D1082nd, D1086th, K1088th, S1089th, N1090th, R1092nd, T1093rd, I1094th, C1095th, A113 8th place, Y1139th, D1141st, T1142nd, F1158th, A1168th, E1190th, E1198th, H1202nd, I1204th, R1205th, I1210th, K1224th, S1 The mutations include any one of positions 232, M1240, V1241, I1242, P1243, G1424, K1248, Q1254, N1257, S1258, T1262, K1263, Y1264, D1266, A1270, K1277, D1284, L1288, V1302, N1316, T1346, and I1374, or a combination thereof.In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain is selected from the group consisting of D11E, D11A, E85D, A88T, T92A, E96D, Y100Q, T109D, D110N, D113N, E115D, R116S, D125E, I127D, K128A, E132K, S147T, I185L, A187T, K228N, Y229N, T232K, M255T, S271T, N273E, A294S, A327V, E355K, K357Q, N3 79G, T380I, S382T, A385N, D439E, R440E, S464A, H469R, Y519F, I528V, N569D, I581V, A607S, K632R, D633E, H6 35Q, E636Q, A647K, D648Q, T703A, P705S, K712E, S713A, A724T, V750I, D882G, S951R, D977E, E979K, S1014P, H1 027R, I1030V, E1081G, D1082E, D1086N, K1088R, S1089T, N1090D, R1092E, T1093K, I1094V, C1095R, A1138V, Y 1139L, D1141E, T1142P, F1158L, A1168T, E1190K, E1198K, H1202Q, I1204V, R1205Q, I1210M, K1224R, S1232T, M 1240I, V1241M, I1242L, P1243S, G1424A, K1248A, Q1254H, N1257G, S1258N, T1262A, K1263E, Y1264H, D1266K, A1270E, K1277E, D1284N, L1288V, V1302A, N1316D, T1346N, I1374L, or a combination thereof. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:714.

[0050] In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises a mutation corresponding to any one of positions D11, E85, A88, T92, E96, Y100, T109, D110, D113, E115, R116, D125, I127, K128, E132, S147, I185, A187, K228, Y229, T232, M255, S271, N273, A294, A327, E355, K357, N379, T380, S382, A385, D439, R440, S464, H469, Y519, I528, N569, I581, A607, K632, D633, H635, E636, A647, D648, T703, P705, K712, S713, A724, V750, D882, S951, D977, E979, S1014, H1027, I1030, E1081, D1082, D1086, K1088, S1089, N1090, R1092, T1093, I1094, C1095, A1138, Y1139, D1141, T1142, F1158, A1168, E1190, E1198, H1202, I1204, R1205, I1210, K1224, S1232, M1240, V1241, I1242, P1243, G1424, K1248, Q1254, N1257, S1258, T1262, K1263, Y1264, D1266, A1270, K1277, D1284, L1288, V1302, N1316, T1346, I1374 or a combination thereof.In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain is selected from the group consisting of D11E, D11A, E85D, A88T, T92A, E96D, Y100Q, T109D, D110N, D113N, E115D, R116S, D125E, I127D, K128A, E132K, S147T, I185L, A187T, K228N, Y229N, T232K, M255T, S271T, N273E, A294S, A327V, E355K, K357Q, N3 79G, T380I, S382T, A385N, D439E, R440E, S464A, H469R, Y519F, I528V, N569D, I581V, A607S, K632R, D633E, H6 35Q, E636Q, A647K, D648Q, T703A, P705S, K712E, S713A, A724T, V750I, D882G, S951R, D977E, E979K, S1014P, H1 027R, I1030V, E1081G, D1082E, D1086N, K1088R, S1089T, N1090D, R1092E, T1093K, I1094V, C1095R, A1138V, Y 1139L, D1141E, T1142P, F1158L, A1168T, E1190K, E1198K, H1202Q, I1204V, R1205Q, I1210M, K1224R, S1232T, M The mutations include those selected from the group consisting of 1240I, V1241M, I1242L, P1243S, G1424A, K1248A, Q1254H, N1257G, S1258N, T1262A, K1263E, Y1264H, D1266K, A1270E, K1277E, D1284N, L1288V, V1302A, N1316D, T1346N, and I1374L, or a combination thereof.

[0051] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Clostridium cellulolyticum Cas9 domain. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 715. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is T4, D10, V9, D20, K21, I27, C33, K36, A47, A49, S64, Q65, E102, L103, T1 22nd place, I124th place, K131st place, D137th place, R163rd place, G166th place, I169th place, F170th place, V183rd place, D184th place, I187th place, E193rd place, K200th place, K208th place, L209th, D221st, N224th, E227th, F228th, S234th, V242nd, K244th, L252nd, T256th, C258th, S261st, V413th, M415 Place, K416th, R417th, K424th, Y426th, K427th, S429th, D430th, A468th, T470th, A472nd, A478th, Q481st, K482nd, L4 85th place, A497th place, L535th place, W540th place, R541, E544th place, G554th place, P556th place, I570th place, Y574th place, M580th place, Y584th place, M585th place, T592nd place, D 593rd place, V606th place, W607th place, I647th place, N650th place, S693rd place, L697th place, E702nd place, S704th place, A713th place, V714th place, I715th place, D776th place, L847th place , G850, G853, A854, R860, I900, H904, M905, I906, E921, Q923, S929, T930, H931, Q939, N994, I997, N1000, K1001, S1002, I1003, K1005, P1008, or a combination thereof.In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is selected from the group consisting of T4S, D10E, V9I, D20N, K21E, I27E, C33I, K36V, A47S, A49P, S64R, Q65H, E102L, L103V, T122V, I124F, K131Q, D137E, R163Q, G166S, I169L, F170L, V183G, D184G, I187T, E193S, K200Q, K208A, L 209Y, D221K, N224Q, E227S, F228S, S234T, V242I, K244N, L252K, T256K, C258T, S261F, V413K, M415L , K416R, R417N, K424Q, Y426I, K427P, S429H, D430Q, A468S, T470S, A472V, A478G, Q481K, K482R, L485 S, A497M, L535H, W540Y, R541K, E544Q, G554F, P556S, I570V, Y574I, M580F, Y584N, M585N, T592A, D5 93A, V606W, W607F, I647R, N650H, S693K, L697F, E702Q, S704N, A713V, V714I, I715V, D776E, L847A, G In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:715.

[0052] In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises a mutation corresponding to any one of positions T4, D10, V9, D20, K21, I27, C33, K36, A47, A49, S64, Q65, E102, L103, T122, I124, K131, D137, R163, G166, I169, F170, V183, D184, I187, E193, K200, K208, L209, D221, N224, E227, F228, S234, V242, K244, L252, T256, C258, S261, V413, M415, K416, R417, K424, Y426, K427, S429, D430, A468, T470, A472, A478, Q481, K482, L485, A497, L535, W540, R541, E544, G554, P556, I570, Y574, M580, Y584, M585, T592, D593, V606, W607, I647, N650, S693, L697, E702, S704, A713, V714, I715, D776, L847, G850, G853, A854, R860, I900, H904, M905, I906, E921, Q923, S929, T930, H931, Q939, N994, I997, N1000, K1001, S1002, I1003, K1005, P1008 or a combination thereof.In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is selected from the group consisting of T4S, D10E, V9I, D20N, K21E, I27E, C33I, K36V, A47S, A49P, S64R, Q65H, E102L, L103V, T122V, I124F, K131Q, D137E, R163Q, G166S, I169L, F170L, V183G, D184G, I187T, E193S, K200Q, K208A, L2 09Y, D221K, N224Q, E227S, F228S, S234T, V242I, K244N, L252K, T256K, C258T, S261F, V413K, M415L, K 416R, R417N, K424Q, Y426I, K427P, S429H, D430Q, A468S, T470S, A472V, A478G, Q481K, K482R, L485S, A 497M, L535H, W540Y, R541K, E544Q, G554F, P556S, I570V, Y574I, M580F, Y584N, M585N, T592A, D593A, V606W, W607F, I647R, N650H, S693K, L697F, E702Q, S704N, A713V, V714I, I715V, D776E, L847A, G850P, The mutations include those selected from the group consisting of G853A, A854P, R860K, I900V, H904D, M905V, I906L, E921Y, Q923E, S929D, T930E, H931Y, Q939P, N994Q, I997P, N1000R, K1001M, S1002N, I1003K, K1005H, and P1008K, or a combination thereof.

[0053] In some embodiments, the RNA-guided nuclease Cas9 domain is a RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO:716.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions K2, D8, I14, D35, K41, F74, V75, K91, I117, R128, T136, Q151, S152, S156, A161, V164, S171, E178, D179, V185, R192, K195, A199, Y204, I207, V208, A212, H215, S219, F227, T260, V261, V271, G274, I276, A278, L279, D282, I287, K289, H293, F299, V302, N307, R313, L317, L318, V331, G337, K341, S348, A354, A355, K356, R359, M372, T377, R380, E395, D399, E404, S416, T441, R445, N464, E504, S508, M515, Q516, E520, G521, V534, L545, K559, T578, K603, T612, L619, S621, N656, N660, L673, D685, I699, N708, N717, R737, V738, S752, D756, Q771, N777, N792, E793, I811, I824, K839, Q845, K848, T849, L895, I902, T908, V929, I943, I946, M948, F990, T995, V1000, Q1014, D1017, S1019, N1020, G1021, S1024, N1030, N1031, R1035, S1036, I1037, V1067, S1071, A1075, I1079 or a combination thereof.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain is selected from the group consisting of K2R, D8E, D8A, I14V, D35E, K41Q, F74V, V75I, K91E, I117V, R128K, T136S, Q151R, S152A, S156G, A161G, V164I, S171A, E178G, D179E, V185I, R192H, K195R, A199S, Y204F, I207M, V208S, A212K, H215N, S219T, F227V, T260I, V261A, V271I , G274S, I276A, A278G, L279P, D282E, I287L, K289E, H293Q, F299Y, V302I, N307R, R313Y, L317I, L318V, V331I, G337D, K34 1Q, S348K, A354K, A355S, K356S, R359L, M372L, T377A, R380H, E395P, D399N, E404N, S416T, T441S, R445K, N464T, E504D, S5 08T, M515T, Q516K, E520D, G521E, V534M, L545H, K559R, T578V, K603R, T612I, L619V, S621T, N656M, N660S, L673F, D685E, I699V, N708E, N717D, ​​R737K, V738I, S752A, D756E, Q771R, N777H, N792D, E793Q, I811V, I824V, K839T, Q845K, K848A, T849S , L895P, I902V, T908K, V929V, I943V, I946M, M948I, F990L, T995I, V1000G, Q1014K, D1017H, S1019G, N1020T, G1021A, S1024E, N1030C, N1031S, R1035S, S1036G, I1037V, V1067L, S1071A, A1075T, I1079V, or a combination thereof. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:716.

[0054] In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions K2, D8, I14, D35, K41, F74, V75, K91, I117, R128, T136, Q151, S152, S156, A161, V164, S171, E178, D179, V185, R192, K195, A199, Y204, I207, V208, A212, H215, S219, F227, T260, V261, V271, G274, I276, A278, L279, D282, I287, K289, H293, F299, V302, N307, R313, L317, L318, V331, G337, K341, S348, A354, A355, K356, R359, M372, T377, R380, E395, D399, E404, S416, T441, R445, N464, E504, S508, M515, Q516, E520, G521, V534, L545, K559, T578, K603, T612, L619, S621, N656, N660, L673, D685, I699, N708, N717, R737, V738, S752, D756, Q771, N777, N792, E793, I811, I824, K839, Q845, K848, T849, L895, I902, T908, V929, I943, I946, M948, F990, T995, V1000, Q1014, D1017, S1019, N1020, G1021, S1024, N1030, N1031, R1035, S1036, I1037, V1067, S1071, A1075, I1079 or a combination thereof.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain is selected from the group consisting of K2R, D8E, D8A, I14V, D35E, K41Q, F74V, V75I, K91E, I117V, R128K, T136S, Q151R, S152A, S156G, A161G, V164I, S171A, E178G, D179E, V185I, R192H, K195R, A199S, Y204F, I207M, V208S, A212K, H215N, S219T, F227V, T260I, V261A, V271I , G274S, I276A, A278G, L279P, D282E, I287L, K289E, H293Q, F299Y, V302I, N307R, R313Y, L317I, L318V, V331I, G337D, K34 1Q, S348K, A354K, A355S, K356S, R359L, M372L, T377A, R380H, E395P, D399N, E404N, S416T, T441S, R445K, N464T, E504D, S5 08T, M515T, Q516K, E520D, G521E, V534M, L545H, K559R, T578V, K603R, T612I, L619V, S621T, N656M, N660S, L673F, D685E, I699V, N708E, N717D, ​​R737K, V738I, S752A, D756E, Q771R, N777H, N792D, E793Q, I811V, I824V, K839T, Q845K, K848A, T849S , L895P, I902V, T908K, V929V, I943V, I946M, M948I, F990L, T995I, V1000G, Q1014K, D1017H, S1019G, N1020T, G1021A, S1024E, N1030C, N1031S, R1035S, S1036G, I1037V, V1067L, S1071A, A1075T, I1079V, or a combination thereof.

[0055] In some embodiments, the RNA-guided nuclease Cas domain is an RNA-guided nuclease Cas12 domain. In some embodiments, the RNA-guided nuclease Cas domain is an RNA-guided nuclease CasX domain. In some embodiments, the I-TEVI nuclease domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 700. In some embodiments, the I-TEVI nuclease domain comprises a mutation at any one of positions corresponding to T11, V16, N14, E25, K26, R27, E36, K37, G38, C39, S41, L45, F49, I60, and E81, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation selected from any one corresponding to T11V, V16I, N14G, E25D, K26R, R27A, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, E81I, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation corresponding to the K26R mutation. In some embodiments, the I-TEVI nuclease domain comprises the amino acid sequence set forth in SEQ ID NO: 700. In some embodiments, the I-TEVI nuclease domain comprises a mutation corresponding to any one of positions T11, V16, N14, E25, K26, R27, E36, K37, G38, C39, S41, L45, F49, I60, and E81, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation selected from mutations corresponding to any one of positions T11V, V16I, N14G, E25D, K26R, R27A, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, and E81I, or a combination thereof. In some embodiments, the I-TEVI nuclease domain comprises a mutation corresponding to the K26R mutation. In some embodiments, the chimeric nuclease further comprises a nuclear localization signal. In some embodiments, the nuclear localization signal comprises an SV40 nuclear localization signal. In some embodiments, the nuclear localization signal comprises a nucleoplasmin nuclear localization signal. In some embodiments, the composition further comprises a donor nucleic acid.In some embodiments, the donor nucleic acid restores non-oncogenic function of a gene containing an oncogenic mutation. In some embodiments, the donor nucleic acid comprises a non-oncogenic version of the oncogenic mutation. In some embodiments, the donor nucleic acid is DNA. In some embodiments, the donor nucleic acid comprises a blunt end and a 3' overhang of at least two nucleotides. In some embodiments, the donor nucleic acid comprises 5' and 3' homology adjacent to the non-oncogenic version of the oncogenic mutation. In some embodiments, the composition does not comprise a donor nucleic acid. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier. In some embodiments, the composition is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic or neutral lipid.

[0056]

[23] In another aspect, the present disclosure provides a nucleic acid or multiple nucleic acids encoding the chimeric nuclease or guide RNA of the present disclosure. In some embodiments, the chimeric nuclease or guide RNA is operably linked to a eukaryotic promoter, enhancer, polyadenylation site, or a combination thereof. In some embodiments, the nucleic acid is an expression vector selected from a plasmid, a lentiviral vector, an adeno-associated viral vector, or an adenoviral vector. In some embodiments, the nucleic acid or multiple nucleic acids further comprise a donor nucleic acid portion.

[0057]

[24] In another aspect, the present disclosure provides a method of targeting an oncogenic mutation in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0058]

[25] In another aspect, the present disclosure provides a use of a composition of the present disclosure in a cell to target an oncogenic mutation in the cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0059]

[0010] In another aspect, the present disclosure provides a method of editing a genome in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual suffering from cancer.

[0060]

[0010] In another aspect, the present disclosure provides a use of a composition of the present disclosure for editing the genome in a cell. In some embodiments, the cell is a cell of an individual suffering from cancer.

[28] In another aspect, the present disclosure provides a method of deleting at least some oncogenic mutations in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0061]

[29] In another aspect, the present disclosure provides a use of a composition of the present disclosure for deleting at least some oncogenic mutations in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0062]

[0010] In another aspect, the present disclosure provides a method of silencing or disrupting at least some oncogenic mutations in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0063]

[31] In another aspect, the present disclosure provides a use of a composition of the present disclosure for silencing or disrupting at least a portion of oncogenic mutations in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0064]

[0010] In another aspect, the present disclosure provides a method of replacing at least a portion of oncogenic mutations in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual suffering from cancer.

[0065]

[33] In another aspect, the present disclosure provides a use of a composition of the present disclosure for replacing at least a portion of an oncogenic mutation in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0066]

[34] In another aspect, the present disclosure provides a method of restoring a non-oncogenic function in a cell, comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[0067]

[35] In another aspect, the present disclosure provides a use of a composition of the present disclosure for restoring a non-oncogenic function in a cell. In some embodiments, the cell is a cell of an individual afflicted with cancer.

[36] In another aspect, the present disclosure provides a method of treating cancer in an individual, comprising administering a composition of the present disclosure to an individual having cancer, thereby treating the cancer in the individual.

[0068]

[37] In another aspect, the present disclosure provides a use of a composition of the present disclosure for treating cancer in an individual.

[38] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. Only illustrative embodiments of the present disclosure have been shown and described herein. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0069] Reference

[39] All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the incorporated publications and patents or patent applications conflict with the disclosure contained herein, it is intended that the present specification supersede and / or supersede any such conflicting material.

[0070]

[40] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and / or advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth specific embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0071] [Figure 1A]

[41] Figure 1A is a schematic representation of the I-TevI ​​domain (1), linker domain (2), and Cas domain (3) within the chimeric nuclease structure. [Figure 1B]

[42] Figure 1B is a flowchart illustrating the steps performed by existing gene editors to create a single break that is repaired by the error-prone non-homologous end joining (NHEJ) pathway, which can generate random indels. [Figure 1C]

[43] Figure 1C is a flowchart illustrating the steps involved in how a chimeric nuclease uses its I-TevI ​​and Cas domains to cleave DNA at two sites, generating a deletion of defined length in cells. The deletion length can be predicted based on the distance between the I-TevI ​​and Cas domains. [Figure 2]

[44] Figure 2A shows the algorithm workflow in which a dataset containing pairs of wild-type and mutant target sites is generated from a database of known disease-causing mutations.

[45] Figure 2B contains sample output data from a wild-type Muc4 gene.

[46] Figure 2C contains sample output data from a mutant Muc4 gene. Bolded columns are found in both the wild-type and mutant copies of the Muc4 gene sequence. The remaining cells in Figure 2C constitute the putative allele-specific TevSaCas9 targets in the Muc4 gene. [Figure 3]

[47] Figure 3A is a diagram showing the target site of TevSaCas9 in an oncogene (bold line) across a large insertion (light gray).

[48] Figure 3B is an agarose gel electrophoresis result showing the cleavage products of TevSaCas9 from an in vitro cleavage assay of wild-type (WT) and mutant (MUT) copies of the Muc4 gene. As shown, TevSaCas9 targeted by the guide RNA of SEQ ID NO: 1685 successfully cleaved the MUT substrate, but not WT Muc4. [Figure 4]

[49] Figure 4A specifically illustrates the EGFR L858R target site. The location of the mutation is indicated by an asterisk ("*").

[50] Figure 4B shows agarose gel electrophoresis results showing the cleavage products of TevSaCas9 from an in vitro cleavage assay of wild-type (WT) and mutant (MUT) copies of the EGFR gene. As shown, TevSaCas9 targeted by the guide RNA of SEQ ID NO: 1686 preferentially cleaved the MUT EGFR L858R substrate over the WT EGFR substrate.

[51] Figure 4C shows the results of a viability assay in which the CLR-5908 cell line containing mutant EGFR L858R and the NuLi-1 cell line containing wild-type EGFR were treated with the TevSaCas9 ribonucleoprotein complex targeting EGFR L858R. As shown, TevSaCas9 targeting EGFR L858R reduced the viability of the CRL-5908 cell line but not the NuLi-1 cell line, demonstrating the allele-specific activity of TevSaCas9 in cells. [Figure 5]

[52] Figure 5 shows a diagram of the mechanism by which TevCas is complexed with multiple guide RNAs and electroporated into cells to disrupt the gene encoding an oncogene and insert a sequence encoding an altered version of the oncogene, thereby restoring wild-type function. DETAILED DESCRIPTION OF THE INVENTION

[0072]

[53] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives may be employed to the embodiments of the invention described herein. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Any reference herein to "or" is intended to encompass "and / or" unless stated otherwise.

[0073]

[54] Unless otherwise defined, all terms of technical, notational, and other scientific terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains. In some cases, for clarity and / or ready reference, terms having commonly understood meanings are defined herein. That is, the inclusion of such definitions herein should not be construed as representing definitions that are substantially different from definitions generally understood in the art.

[0074]

[55] Within the framework of this specification and the claims that follow, unless otherwise indicated, all numbers expressing amounts, quantities, percentages, etc., are to be understood to be preceded in all instances by the term "about." As used herein, the term "about" is defined as ±5%. Also, all ranges of numerical entities include, in addition to the numerical values ​​specifically set forth hereinafter, all possible combinations of maximum and minimum numerical values, and all possible intermediate ranges therebetween.

[0075]

[56] The term "and / or" as used herein is defined as the possibility of having one or the other or both. For example, "A and / or B" provides for the scenario of having only A, or only B, or a combination of A and B. If a claim reads A and / or B and / or C, the composition may contain as components only A, only B, only C, A and B but not C, B and C but not A, A and C but not B, or all three of A, B, and C.

[0076]

[57] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. These definitions should be read in light of the present disclosure and should be understood by one of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0077]

[58] The articles "a" and "an" are used herein to mean one or more than one (e.g., at least one) of the grammatical object of the article. The term "and / or," as used herein, defines the possibility of having one or the other or both. For example, "A and / or B" provides for scenarios having only A, or only B, or a combination of A and B. If a claim reads A and / or B and / or C, the composition could contain as components only A, only B, or only C, A and B but not C, B and C but not A, A and C but not B, or all three of A, B, and C.

[0078]

[59] The term "donor DNA," as used herein, means DNA that differs, in whole or in part, from the original target DNA sequence and can be introduced into an oncogene to restore wild-type or non-oncogenic function to the oncogene.

[0079]

[60] The term "flexible linker," as used herein, refers to an amino acid linker domain that ensures the flexibility of the I-TevI ​​domain when the RNA-guided Cas nuclease domain is bound to the target DNA sequence, allowing it to remain flexible under physiological conditions of the cell (typically pH about 7.2, temperature about 37°C, [K + ] Approximately 140mM, [Na + ] Approximately 5~15mM, [Cl - ]about 4mM, [Ca ++ [0001 mM] (approximately 0.0001 mM) allows recognition, binding, and cleavage of its target sequence. The length of the amino acid linker can influence how many nucleotides are preferred between the Cas target site and the I-TevI ​​target site. Certain amino acids in the linker may also make specific contacts with the DNA sequence targeted by TevCas. These linker-DNA contacts may affect the flexibility of the I-TevI ​​domain. Amino acid substitutions in the linker domain may affect the linker domain's ability to contact DNA.

[0080]

[61] The term "including" as used herein is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0081]

[62] The terms "patient," "individual," "subject," or "host" treated by the subject method can refer to a human or a non-human animal. Non-human animals include companion animals (e.g., cats, dogs) and animals raised for consumption (i.e., food animals), such as cows, pigs, and chickens.

[0082]

[63] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any subject composition or its components from one organ or body part to another. Each carrier is "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as dextrose, lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as microcrystalline cellulose, sodium carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), and cellulose acetate; (4) glycols, such as propylene glycol; (5) polyols, such as glycerin, sodium carbonate; Examples of suitable excipients include: (1) sorbitol, mannitol, and polyethylene glycol; (2) esters, such as ethyl oleate, glyceryl behenate, and ethyl laurate; (3) buffers, such as monobasic and dibasic phosphates, Tris / borate / EDTA, and Tris / acetate / EDTA; (4) pyrogen-free water; (5) isotonic saline; (6) Ringer's solution; (7) ethyl alcohol; (8) phosphate buffer; (9) polysorbates; (10) polyphosphates; and (11) other non-toxic, compatible substances employed in pharmaceutical formulations. The disclosed excipients may serve more than one function. For example, a solubilizer may also be a suspension aid, emulsifier, preservative, etc.

[0083]

[64] In certain preferred embodiments, the pharmaceutically acceptable excipient is a crystalline bulking excipient. As used herein, the term "crystalline bulking excipient" or "crystalline bulking agent" refers to an excipient that provides bulk and structure to the lyophilized cake. These crystalline bulking agents are inert and do not react with proteins or nucleic acids. Furthermore, the crystalline bulking agents are capable of crystallizing under the conditions of lyophilization. Examples of suitable crystalline bulking agents include hydrophilic excipients, such as water-soluble polymers, sugars, such as mannitol, sorbitol, xylitol, glucitol, ducitol, inosithiol, arabinitol, arabitol, galactitol, iditol, allitol, maltitol, fructose, sorbose, glucose, xylose, trehalose, allose, dextrose, altrose, lactose, glucose, fructose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose, lactulose, Examples of suitable crystalline bulking agents include fucose, rhamnose, melezitose, maltotriose, raffinose, altritol, their optically active forms (D- or L-forms), and the corresponding racemates; inorganic salts of minerals and organic minerals, such as calcium salts, e.g., lactate, gluconate, glyceryl phosphate, citrate, monobasic or dibasic phosphates, succinate, sulfate, and tartrate, as well as aluminum and magnesium salts; carbohydrates, such as conventional monosaccharides and disaccharides, and the corresponding polyhydric alcohols; proteins, e.g., albumin; amino acids, e.g., glycine; emulsifiable fats; and polyvinylpyrrolidone. Crystalline bulking agents may be selected from any one or combination of glycine, mannitol, dextran, dextrose, lactose, sucrose, polyvinylpyrrolidone, trehalose, and glucose. Particularly useful bulking agents include dextran.

[0084]

[65] The term "pharmaceutically acceptable salts," as used herein, refers to art-recognized, relatively non-toxic inorganic and organic acid addition salts or inorganic or organic base addition salts of compounds, including, for example, those contained in the compositions of the present invention. Some examples of pharmaceutically acceptable salts include: (1) calcium chloride, (2) sodium chloride, (3) sodium citrate, (4) sodium hydroxide, (5) sodium phosphate, (6) sodium ethylenediaminetetraacetate, (7) potassium chloride, (8) potassium phosphate, and (9) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0085]

[66] The term "substitution," as used herein, refers to the replacement of an amino acid in a sequence with a different amino acid. As used herein, the shorthand expression X10Y indicates "substituting" amino acid Y for amino acid X at the 10th position of a sequence. As an example, W26C indicates that amino acid tryptophan 26 (Trp, W) has been changed to cysteine ​​(Cys). Similarly, the notation AA X indicates that AA is the amino acid that replaces the amino acid at position X. For example, Lys 26 represents a substitution of lysine at amino acid position 26 in the sequence. The use of either shorthand notation is interchangeable. Additionally, the use of one-letter or three-letter abbreviations for amino acids is also interchangeable.

[0086]

[67] The term "therapeutic agent," as used herein, means any chemical or biochemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as "drugs," are described in well-known references, such as the Merck Index, the Physician's Desk Reference, and The Pharmacological Basis of Therapeutics, and include, but are not limited to, medicines, vitamins, mineral supplements, substances used to treat, prevent, diagnose, cure, or mitigate a disease or illness, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment.

[0087]

[68] The term "hybridization," as used herein, generally refers to and / or includes the ability and / or capacity of a first nucleic acid molecule to non-covalently bind (e.g., form Watson-Crick base pairs and / or G / U base pairs), anneal, and / or hybridize to a second nucleic acid molecule under appropriate or certain in vitro and / or in vivo conditions of temperature, pH, and / or solution ionic strength. Generally, standard Watson-Crick base pairing includes adenine (A) and thymidine (T), adenine (A) and uracil (U), and guanine (G) and cytosine (C). In some embodiments, hybridization involves at least two nucleic acids comprising complementary sequences (e.g., fully complementary, substantially complementary, or partially complementary). In certain embodiments, hybridization involves at least two nucleic acids comprising fully complementary sequences. In certain embodiments, hybridization involves at least two nucleic acids that comprise substantially complementary sequences (e.g., greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% complementarity). In certain embodiments, hybridization involves at least two nucleic acids that comprise partially complementary sequences (e.g., greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 70% complementarity). In certain embodiments, partially complementary sequences comprise one or more regions of fully or substantially complementary sequences. In certain embodiments, partially complementary sequences comprise one or more regions of fully or substantially complementary sequences, even if the overall complementarity is low (e.g., less than about 50%, less than about 40%, less than about 30%, or less than about 20% total complementarity). Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. For example, the greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of hybrids of nucleic acids having those sequences. For hybridization between nucleic acids having short stretches of complementarity (e.g., complementarity over 35 or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides), the position of mismatches becomes important (see Sambrook et al., supra, pp. 11.7-11.8).

[0088]

[69] The terms "complementary" or "complementarity," as used herein, generally refer to a polynucleotide comprising a nucleotide sequence that can selectively anneal to an identified region of a target polynucleotide under certain conditions. As used herein, the term "substantially complementary" and grammatical equivalents are intended to refer to a polynucleotide comprising a nucleotide sequence that can specifically anneal to an identified region of a target polynucleotide under certain conditions. Annealing refers to the nucleotide base-pairing interaction between one nucleic acid and another, resulting in the formation of a duplex, triplex, or other higher-order structure. Primary interactions are typically nucleotide base-specific through Watson-Crick and Hoogsteen-type hydrogen bonding, e.g., A:T, A:U, and G:C. In certain embodiments, base stacking and hydrophobic interactions may also contribute to duplex stability. Conditions under which a polynucleotide anneals to a complementary or substantially complementary region of a target nucleic acid are well known in the art and are described, for example, in Nucleic Acid Hybridization, A Practical Approach, edited by Hames and Higgins, IRL Press, Washington, DC (1985), and Wetmur and Davidson, Mol. Biol. 31:349 (1968). Annealing conditions depend on the particular application and can be routinely determined by one skilled in the art without undue experimentation. Hybridization generally refers to the process by which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide.

[0089]

[70] Temperature and solution salt concentration are generally recognized as factors that facilitate hybridization and may be adjusted as necessary depending on factors such as the length of the complementary region and the degree of complementarity. Hybridization and washing conditions are well known and are exemplified in Sambrook, I., Fritsch, E:F., and Maniatis, T., Molecular Cloning: A Laboratory Manual—Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1; and Sambrook, J., and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the stringency of hybridization. In some embodiments, hybridization is measured at physiological temperature (e.g., 37°C) and salt concentration (e.g., 0.15 molar or 0.9% salt in solution).

[0090]

[71] The term "treat," as used herein, includes any effect, such as lowering, reducing, modulating, or eliminating, that results in the improvement of a condition, disease, disorder, etc. As used herein, "treat" can include both prophylactic and therapeutic treatment.

[0091]

[72] As used herein, a "buffer" refers to any combination of acids or salts that are pharmaceutically acceptable and capable of maintaining the compositions of the present invention within a desired pH range. The buffers in the disclosed compositions maintain a pH within a range of about 2 to about 8.5, about 5.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 7.5, or about 6.5. Suitable buffers include any pharmaceutically acceptable buffers capable of maintaining the above pH ranges, such as acetate, tartrate phosphate, or citrate buffers. In one embodiment, the buffer is a phosphate buffer. In another embodiment, the buffer is an acetate buffer. In one embodiment, the buffer is disodium hydrogen phosphate, sodium chloride, potassium chloride, and monobasic potassium phosphate.

[0092]

[73] In the disclosed compositions, the concentration of the buffering agent is typically within the range of about 0.1 mM to about 1000 mM, about 0.2 mM to about 200 mM, about 0.5 mM to about 50 mM, about 1 mM to about 10 mM, or about 6.0 mM.

[0093]

[74] As used herein, a stabilizer is a composition that maintains the chemical and biological stability of a chimeric nuclease. Examples of stabilizers include polyols, including sugars, preferably monosaccharides or disaccharides such as glucose, trehalose, raffinose, or sucrose, sugar alcohols such as mannitol, sorbitol, or inositol, polyhydric alcohols such as glycerin or propylene glycol, or mixtures thereof, and albumin.

[0094]

[75] Pharmaceutically acceptable salts are salts suitable for administration to a subject, e.g., a human. The chimeric nucleases of the present invention may have one or more protons that are sufficiently acidic to react with a suitable organic or inorganic base to form a base addition salt. Base addition salts include salts derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc., and organic bases, such as alkoxides, alkylamides, alkyl and arylamines, etc. Thus, such bases useful in preparing the salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc. The chimeric nucleases of the present invention having a sufficiently basic group, e.g., an amine, may react with an organic or inorganic acid to form an acid addition salt. Acids commonly employed to form acid addition salts from compounds having a basic group include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1, These include 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and others.

[0095]

[76] As used herein, "cell" may generally refer to a biological cell. A cell may be the basic structural, functional, and / or biological unit of a living organism. A cell may originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants, animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), and others. A cell may not be derived from a natural organism (e.g., a cell may be synthetically produced, sometimes referred to as an artificial cell). In certain embodiments, a cell refers to a human cell.

[0096]

[77] The terms "protein" and "polypeptide" can be used interchangeably to refer to a polymer of two or more amino acids linked by covalent bonds (e.g., amide bonds) and capable of adopting a three-dimensional conformation. In some embodiments, a protein or polypeptide comprises at least 10, 15, 20, 30, or 50 amino acids linked by covalent bonds (e.g., amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, a zymogen protein, a regulatory protein, a structural protein, a receptor, a nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody. In some embodiments, a protein can be a full-length protein (e.g., a fully processed protein that has a certain biological function). In some embodiments, a protein can be a variant or fragment of a full-length protein. For example, in some embodiments, a Cas9 protein domain comprises an H840A amino acid substitution compared to the naturally occurring S. pyogenes Cas9 protein. A variant of a protein or enzyme, e.g., a variant reverse transcriptase, includes a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of the reference protein.

[0097]

[78] In some embodiments, a protein comprises a functional variant or functional fragment of a full-length wild-type protein. "Functional fragment" or "functional portion," as used herein, refers to any portion of a reference protein (e.g., a wild-type protein) that encompasses less than the entire amino acid sequence of the reference protein while retaining one or more functions, e.g., catalytic or binding functions. For example, a functional fragment of a Cas or I-TevI ​​protein may encompass less than the entire amino acid sequence of a wild-type Cas or I-TevI ​​protein but retain the ability to catalyze the cleavage of a polynucleotide sequence. Where the reference protein is a fusion of multiple functional domains, the functional fragment may retain one or more functions of at least one of the functional domains. For example, a functional fragment of Cas may encompass less than the entire amino acid sequence of wild-type Cas but retain its DNA-binding ability and partially or completely lack its nuclease activity. In certain embodiments, a functional fragment comprises one or more deletions from the N-terminus or C-terminus of a protein, polypeptide, or domain described herein. In certain embodiments, a functional fragment comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 amino acids from the N-terminus or C-terminus of a protein, polypeptide, or domain described herein.

[0098]

[79] "Functional variant" or "functional mutant," as used herein, refers to any variant or mutant of a reference protein (e.g., a wild-type protein) that contains one or more changes to the amino acid sequence of the reference protein while retaining one or more functions, such as catalytic or binding functions. In some embodiments, the one or more changes to the amino acid sequence include amino acid substitutions, insertions, or deletions, or any combination thereof. In some embodiments, the one or more changes to the amino acid sequence include amino acid substitutions. For example, a functional variant of a Cas or I-TevI ​​protein may contain one or more amino acid substitutions compared to the amino acid sequence of a wild-type Cas or I-TevI ​​protein, but retain the ability to catalyze the cleavage of a polynucleotide sequence. In cases where the reference protein is a fusion of multiple functional domains, the functional variant may retain one or more functions of at least one of the functional domains. For example, in some embodiments, a functional fragment of Cas9 may contain one or more amino acid substitutions in the nuclease domain compared to the amino acid sequence of wild-type Cas9, e.g., the H840A amino acid substitution, but retain DNA binding activity and partially or completely lack nuclease activity.

[0099]

[80] As used herein, the terms "homologous," "homology," or "% homology" refer to the degree of sequence identity between an amino acid and a corresponding reference amino acid sequence, or between a polynucleotide sequence and a corresponding reference polynucleotide sequence. "Homology" can refer to similar polymeric sequences, such as polypeptide or DNA sequences. Homology can refer to, for example, nucleic acid sequences having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In other embodiments, a "homologous sequence" of a nucleic acid sequence can exhibit 93%, 95%, or 98% sequence identity to a reference nucleic acid sequence. For example, a "region of homology to a genomic region" can be a region of DNA having a similar sequence to a given genomic region in a genome. The region of homology can be of any length sufficient to facilitate binding of a spacer, primer binding site, or protospacer sequence to the genomic region. For example, the region of homology can be at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 350, 400, 450, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3500, 4000, 4500, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 950 ... It may comprise 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases in length.

[0100]

[81] The terms "identity" or "homology," as used interchangeably herein, may refer to the calculation of "identity," "homology," or "% homology" between two or more nucleotide or amino acid sequences, which can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence). Nucleotides at corresponding positions can then be compared, and the % identity between the two sequences can be a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100). For example, a position in a first sequence can be occupied by the same nucleotide as the corresponding position in a second sequence, such that the molecules are identical at that position. The % homology between two sequences can be a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. In some embodiments, the length of sequences aligned for comparison purposes may be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. Homology between two sequences may be determined by a BLAST® search. The two sequences may be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished using well-known methods, such as mathematical algorithms. Non-limiting examples of such mathematical algorithms are described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such an algorithm may be incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, any of the associated parameters of the respective programs (e.g., NBLAST) can be used.For example, parameters for sequence comparison may be set at score=100, word length=12, or may be varied (e.g., W=5 or W=20). Other examples include the Myers and Miller algorithm, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In another embodiment, the percent identity between two amino acid sequences can be achieved using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0101]

[82] When specifying the percentage of sequence homology or identity in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of the two or more sequences over a portion of their length when compared and aligned for maximum correspondence. If a position in the compared sequences can be occupied by the same base or amino acid, the molecules can be homologous at that position. Unless otherwise stated, sequence homology or identity is assessed over a specified length of the nucleic acid, polypeptide, or portion thereof. In some embodiments, homology or identity is assessed over a functional or specified portion of that length.

[0102]

[83] Alignment of sequences to assess sequence homology can be performed using algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm described in Altschul et al., J. Mol. Biol. 215:403-410, 1990. A publicly available internet interface for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Further known algorithms include those published by Smith & Waterman, "Comparison of Biosequences," Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, "A general method applicable to the search for similarities in the amino acid sequence of two proteins," J. Mol. Biol. 48:443, 1970; Pearson & Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci. USA 85:2444, 1988, or by automated implementations of these or similar algorithms. Global alignment programs can also be used to align similar sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss_needle / ), which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000;16:276-277), and the GGSEARCH program (https: / / fasta.bioch.virginia.edu / fasta_www2 / ), which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85:2444-2448).Both of these programs are based on the Needleman-Wunsch algorithm, which is used to find the optimal alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

[0103]

[84] One of skill in the art will understand that amino acid (or nucleotide) positions can be determined in homologous sequences based on alignment, e.g., "H840" in a reference Cas9 sequence may correspond to H839 or another position in a Cas9 homolog.

[0104]

[85] The term "polynucleotide" or "nucleic acid molecule" refers to any polymeric form of nucleotide, including DNA, RNA, hybrids thereof, or RNA-DNA chimeric molecules. In some embodiments, the polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA. In some embodiments, the polynucleotide is double-stranded, e.g., double-stranded DNA in a gene. In some embodiments, the polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or mRNA. In some embodiments, the polynucleotide is a cell-free nucleic acid molecule. In some embodiments, the polynucleotide circulates in the blood. In some embodiments, the polynucleotide is a cellular nucleic acid molecule. In some embodiments, the polynucleotide is a cellular nucleic acid molecule within a cell circulating in the blood.

[0105]

[86] A polynucleotide can have any three-dimensional structure. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), exon, intron, intergenic DNA (including, but not limited to, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA, isolated RNA, sgRNA, guide RNA, nucleic acid probe, primer, snRNA, long non-coding RNA, snoRNA, siRNA, miRNA, tRNA-guided small RNA (tsRNA), antisense RNA, shRNA, or small rDNA-guided RNA (srRNA).

[0106]

[87] In some embodiments, a polynucleotide comprises deoxyribonucleotides, ribonucleotides, or their analogs. In some embodiments, a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0107]

[88] In some embodiments, a polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and, if the polynucleotide is RNA, uracil (U) in place of thymine. In some embodiments, a polynucleotide may also contain one or more other nucleotide bases, such as inosine (I), which is read as guanine (G) by the translation machinery.

[0108]

[89] In some embodiments, a polynucleotide can be modified. As used herein, the term "modified" or "modification" refers to a chemical modification of A, C, G, T, and U nucleotides. In some embodiments, the modification can be in the nucleoside base and / or sugar moiety of the nucleosides that make up the polynucleotide. In some embodiments, the modification can be in the internucleoside linkage (e.g., the phosphate backbone). In some embodiments, the modified nucleic acid molecule includes multiple modifications. In some embodiments, the modified nucleic acid molecule includes a single modification.

[0109]

[90] As used herein, the term "mutation" refers to an alteration and / or change in the amino acid sequence of a protein or the nucleic acid sequence of a polynucleotide. Such alteration and / or change may include a substitution, insertion, deletion, and / or truncation of one or more amino acids, in the case of an amino acid sequence, and / or nucleotides, in the case of a nucleic acid sequence, compared to a reference amino acid or reference nucleic acid sequence. In some embodiments, the reference sequence is a wild-type sequence. In some embodiments, a mutation in the nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide. In some embodiments, a mutation in the amino acid sequence of a polypeptide or a mutation in the nucleic acid sequence of a polynucleotide is a mutation associated with a disease state.

[0110]

[91] The terms "subject" or "individual" may be used interchangeably. As used herein, grammatical equivalents may refer to a human or a non-human. An individual may be a mammal. A human individual may be male or female. A human individual may be of any age. An individual may be a human embryo. A human individual may be a newborn, infant, child, adolescent, or adult. A human individual may be in need of treatment for a genetic disease or disorder. In some embodiments, the individual has, is susceptible to, or is at risk for advanced cancer.

[0111]

[92] The term "oncogene" refers to a gene that, upon mutation, disruption, or overexpression, can cause uncontrolled cell division, leading to the formation of a tumor or cancer. An oncogene before mutation is known as a proto-oncogene. An "oncogenic mutation" is any mutation that results in the conversion of a proto-oncogene to an oncogene. An oncogene can have a non-oncogenic function that is restored by editing the gene's function back to a wild-type or non-pathogenic state. Such editing can restore the wild-type nucleotide or amino acid sequence, or a sequence (amino acid or nucleotide) that differs from the wild-type but restores the wild-type or non-pathogenic function.

[0112]

[93] While specific embodiments of the present disclosure have been discussed, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification and such variations.

[0113]

[94] The term "chimeric nuclease" refers to a fusion protein comprising an I-TevI ​​nuclease domain and a Cas nuclease domain. In some cases, the I-TevI ​​nuclease domain and the Cas nuclease domain are operably linked. The target gene of a chimeric nuclease may comprise a double-stranded DNA molecule having two complementary strands, a first strand referred to as the "coding strand" and a second strand referred to as the "non-coding strand." In some embodiments, the chimeric nuclease uses a guide RNA sequence that is complementary or substantially complementary to a specific sequence on the target strand. In some cases, the guide RNA hybridizes or substantially hybridizes to a specific sequence on the target strand. In some embodiments, the guide RNA sequence anneals to the target strand at the interrogation target sequence.

[0114]

[95] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0115]

[96] This disclosure describes chimeric nucleases and methods for targeting oncogenes with chimeric nucleases. For example, if a chimeric nuclease cleaves at two sites, it excises a precise length of DNA (e.g., approximately 30-38 bases, depending on the sites targeted by the I-TevI ​​and Cas domains). That is, target sites can be selected within a gene to generate a precise length deletion that knocks the gene out of frame in a cell. In one embodiment of the described nuclease, the nuclease targets and cleaves a target oncogene.

[0116]

[97] The present disclosure describes methods of using the described nucleases to engineer cell populations to target, contact, edit, silence, disrupt, restore, insert, modify, delete, or replace nucleotide sequences at genomic locations. The present disclosure describes forming a complex of a chimeric nuclease and a guide RNA and administering the chimeric nuclease and guide RNA complex to cells. This administration may be performed using one or more methods of electroporation or lipid-mediated transfection (e.g., cationic lipids). Alternatively, a nucleic acid or nucleic acids encoding the guide RNA and / or chimeric nuclease can be introduced into cells using a method selected from electroporation, viral transduction, and / or lipid-mediated transfection. In embodiments in which genomic sequences are added to modify the genome, donor DNA may be administered to affect the insertion or modification. The donor DNA may suitably be provided as linearized DNA, plasmid DNA, or a viral vector.

[0117]

[98] The method described herein targets oncogenes in cells or individuals using chimeric nucleases containing an I-TevI ​​nuclease domain and a Cas nuclease domain. As shown in Figure 5A, purified chimeric nuclease (1) can be mixed and complexed with multiple guide RNAs (2) to form a ribonucleoprotein complex (3). Donor DNA (4), encoding exogenous donor DNA containing ends complementary to one or more of the sites targeted by the chimeric nuclease, can also be mixed with the ribonucleoprotein complex (3). As shown in Figure 5B, a population of cells (5) encoding an oncogene expressed from genomic DNA (7) can be exposed to the mixture of ribonucleoprotein complex (3) and donor DNA (4). An electric pulse (8) can be applied to the mixture to permeabilize the cell membrane (9) (Figure 5C). As shown in Figure 5D, the ribonucleoprotein complex (3) and donor DNA (4) can enter the cells through the permeabilized cell membrane. The ribonucleoprotein complex (3) can be targeted to the nucleus (10) through one or more nuclear localization sequences ("NLSs"). As shown in Figure 5E, the ribonucleoprotein complex (3) can bind to its target site on genomic DNA (7). As shown in Figure 5F, the ribonucleoprotein complex (3) can cleave the genomic DNA (7) to create a deletion (12) of a defined length, or, if a matching donor DNA (13) is present, insert donor DNA (13) into the cleaved site. The deleted region of genomic DNA (7) disrupts the gene encoding the oncogene (6), and the donor DNA (13) encodes a modified version of the oncogene, thereby restoring wild-type function.

[0118]

[99] The present disclosure further relates to a method for targeted gene disruption (e.g., insertion, editing, deletion, modification, or replacement) of all or a portion of a DNA sequence in the genome of a human cell to knock out the gene out of frame, comprising: (a) exposing the cells to a nuclease ex vivo; and (b) applying a current of 1000-2500 V to the cell population to permeabilize the membrane and allow passage of the claimed nuclease into the cells. Other ranges of current applied may be 1000-1500 V, 1501-1700 V, 1701-1900 V, 1901-2100 V, or 2101-2500 V. The nuclease may be delivered to the cells using lipofection or polymer-based transfection, or using a viral vector such as an adeno-associated virus or lentivirus. Nucleases may also be delivered as ribonucleoprotein complexes, DNA encoding the nuclease, or messenger RNA encoding the nuclease. In eukaryotic cells, the chimeric nucleases of the present disclosure can be targeted to the nucleus of the cell through one or more nuclear localization sequences ("NLS"). For applications involving the generation of oncogene knockouts in cells, a mixture of nucleases can be applied to target one or more oncogenes in a cell population. Specific guide RNAs that target the chimeric nuclease to a precise genomic location may be included along with the nuclease or messenger RNA encoded by the nucleic acid. For applications involving the replacement, repair, or insertion of DNA into a genomic location, the donor nucleic acid may be included as an isolated and purified nucleic acid, via a linear double-stranded nucleic acid, via a plasmid or viral vector. The donor nucleic acid may be delivered together with the nuclease and guide RNA, or separately in a separate formulation, or may be delivered by a different method compared to the delivery of the nuclease and guide RNA. In the presence of the donor nucleic acid, the cell can insert the donor nucleic acid sequence (in whole or in part) between the two cleaved sites in the target genomic DNA by directed ligation by non-homologous end joining.

[0119]

[0100] The present disclosure relates to chimeric nucleases comprising various combinations of I-TevI ​​domains and Cas domains. In some embodiments, the chimeric nuclease further comprises a linker domain. In some embodiments, the chimeric nuclease further comprises a guide RNA.

[0120]

[0101] A chimeric nuclease targeting an oncogene may comprise (a) an I-TevI ​​domain and a Cas domain, and (b) a guide RNA. In some embodiments, the guide RNA comprises an RNA sequence that hybridizes to or is sufficiently complementary to at least a portion of a sequence selected from any one of SEQ ID NOs: 1-683, or a combination thereof, or comprises a nucleotide sequence set forth in SEQ ID NOs: 1001-1686, or a combination thereof. In some embodiments, a chimeric nuclease targeting KRAS comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising a nucleic acid sequence that hybridizes to a target nucleic acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 37, 42, 51, 52, 62, 63, or 77. In some embodiments, a chimeric nuclease targeting PI3KCA comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising a nucleic acid sequence that hybridizes to a target nucleic acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5, 6, 7, 8, 33, 202, 204, 209, or 210. In some embodiments, a chimeric nuclease targeting MUC-4 comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising a nucleic acid sequence that hybridizes to a target nucleic acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 676, 677, 678, 679, or 682. In some embodiments, the chimeric nuclease targeting EGFR comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising a nucleic acid sequence that hybridizes to a target nucleic acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 45, 130, 141, or 683.

[0121]

[0102] The chimeric nuclease targeting an oncogene comprises (a) an I-TevI ​​domain and a Cas domain, and (b) a guide RNA. In some embodiments, the guide RNA comprises an RNA sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to or identical to a sequence selected from any one of SEQ ID NOs: 1001-1686, or a combination thereof. In some embodiments, the chimeric nuclease targeting KRAS comprises (a) the chimeric nuclease described above, and (b) a guide RNA that comprises an RNA sequence that is at least about 90%, 95%, 97%, 98%, or 99% identical to or identical to a sequence selected from any one of SEQ ID NOs: 1037, 1042, 1051, 1052, 1062, 1063, and 1077, or a combination thereof. In some embodiments, a chimeric nuclease that targets PI3KCA comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising an RNA sequence that has at least about 90%, 95%, 97%, 98%, or 99% identity to or is identical to a sequence selected from any one of SEQ ID NOs: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, 1210, or a combination thereof. In some embodiments, a chimeric nuclease that targets MUC-4 comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising an RNA sequence that has at least about 90%, 95%, 97%, 98%, or 99% identity to or is identical to a sequence selected from any one of SEQ ID NOs: 1676, 1677, 1678, 1679, 1682, or a combination thereof. The chimeric nuclease that targets EGFR comprises (a) the chimeric nuclease described above, and (b) a guide RNA comprising an RNA sequence that has at least about 90%, 95%, 97%, 98%, or 99% identity to or is identical to a sequence selected from any one of SEQ ID NOs: 1683, 1684, or a combination thereof.

[0122] In some embodiments, chimeric nucleases are used to simultaneously edit multiple genes to generate multiple oncogene knockouts in a cell population. For example, multiple chimeric nucleases can be used to target different oncogenes in an individual. In some embodiments, at least one chimeric nuclease can be used to target oncogenic mutations. In some embodiments, at least two chimeric nucleases can be used to target oncogenic mutations. In some embodiments, at least three chimeric nucleases can be used to target oncogenic mutations. In some embodiments, at least four chimeric nucleases can be used to target oncogenic mutations. In some embodiments, at least five chimeric nucleases can be used to target oncogenic mutations. In particular, the composition is directed to a mixture of chimeric nucleases discussed above in the preceding paragraph combined with a mixture of guide RNAs according to SEQ ID NOs: 1001-1686 in an equimolar ratio to the chimeric nucleases. In another embodiment, the composition relates to a mixture of chimeric nucleases as discussed above in the preceding paragraph combined with a mixture of guide RNAs according to SEQ ID NOs: 1001-1686 in an equimolar ratio with the chimeric nuclease.

[0123] In some embodiments, other chimeric nuclease compositions include various combinations of I-TevI ​​domains and RNA-guided nuclease domains. In particular, the compositions relate to chimeric nucleases of SEQ ID NOs: 730-736, 740-755, or 756, wherein the chimeric nuclease comprises a wild-type I-TevI ​​domain or a variant thereof, and a wild-type Cas domain or a variant thereof.

[0124]

[0105] The chimeric nucleases described herein can be formed from two different nucleases, and are useful for the ex vivo gene editing applications and in vivo applications described herein.

[0125] Chimeric nuclease The chimeric nucleases of the present disclosure may comprise different combinations of I-TevI ​​domains and Cas domains. In some embodiments, the Cas domain may be a Cas9 domain. In some embodiments, the Cas9 domain is derived from Staphylococcus aureus, Streptococcus pyogenes, Neisseria meningitidis, Campylobacter jejuni, Streptococcus pasteurianus, Clostridium cellulolyticum, or Geobacillus thermodenitrificans T1.

[0126] In some embodiments, the chimeric nuclease further comprises a linker domain. In some embodiments, the chimeric nuclease further comprises a guide RNA, wherein the guide RNA targets the oncogenic mutation. In some embodiments, the chimeric nuclease can be used to target the oncogenic mutation. In some embodiments, the oncogenic mutation is a single polynucleotide polymorphism or SNP. In some embodiments, the oncogenic mutation is an insertion of one or more nucleotides. In some embodiments, the oncogenic mutation is a substitution or deletion of 10 or fewer nucleotides. In some embodiments, the oncogenic mutation comprises a deletion of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide. In some embodiments, the oncogenic mutation is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 1-628, or a combination thereof. In some embodiments, the oncogenic mutation is ABL1, AFF4 / MLLT11, AKT2, ALK, ALK / NPM, RUNX1 (AML1), RUNX1 / MTG8 (ETO), AXL, BCL-2, BCL-3, BCL-6, BCR / ABL, MYC (c-MYC), MCF2 (DBL), DEK / NUP214, TCF3 / PBX1, EGFR, MLLT11, ERG / FUS, ERBB2, ETS1, EWSR1 / FLI1, CSF1R, FOS, FES, GLI1, GNAS (GSP), HER2 / neu, TLX1, FGF4, IL3, FGF3 (INT-2), JUN, KIT, FGF4 (KS3), K-SAM, AKAP1 3, occurs in at least one of LCK, LMO1, LMO2, MYCL, LYL1, NFKB2, NFKB2 / Cα1, MAS1, MDM2, MLLT11, MOS, MUC4, RUNX1T1, MYB, MYH11 / CBFB, NEU, MYCN, MCF2L(OST), PAX-5, PBX1 / E2A, PIM1, PIK3CA, CCND1, RAF1, RARA / PML, HRAS, KRAS, NRAS, REL / NRG, RET, RHOM1, RHOM2, ROS1, SKI, SIS (also known as PDGFB), SET / CAN, SRC, TAL1, TAL2, NOTCH1(TAN1), TIAM1, TSC2, or NTRK1.In some embodiments, the oncogenic mutation occurs in at least one of Muc4, PIK3CA, EGFR, or KRAS.

[0127] In some embodiments, the oncogenic mutation occurs in EGFR (e.g., UniProt accession number P00533). In some embodiments, the oncogenic mutation in EGFR is not a deletion in exon 19 of EGFR. In some embodiments, the oncogenic mutation in EGFR is P3L, S4A, G5A, T6A, A7P, A7D, L858R, V769_D770insASV, G8R, A10G, A13V, A16S, L18F, P20L, P20Q, A21S, A21T, A24T, K29T, T34M, T39M, D46E, R53K, E59K, E6K, Q18R, Q71R, T28A, T81 In some embodiments, the oncogenic mutation comprises at least one mutation corresponding to any one of: A, Q83E, Q30E, E31Q, E84Q, E84D, E31D, A33V, A86V, L37F, L90F, N41S, N94S, V43M, V96M, R98Q, R45Q, Q52H, Q105H, R108G, R55G, R108K, R55K, G109A, G56A, M111T, M58T, or a combination thereof. In some embodiments, the oncogenic mutation comprises a mutation in EGFR corresponding to L858R. In some embodiments, the oncogenic mutation in EGFR is a mutation corresponding to V769_D770insASV.

[0128] In some embodiments, the oncogenic mutation occurs in Muc4 (e.g., UniProt accession number Q99102). In some embodiments, the Muc4 mutation is an in-frame deletion of exon 2 or an in-frame deletion of exon 3. In some embodiments, the Muc4 mutation is a mutation corresponding to any one of positions P1542, P1680, T1711, V1721, P1826, A1830, S3560, A1833, D2253, V2281, P3088, T3119, T3183, V3817, A3902, or any combination thereof. In some embodiments, the Muc4 mutation is selected from mutations corresponding to P1542L, P1680S, T1711I, V1721A, P1826H, A1830T, S3560S, A1833V, D2253H, V2281AM, P3088L, T3119T, T3183M, V3817A, A3902V, or a combination thereof.

[0129] In some embodiments, the oncogenic mutation occurs in KRAS (e.g., UniProt accession number P01116). In some embodiments, the KRAS mutation comprises a mutation corresponding to any one of positions A59, D119, D33, G21, G12, G13, Q61, A146, K117, or any combination thereof. In some embodiments, the KRAS mutation is a mutation corresponding to any one of A59T, A59E, A59T, D119N, D33E, G21C, G12C, G12D, G12V, G12R, G12A, G12S, G13D, G13C, G13V, G13R, Q61R, Q61V, Q61L, Q61K, Q61H, Q61A, Q61P, Q61E, A146T, A146V, K117N, K117R, or a combination thereof.

[0130] In some embodiments, the oncogenic mutation occurs in PIK3CA (e.g., UniProt Accession No. P42336). In some embodiments, the PIK3CA mutation is a mutation corresponding to positions H1047, E542, E545, N345, C1636, G1624, G1633, A3140, C3075, A1634, A1173, or a combination thereof. In some embodiments, the PIK3CA mutation is a mutation corresponding to any one of positions H1047R, H1047L, E542K, E545K, N345K, C1636A, G1624A, G1633A, A3140T, A3140G, C3075T, A1634C, A1173G, or a combination thereof.

[0131] This disclosure describes chimeric nucleases and methods for using them to target oncogenic mutations. While cleavage by current single-cutting endonucleases can leave compatible DNA ends at the target site (Figure 1B), cleavage by chimeric nucleases can leave a blunt end at the Cas site and a 3' overhang at the I-TevI ​​site (Figure 1C). When a chimeric nuclease cleaves at two sites, it excises a precise length of DNA (e.g., about 30-40 bases, depending on the sites targeted by I-TevI ​​and SaCas9). The methods described herein can generate precise deletions of at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at most about 40 nucleotides from the genome. The chimeric nucleases described herein can generate precise deletions of at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or at most about 40 nucleotides from a genome. That is, target sites can be selected within a gene to generate deletions of precise lengths that knock an oncogene out of frame in a cell or organism. In some embodiments, the chimeric nucleases of the present disclosure require four elements within the target site to bind and cleave: 1) an I-TevI ​​site, 2) a spacer sequence of about 14-19 nucleotides, 3) a protospacer sequence of about 19-21 nucleotides, and 4) a protospacer adjacent motif (PAM), where N is any nucleotide, R is an A or G nucleotide, and K is a T or G nucleotide. Exemplary I-TevI ​​motifs and PAMs are shown in Tables 1 and 2, respectively. Given the requirement for a unique target site for the chimeric nuclease, a target site can be chosen where the oncogenic mutation alters one of these elements to preferentially or selectively target that mutation. In one embodiment, the described chimeric nuclease generates a precise length deletion in the oncogene, but not in the wild-type gene.

[0132]

[0113] Table 1 lists various I-TevI ​​variants. Different mutations to I-TevI ​​can alter the specificity of the binding site and change the consensus sequence.

[0133] Oncogenic mutations Oncogenic mutations other than those described can also be allele-specifically targeted by chimeric nucleases. Other allele-specific oncogenic mutations can be targeted as a result of alterations in the binding site of the I-TevI ​​domain, alterations in the spacer DNA sequence between the I-TevI ​​site and the Cas site, alterations in the Cas protospacer sequence, or alterations in the Cas protospacer adjacent motif sequence.

[0134] Epidermal growth factor- EGFR In some embodiments, the oncogenic mutation occurs in EGFR. In some embodiments, the oncogenic mutation in EGFR is not a deletion in exon 19 of EGFR. In some embodiments, the oncogenic mutation in EGFR is a mutation corresponding to L858R. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 45, 130, or 141, or comprises a nucleotide sequence set forth in SEQ ID NO: 1045, 1130, 1141, or 1686. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 45, 130, or 141, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1045, 1130, 1141, or 1686. In some embodiments, the oncogenic mutation in EGFR is a mutation corresponding to V769_D770insASV. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 683, or comprises a nucleotide sequence set forth in SEQ ID NO: 1683 or 1684. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 683, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1683 or 1684.

[0135] Mucin 4 - Muc4 Mucin 4 (Muc4) is a mucin protein encoded by the MUC4 gene in humans. Like other mucins, MUC-4 is a high molecular weight glycoprotein. MUC-4 is membrane-anchored and belongs to the human mucin family, which can range in molecular weight from 550 to 930 kDa for the actual protein to 4,650 kDa if glycosylated. MUC4 may also be referred to as ASGP, HSA276359, MUC-4, or mucin 4.

[0136] MUC4 is an O-glycoprotein that can extend up to 2 μm outside the cell. MUC4 mucin consists of a large, highly glycosylated extracellular α subunit and a β subunit that is anchored to the cell membrane and extends into the cytosol. The β subunit is considered an oncogene, and its role in cancer is increasingly recognized, particularly due to its involvement in signal transduction pathways, particularly in conjunction with ErbB2 (Her2).

[0137] The two subunits of MUC4 are transcribed from a single gene consisting of 25 exons with an exon / intron structure identical to that of the mouse gene. More than 24 splice variants of MUC4 have been found using commercially available mRNA or total RNA extracted from cancer cell lines.

[0138] MUC-4 is thought to play a role in cancer progression by inhibiting apoptosis and consequently increasing tumor cell proliferation. The molecular mechanism is thought to be through a complex between MUC-4 and the ERBB2 receptor, which alters downstream signaling and downregulates CDKN1B. The β subunit of MUC-4 appears to act as a ligand that triggers ErbB2 phosphorylation but does not activate the MAPK or AKT pathways. MUC-4 also influences HER2 signaling, potentially resulting in its stabilization. As a mucin, MUC-4 also alters the adhesive properties of cells. When overexpressed, mucin disorganization can reduce adhesion to other cells and the extracellular matrix, promoting cancer cell migration and metastasis.

[0139] The chimeric nucleases of the present disclosure can target oncogenic mutations, such as Muc-4. In some embodiments, the oncogenic mutation occurs in Muc4. In some embodiments, the Muc4 mutation is an in-frame deletion of exon 2 or an in-frame deletion of exon 3. In some embodiments, the Muc4 mutation is a mutation corresponding to any one of positions P1542, P1680, T1711, V1721, P1826, A1830, S3560, A1833, D2253, V2281, P3088, T3119, T3183, V3817, A3902, or any combination thereof. In some embodiments, the Muc4 mutation is P1542L, P1680S, T1711I, V1721A, P1826H, A1830T, S3560S, A1833V, D2253H, V2281AM, P3088L, T3119T, T3183M, V3817A, A3902V, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 676, 677, 678, 679, or 682, or comprises a nucleotide sequence set forth in SEQ ID NO: 1676, 1677, 1678, 1679, 1682, or 1685. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 676, 677, 678, 679, or 682, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1676, 1677, 1678, 1679, 1682, or 1685.

[0140] K-Ras - KRAS K-Ras is part of the RAS / MAPK pathway. KRAS is also known as KRAS, CK-RAS, CFC2, K-RAS2A, K-RAS2B, K-RAS4A, K-RAS4B, KI-RAS, KRAS1, KRAS2, NS, NS3, RALD, RASK2, K-ras, KRAS proto-oncogene, GTPase, c-Ki-ras2, OES, c-Ki-ras, K-Ras 2, CK-RAS, K-Ras, Kirsten rat sarcoma virus, or Kirsten rat sarcoma virus. This protein transmits signals from outside the cell to the cell's nucleus. These signals direct the cell to grow and divide (proliferation) or to mature and take on specialized functions (differentiation). It was first identified as a viral oncogene in the Kirsten rat sarcoma virus, hence the name KRAS. Because the oncogenes identified were derived from the cellular genome, KRAS is referred to as a proto-oncogene when found within the cellular genome.

[0141] KRAS acts as a molecular on / off switch. Once allosterically activated, it recruits and activates proteins required for growth factor propagation, as well as other cell signaling receptors such as c-Raf and PI3-kinase. KRAS upregulates the GLUT1 glucose transporter, thereby contributing to the Warburg effect in cancer cells. KRAS binds to GTP in its active state. It also has the intrinsic enzymatic activity of cleaving the terminal phosphate of nucleotides and converting them to GDP. Upon conversion of GTP to GDP, KRAS is inactivated. The rate of conversion is usually slow but can be dramatically increased by accessory proteins of the GTPase-activating protein (GAP) class, such as RasGAP. Subsequently, KRAS can bind to proteins of the guanine nucleotide exchange factor (GEF) class (e.g., SOS1), which forces the release of the bound nucleotide (GDP). KRAS then binds to GTP present in the cytosol, releasing the GEF from ras-GTP.

[0142] In some embodiments, the oncogenic mutation occurs in KRAS. In some embodiments, the KRAS mutation comprises a mutation corresponding to any one of positions A59, D119, D33, G21, G12, G13, Q61, A146, K117, or any combination thereof. In some embodiments, the KRAS mutation is a mutation corresponding to any one of A59T, A59E, A59T, D119N, D33E, G21C, G12C, G12D, G12V, G12R, G12A, G12S, G13D, G13C, G13V, G13R, Q61R, Q61V, Q61L, Q61K, Q61H, Q61A, Q61P, Q61E, A146T, A146V, K117N, K117R, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 37, 42, 51, 52, 62, 63, or 77, or comprises a nucleotide sequence set forth in SEQ ID NO: 1042, 1051, 1052, 1062, 1063, or 1077. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 37, 42, 51, 52, 62, 63, or 77, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1037, 1042, 1051, 1052, 1062, 1063, or 1077.

[0143] Phosphatidylinositol 3-kinase catalytic subunit - PIK3CA Phosphatidylinositol 3-kinase catalytic subunit (PIK3CA) is one of the most commonly mutated genes in breast cancer and has been found to be important in several cancer types. PIK3CA, a key part of the PI3K pathway, has long been described as an oncogene and has two major hotspots for activating mutations: the 542 / 545 region of the helical domain and the 1047 region of the kinase domain.

[0144] Phosphatidylinositol-4,5-bisphosphate 3-kinase (also called phosphatidylinositol 3-kinase (PI3K)) is composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit (PIK3CA). The protein encoded by this gene represents the catalytic subunit, which uses ATP to phosphorylate phosphatidylinositols (PtdIns), namely PtdIns4P and PtdIns(4,5)P2. PIK3CA may also be referred to as CLOVE, CWS5, MCAP, MCM, MCMTC, PI3K, p110-α, PI3K-α, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α, CLAPO, or CCM4.

[0145] In some embodiments, the oncogenic mutation occurs in PIK3CA. In some embodiments, the PIK3CA mutation corresponds to any one of positions H1047, E542, E545, N345, C1636, G1624, G1633, A3140, C3075, A1634, and A1173, or a combination thereof. In some embodiments, the PIK3CA mutation corresponds to any one of positions H1047R, H1047L, E542K, E545K, N345K, C1636A, G1624A, G1633A, A3140T, A3140G, C3075T, A1634C, and A1173G, or a combination thereof. In some embodiments, the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 5, 6, 7, 8, 33, 202, 204, 209, or 210, or comprises a nucleotide sequence set forth in SEQ ID NO: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, or 1210. In some embodiments, the guide RNA comprises a nucleic acid sequence that hybridizes to a target nucleic acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5, 6, 7, 8, 33, 202, 204, 209, or 210, or comprises a nucleotide sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1005, 1006, 1007, 1008, 1033, 1202, 1204, 1209, or 1210.

[0146] I-TevI ​​nuclease

[0127] A chimeric nuclease of the present disclosure can comprise an I-TevI ​​nuclease domain. The unmodified full-length I-TevI ​​protein comprises a sequence according to SEQ ID NO: 702. MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRN (SEQ ID NO: 702)

[0147] The sequence provided by SEQ ID NO:700, 702, or 704 is the wild-type version of I-TevI, except for the insertion of a glycine at position 2, which increases protein stability and prevents N-terminal degradation. For specific substitutions referred to herein, the numbering corresponds to the wild-type version of the protein lacking glycine stabilization. That is, in the stabilized version of I-TevI, the lysine at position 27 of SEQ ID NO:700, 702, or 704 is designated K26, which corresponds to the wild-type position without glycine at position 2. There are several I-TevI ​​substitutions in the I-TevI ​​domain that are known to have little effect on the nuclease activity of I-TevI. The nuclease activity of I-TevI ​​can be assessed by mixing a chimeric nuclease containing the I-TevI ​​domain with linear DNA containing a known I-TevI ​​target and running the products of the cleavage reaction on an agarose gel. If the I-TevI ​​nuclease is active, a product of the predicted size will be present.

[0148]

[0129] The chimeric nuclease of the present disclosure can have an I-TevI ​​nuclease domain. In some embodiments, the I-TevI ​​nuclease domain is derived from Enterobacteria phage T4. The I-TevI ​​domain can include the 93 amino acid I-TevI ​​domain of Enterobacteria phage T4 according to the following sequence: MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIA (SEQ ID NO: 700)

[0149] In some embodiments, an exemplary I-TevI ​​nuclease domain is shown in SEQ ID NO: 700. In some embodiments, the mutation can correspond to any one of SEQ ID NO: 700. In some embodiments, the I-TevI ​​nuclease domain may comprise at least one mutation compared to SEQ ID NO: 700, 702, or 704. In some embodiments, the I-TevI ​​nuclease domain comprises a mutation corresponding to any one of positions selected from any one of T11, V16, N14, E25, K26, R27, E36, K37, G38, C39, S41, L45, F49, I60, E81, or a combination thereof. In some embodiments, the I-TevI ​​nuclease domain comprises a mutation corresponding to any one of positions selected from any one of T11V, V16I, N14G, E25D, K26R, R27A, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, E81I, or a combination thereof. In some embodiments, the I-TevI ​​nuclease domain comprises at least a K26R mutation compared to the wild-type sequence. In some embodiments, the I-TevI ​​nuclease domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 700, 702, or 704.

[0150] Other versions of the I-TevI ​​nuclease domain may contain various combinations of mutations that alter the site targeted by the I-TevI ​​domain or the activity of the I-TevI ​​domain, for example, mutations that alter the sequence recognized by I-TevI, such as K26 and / or C39. Other versions of the nuclease may replace the I-TevI ​​domain with other GIY-YIG nuclease domains, such as I-BmoI, Eco29kI, etc. Some versions of I-TevI ​​do not contain Met1 as a result of processing when expressed in E. coli.

[0151] The unmodified full-length I-TevI ​​nuclease comprises a nuclease domain comprising positions 1 to 93 and a linker domain comprising positions 94 to 169. The positions of the mutations correspond to positions in the unmodified full-length I-TevI ​​nuclease or to SEQ ID NO: 700, 702, or 704.

[0152] Table 1 summarizes a list of exemplary wild-type I-TevI ​​and its variants. Additionally, the table notes the different cleavage motifs used by the different I-TevI ​​proteins.

[0153] [Table 1-1]

[0154] [Table 1-2]

[0155] Linker Domain

[0135] The chimeric nuclease of the present disclosure may further comprise a linker domain. The linker may comprise a flexible amino acid linker comprising at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. The linker may comprise a flexible amino acid linker comprising no more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids. In some embodiments, the linker domain may be unstructured and may comprise a Gly-Ser linker. Longer linkers can generally relax the spacing requirement of I-TevI ​​of 14-19 base pairs at the target site, while shorter linkers generally restrict this. Useful linkers include, but are not limited to, (GS) n (SEQ ID NO: 900), (GSGGS) n (SEQ ID NO: 901), (GGGGS) n (SEQ ID NO: 902), and (GGGS) n(SEQ ID NO: 903), where n is an integer of at least 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Exemplary linkers for linking antibody fragments or single-chain variable fragments may include AAEPKSS (SEQ ID NO: 904), AAEPKSSDKTHTCPPCP (SEQ ID NO: 904), GGGG (SEQ ID NO: 905), or GGGGDKTHTCPPCP (SEQ ID NO: 906). Alternatively, various non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, also find use as linkers. In some embodiments, the I-TevI ​​nuclease domain is connected to the Cas domain by a linker domain. The linker domain may comprise the I-TevI ​​linker (amino acids 93-169 of SEQ ID NO: 701). In some embodiments, the linker is DATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGS (SEQ ID NO: 703) The amino acid sequence is as follows:

[0156] In some embodiments, an exemplary linker is set forth in SEQ ID NO: 702 or 704. In some embodiments, the mutation may correspond to any one of SEQ ID NOs: 702 or 704. In some embodiments, the linker comprises a mutation corresponding to a position selected from any one of T95, S101, A119, K120, K135, P126, D127, N140, T147, Q158, A161, V117, S165, or a combination thereof. In some embodiments, the linker comprises a mutation corresponding to any one of T95S, S101Y, A119D, K120N, K135N, K135R, P126S, D127K, N140S, T147I, Q158R, A161V, V117F, S165G, or a combination thereof. In some embodiments, the linker comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 701, 702, 703, or 704. In some embodiments, the linker comprises a mutation corresponding to K135R and / or N140S. In some embodiments, the linker comprises a mutation corresponding to V117F. In some embodiments, the linker comprises a mutation corresponding to V117F, K135R, and / or N140S.

[0157] Cas protein domain

[0138] CRISPR systems generally contain two components: guide RNA (gRNA or sgRNA) and CRISPR-associated endonuclease (Cas protein). In nature, CRISPR / CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNA (crRNA) to guide the silencing of invading nucleic acids. CRISPR-Cas is an RNA-mediated adaptive defense system that relies on small RNA molecules for sequence-specific detection and silencing of foreign nucleic acids. The CRISPR-Cas system consists of a CRISPR array consisting of cas genes organized into an operon and genomic target sequences (termed spacers).

[0158]

[0139] A CRISPR-Cas system generally refers to and may include an enzyme system comprising a guide RNA sequence, which comprises a nucleotide sequence complementary or substantially complementary to a region of a target polynucleotide (e.g., a template nucleic acid such as HSV genomic DNA), and a protein with nuclease activity. CRISPR-Cas systems may include type I CRISPR-Cas systems, type II CRISPR-Cas systems, type III CRISPR-Cas systems, and derivatives thereof. CRISPR-Cas systems include engineered and / or programmed nuclease systems derived from naturally occurring CRISPR-Cas systems. CRISPR-Cas systems may include engineered and / or mutated Cas proteins. In certain embodiments, a nuclease generally refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of a nucleic acid. In some embodiments, an endonuclease generally refers to an enzyme capable of cleaving phosphodiester bonds in a polynucleotide chain.

[0159] In some embodiments, the CRISPR-Cas system used herein may be a Type I, Type II, or Type III system. Non-limiting examples of suitable CRISPR-Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, CasF, CasG, CasH, CasX, CasΦ, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or Cas B), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CasX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. In some embodiments, the CRISPR-Cas protein or endonuclease is Cas9. In some embodiments, the CRISPR-Cas protein or endonuclease is Cas12. In some embodiments, the CRISPR-Cas protein or endonuclease is CasX.

[0160]

[0141] In some embodiments, the Cas9 protein is derived from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus desulforudis, Clostridium botulinum, Clostridium difficile, Fine goldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

[0161] In some embodiments, the CRISPR-Cas-like protein may be a wild-type CRISPR-Cas protein or a modified CRISPR-Cas protein. In some embodiments, the CRISPR-Cas-like protein can be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or alter another property of the protein. For example, the nuclease (i.e., DNase, RNase) domain of the CRISPR-Cas-like protein can be modified, deleted, or inactivated. Alternatively, in some embodiments, the CRISPR-Cas-like protein can be truncated to remove domains that are not essential for the function of the Cas protein. In some embodiments, the CRISPR-Cas-like protein can be truncated or modified to optimize the activity of the effector domain of the Cas protein.

[0162] In some embodiments, the CRISPR-Cas-like protein can be derived from a wild-type Cas protein or a fragment thereof. In certain embodiments, the CRISPR-Cas-like protein is a modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified relative to the wild-type or another Cas protein to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.). Alternatively, in some embodiments, domains of the Cas9 protein that are not involved in RNA-guided cleavage can be removed from the protein, such that the modified Cas9 protein is smaller than the wild-type Cas9 protein.

[0163]

[0144] The chimeric nuclease of the present disclosure may comprise a Cas domain. In some embodiments, the Cas domain is a Cas9 domain. In some embodiments, the Cas domain is a Cas12 domain.

[0164] In some embodiments, the Cas9 domain is derived from a bacterial organism, such as Staphylococcus aureus, Streptococcus pyogenes, Neisseria meningitidis, Campylobacter jejuni, Streptococcus pasteurianus, Clostridium cellulolyticum, or Geobacillus thermodenitrificans T1. In some embodiments, the Cas9 domain is derived from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 domain is derived from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 domain is derived from Neisseria meningitidis (NmCas9). In some embodiments, the Cas9 domain is derived from Campylobacter jejuni (CjCas9). In some embodiments, the Cas9 domain is derived from Streptococcus pasteurianus (SpCas9). In some embodiments, the Cas9 domain is derived from Clostridium cellulolyticum (CcCas9). In some embodiments, the Cas9 domain is derived from Geobacillus thermodenitrificans T1 (GtCas9).

[0165] In some embodiments, exemplary RNA-guided nuclease Cas9 domains are set forth in SEQ ID NOs: 710, 711, 712, 713, 714, 715, or 716. In some embodiments, the mutation or amino acid substitution may correspond to any one of SEQ ID NOs: 710, 711, 712, 713, 714, 715, or 716.

[0166] In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises or is identical to an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 710. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises or is identical to an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 710. The Cas9 domains are D10, H557, N580, H840, D1135, R1335, T1337, T267, L325, V327, D333, A336, I341, E345, D348, K352, S360, T368, N369, N371, S372, E373, K386, N393, H408, N410, I414, A415, T438, Y467, N471, D485, M489, E506, R409, T510, N515, Y518, A539, ​​F550, N551, S596, T602, A611, I617, T620, G654, N667, R685, K695, I706, K722, A723, K724, M731, F732, K73 5, S739, P741, E742, E746, Q747, I754, T755, H757, K760, H761, P778, E781, I783, N784, D785, T786L, L787, Y788, K792, D 794, T798, L799, V801, N803, L804, N805, G806, D813, K814, L818, I819, S822, E824, L841, G847, D848, Y857, V875, I876 , N884, A888, L890, D894, D895, P897, V903, G920, F924, N929, E936, N937, V941, N942, S943, C945, E947, K951, L952, S95 6, N957, Q958, A959, N974, G975, V983, N984, N985, D986, I991, V993, M995, I996, T999, Y1000, R1001, E1002, L1004, E10 05, N1006, M1007, D1009, K1010, R1011, P1012, P1013, I1015, I1016, A1020, S1021, Q1024, K1027, E1039, H1045, I0148,In some embodiments, the RNA-guided nuclease comprises a mutation or amino acid substitution corresponding to a position selected from any one of Staphylococcus aureus K1050 and K1050, or a combination thereof. The Cas9 domains are D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, T267A, L325F, V327I, D333G, A336S, I341L, E345D, D348N, K352E, S360A, T368A, N369E, N371E, S372P, E373K, K386T, N393R, H408N, N410S, I414M, A415T, T438S, Y467F, N471K, D485E, M489F, E506K, and R409. K, T510E, N515K, Y518F, A539P, F550Y, N551H, S596A, T602I, A611S, I617V, T620K, G654E, N667D, R685K, K695Q, I706V, K722T, A723T, K72 4N, M731T, F732V, K735Q, S739N, P741L, E742G, E746D, Q747D, I754D, T755I, H757R, K760Q, H761S, P778I, E781K, I783V, N784D, D785E, T7 86L, L787V, Y788H, K792E, D794T, T798R, L799I, V801I, N803S, L804I, N805K, G806N, D813G, K814E, L818I, I819F, S822P, E824G, L841T, G847S, D848N, Y857H, V875I, I876V, N884K, A888V, L890R, D894G, D895H, P897L, V903I, G920D, F924L, N929Y, E936D, N937G, V941I, N942D , S943L, C945A, E947K, K951R, L952Q, S956N, N957E, Q958K, A959S, N974D, G975K, V983A, N984S, N985D, D986G, I991V, V993L, M995F, I996 V, T999N, Y1000K, R1001E, E1002D, L1004I, E1005K, N1006M, M1007N, D1009L, K1010S, R1011T, P1012S, P1013F, I1015L, I1016R, A1020G,The modified I-TevI ​​nuclease domain comprises a mutation or substitution corresponding to one or more of S1021K, Q1024K, K1027S, E1039K, H1045K, I0148M, K1050M, or a combination thereof. In certain embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation or substitution corresponding to a D10E substitution. In certain embodiments, the modified I-TevI ​​nuclease domain comprises SEQ ID NO: 700, the linker comprises any one of SEQ ID NOs: 701 or 703, and the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises any one of SEQ ID NOs: 710-715 or 716. In certain embodiments, the modified I-TevI ​​nuclease domain comprises or is identical to an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 700, the linker domain comprises or is identical to an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 701 or 703, and the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 710-715 or 716.

[0167] In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 710. In some embodiments, the RNA-guided nuclease comprises an amino acid sequence that has 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 710. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation corresponding to any one of positions D10, H557, N580, H840, D1135, R1335, T1337, or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation corresponding to positions D10, H557, N580, H840, D1135, R1335, T1337, or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation corresponding to any one of positions D10A, D10E, H557A, N580A, H840A, D1135E, R1335Q, T1337R, or a combination thereof. In some embodiments, the RNA-guided nuclease Staphylococcus aureus Cas9 domain (saCas9) comprises a mutation corresponding to the D10E mutation. In some embodiments, saCas9 comprises a mutation corresponding to the D10E and / or N580A mutations. In some embodiments, saCas9 comprises mutations corresponding to D10A and / or N580A mutations. In some embodiments, saCas9 comprises mutations corresponding to D10E, D1135E, R1335Q, and / or T1337R mutations. In some embodiments, saCas9 comprises mutations corresponding to D10E, D1135E, R1335Q, T1337R, and / or H840A mutations. In some embodiments, saCas9 comprises mutations corresponding to D10E and / or H557A mutations. In some embodiments, saCas9 comprises mutations corresponding to D10E, H840A, D1135E, R1335Q, and / or T1337R mutations.

[0168] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Streptococcus pyogenes Cas9 domain. In some embodiments, the RNA-guided nuclease Streptococcus pyogenes Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO:711.

[0169]

[0150] Part of the Staphylococcus pyogenes, RNA-inducible Staphylococcus pyogenes Cas9 demethylation is, D10, S29, F32, D39, R40, H41, S42, I48, C80, S87, K112, H113, K132, K141, D147, L158, E171, P176, I186, V189, Q190, Q194, N199, I201, N202, A203, S204, R205, A210, Q228, L229, G231, S245, T249, S254, D261, T270, N295, T300, D3 04, V308, N309, I312, T333, A337, E345, F352, Q354, S355, K356, G366, A367, E396, L398, I414, D428, F429, D435, K468, S469, E470, T472, E480, A486, S490, F498, K500, N501, N504, K528, V530, E532, G533, A538, T555, K570, F575, D605, E611 , R629, E634, T638, R655, R664, R671, K705, E706, Q709, K710, S714, G7115, G717, H721, H723, A725, N726, V743, L747, V748, K772, K775, N776, I788, G792, K797, Y799, T804, N808, L811, R820, N831, R832, ​​V842, L847, N869, E874, N881, Q885, N 888, T893, L911, Y945, D946, L949, E952, A1023, Y1036, G1067, G1077, R1078, N1093, R1114, N1115, D1117, A1121, D1125, P1128, K1129, V1146, S1154, S1159, L1164, S1172, N1177, P1178, I1179, D1180, K1211, M1213, G1218, N1234, E1243, K1244,The mutations include any one of positions E1253, E1260, K1263, H1264, E1271, Q1272, E1275, V1290, L1291, S1292, A1293, N1295, H1297, R1298, D1299, K1300, R1303, E1307, N1308, I1309, I1310, H1311, L1312, L1315, T1316, N1317, Y1326, D1328, V1342, A1345, I1360, and S1363, or a combination thereof. In some embodiments, the RNA-guided nuclease Streptococcus pyogenes Cas9 domain is selected from the group consisting of D10E, D10A, S29T, F32M, D39N, R40K, H41Q, S42T, I48L, C80R, S87A, K112D, H113N, K132N, K141E, D147E, L158V, E171Q, P176S, I186K, V189L, Q190H, Q194E, N199R, I201L, N202E, A203E, S204I, R205K, A206K, A207K, A208K, A210K, A211K, A212K, A213K, A214K, A215K, A216K, A217K, A218K, A219K, A220K, A221K, A222K, A223K, A224K, A225K, A226K, A227K, A228K, A229K, A230K, A231K, A232K, A233K, A234K, A235K, A236K, A237K, A238K, A239K, A240K, A241K, A242K, A243K, A244K, A245K, A246K, A247K, A248K, A249K, A250K, A251K, A252K, A253K, A254K, A255K, A256K, A257K, A258K, A25 10G, Q228A, L229F, G231N, S245A, T249M, S254A, D261N, T270S, N295K, T300I, D304G, V308A, N309D, I312V, T333A, A337V, E345K, F352S, Q354K, S355T, K356T, G366K, A367T, E396D, L398F, I414V, D428A, F429Y, D435 E, K468Q, S469R, E470N, T472A, E480D, A486T, S490L, F498V, K500E, N501H, N504T, K528R, V530I, E532D, G 533E, A538E, T555A, K570Q, F575C, D605E, E611D, R629K, E634K, T638K, R655H, R664K, R671K, K705V, E706D , Q709K, K710A, S714F, G7115E, G717K, H721K, H723Q, A725S, N726A, V743I, L747I, V748I, K772Q, K775R, N7 76R, I788M, G792R, K797E, Y799H, T804A, N808D, L811R, R820K, N831D, R832H, V842I, L847I, N869D, E874A,N881S, Q885R, N888K, T893S, L911A, Y945H, D946G, L949P, E952A, A1023G, Y 1036R, G1067E, G1077E, R1078K, N1093T, R1114G, N1115E, D1117A, A1121P, D 1125G, P1128T, K1129T, V1146I, S1154T, S1159P, L1164V, S1172N, N1177D, P1178S, I1179V, D1180S, K1211R, M1213L, G1218T, N1234H, E1243D, K1244T, The mutations include those corresponding to any one of E1253K, E1260D, K1263Q, H1264Y, E1271D, Q1272W, E1275H, V1290L, L1291R, S1292A, A1293T, N1295E, H1297N, R1298T, D1299H, K1300L, R1303S, E1307D, N1308S, I1309M, I1310L, H1311N, L1312A, L1315F, T1316S, N1317R, Y1326F, D1328N, V1342I, A1345S, I1360L, and S1363N, or a combination thereof. In some embodiments, the RNA-guided nuclease Streptococcus pyogenes Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 711. In some embodiments, the RNA-guided nuclease Streptococcus pyogenes Cas9 domain comprises an amino acid sequence having 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 711.

[0170] In some embodiments, the RNA-guided nuclease Cas9 domain is a RNA-guided nuclease Neisseria meningitidis Cas9 domain. In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 712. Other Neisseria meningitidis Cas9s can be found at www.uniprot.org / uniprot / under accession numbers C9X1G5, A1IQ68, E0NB23, A9M1K5, or C6S593.Neisseria meningitidis Cas9ドメインは, I9 bit, D16 bit, D30 bit, E31 bit, A94 bit, I103 bit, P124 bit, N164 bit, I213 bit , G229 bit, T241 bit, S376 bit, E393 bit, G454 bit, K471 bit, G490 bit, D660 bit, C665 bit, K76 4-bit, T770-bit, P803-bit, A841-bit, H842-bit, K843-bit, D844-bit, L846-bit, R847-bit, K854-bit, H855-bit, N856-bit, K858-bit, K862-bit, W865-bit, E868-bit, I869-bit, A872-bit, D873-bit, N876 , Y880, G883, I886, E887, E890, R895, A898, Y899, G900, G901, N902, A903, K904, Q905, D908, N912, K917, G919, L921, V927, K929, T930, E932, S933, L936, L937, N938, K939, K940, Y943, T944, G949, D950, C958, K965, N966, Q967, F969, A975, E980, N981, I986, D987, C988, K989, G990, Y991, R992, I993, D994, Y997, T998, C1000, S1002, H1004, K1005, Y1006, A1010, F1011, Q1012, K1013, D1014, E1015, K1018, V1019, E1020, F1021, A1022, Y1024, I1025, N1026, C1027, D1028 , S1029, S1030, N1031, R1033, F1034, Y1035, L1036, A1037, W1038, K1041, G1042, K1044, E1045, Q1046, Q1047, F1048, R1 049 bit, I1050 bit, S1051 bit, T1052 bit, Q1053 bit, N1054 bit, L1055 bit, V1056 bit, L10 57 bits, I1058 bits, Y1061 bits, V1063 bits, and N1064 bits are all the same.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain is selected from the group consisting of I9M, D16E, D30E, E31K, A94D, I103V, P124C, N164D, I213N, G229D, T241A, S376T, E393K, G454C, K471E, G490C, D660E, C665R, K764E, T770A, P803S, A841Q, H842G, K843H, D844E, L846V, R847K, K854R, H855L, N856D, K858G, K862L, W865P, E868Q, I869L, A872K, D873G, N876 K, Y880R, G883E, I886P, E887K, E890E, R895Q, A898T, Y899H, G900K, G901D, N902D, A903P, K904T, Q905K, D908A, N912E, K917Y, G919T, L921Q , V927I, K929Q, T930V, E932K, S933T, L936W, L937V, N938R, K939N, K940H, Y943N, T944G, G949A, D950T, C958E, K965G, N966G, Q967K, F969Y, A975S, E980K, N981G, I986R, D987A, C988V, K989V, G990A, Y991F, R992K, I993D, D994E, Y997F, T998E, C1000R, S1002I, H1004Y, K1005A, Y10 06N, A1010K, F1011L, Q1012T, K1013A, D1014K, E1015K, K1018N, V1019E, E1020F, F1021L, A1022G, Y1024F, I1025V, N1026S, C1027L, D1028N , S1029R, S1030A, N1031T, R1033A, F1034I, Y1035D, L1036I, A1037R, W1038T, K1041T, G1042D, K1044T, E1045K, Q1046G, Q1047E, F1048Q, R1049S, I1050V, S1051G, T1052V, Q1053K, N1054T, L1055A, V1056L, L1057S, I1058F, Y1061N, V1063I, N1064D, or a combination thereof.In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 712. In some embodiments, the RNA-guided nuclease Neisseria meningitidis Cas9 domain comprises an amino acid sequence having 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 712.

[0171] In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain is an RNA-guided nuclease Campylobacter jejuni Cas9 domain. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 713. Other Campylobacter jejuni Cas9s can be found at www.uniprot.org / uniprot / under accession numbers Q0P897, A7H5P1, A0A2U0QR81, A0A5Y4VLH1, or A0A381CRM8. In some embodiments, the RNA-guided nuclease Campylobacter jejuni The Cas9 domain is L5, A6, D8, I9, S12, S13, F18, S19, L24, K25, I31, T40, E42, L50, L58, A59. 1st, R61st, L58th, L65th, H67th, N74th, K77th, L98th, I99th, P101st, N110th, L113th, A119th, A126th, R128th, I134th, K140th, A144th, K147th, Q151st, L156th, V184th, S190th, F199th, D202nd, G203rd, R212th, F214th, K221st, E223rd, Y232nd, A235th, V243rd, S247th, D251st, P256th, L261st, T269th, N276th, N277th, L285th, T287th, L291st, K300th, T305th, Q308th, L312th, G314th, Y335th, K336th, I339th, H345th, D351st, N353rd, E354th, I362nd, K370th, D383rd, S384th, K391st, I396th, L403rd, T405th, K413th, N419th, L421st, D430th, K432nd, A437th, L453rd, K457th, V462nd, A465th, K472nd, N477th, A492nd, E495th, L525th, K526th, L527th, K531st, E532nd, E542nd, Q550th, E556th, H559th, Y561st, S564th, M572nd, V577th, Q581st, N587th, N596th, K600th, Q602nd, K603rd, Q616th, K617th, N623rd, Y624th,K633rd, D634th, Y642nd, N649th, D656th, L660th, D662nd, K667th, V677th, E680th, K682nd, L686th, H692nd, T693rd, V712th, I714th, V722nd, K723rd, S736th, L739th, K742nd, L747th, N751st, F756th, R763rd, Q764th, E772nd, K777th, A In some embodiments, the RNA-guided nuclease comprises a mutation corresponding to any one of positions 786, E790, F792, Q800, S801, G804, L812, E813, V833, I835, T841, Y845, A855, L856, A863, V864, D879, E883, D900, Q902, K927, F928, V971, T972, or a combination thereof. The Cas9 domains are L5I, A6G, D8N, D8E, I9L, S12A, S13N, F18L, S19R, L24I, K25I, I31V, T40N, E42N, L50E, L58V, A59K, R61K, L58V, L65M, H67A, N74K, K77N, L98T, I99Q, P101I, N110S, L113I, A119S, and A126V, R128H, I134S, K140N, A144T, K147E, Q151K, L156M, V184I, S190D, F199L, D202Q, G203E , R212K, F214L, K221K, E223K, Y232F, A235P, V243I, S247I, D251N, P256A, L261S, T269G, N276 K, N277S, L285V, T287E, L291I, K300D, T305S, Q308K, L312I, G314N, Y335L, K336N, I339K, H34 5T, D351I, N353D, E354S, I362T, K370E, D383E, S384K, K391N, I396L, L403Q, T405I, K413R, N4 19E, L421C, D430E, K432S, A437L, L453I, K457C, V462L, A465D, K472S, N477H, A492K, E495I, L 525Q, K526I, L527V, K531E, E532D, E542L, Q550D, E556V, H559Y, Y561R, S564N, M572S, V577T,Q581L, N587G, N596E, K600L, Q602A, K603E, Q616R, K617F, N623F, Y624F, K633T, D634E, Y642W, N649S, D656S, L660I, D66 2E, K667A, V677Q, E680V, ​​K682S, L686I, H692N, T693F, V712I, I714V, V722I, K723F, S736K, L739F, K742N, L747S, N751L, F The mutations include any one of 756L, R763K, Q764E, E772N, K777H, A786T, E790L, F792P, Q800N, S801T, G804D, L812V, E813K, V833S, I835L, T841K, Y845H, A855S, L856T, A863T, V864P, D879N, E883N, D900G, Q902K, K927N, F928Y, V971L, and T972S, or a combination thereof. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 713. In some embodiments, the RNA-guided nuclease Campylobacter jejuni Cas9 domain comprises an amino acid sequence having 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 713.

[0172] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Streptococcus pasteurianus Cas9 domain. In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 714. Other Streptococcus pasteurianus Cas9s can be found at www.uniprot.org / uniprot / under accession number F5X275.

[0173]

[0153] In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises a mutation corresponding to any one of positions D11, E85, A88, T92, E96, Y100, T109, D110, D113, E115, R116, D125, I127, K128, E132, S147, I185, A187, K228, Y229, T232, M255, S271, N273, A294, A327, E355, K357, N379, T380, S382, A385, D439, R440, S464, H469, Y519, I528, N569, I581, A607, K632, D633, H635, E636, A647, D648, T703, P705, K712, S713, A724, V750, D882, S951, D977, E979, S1014, H1027, I1030, E1081, D1082, D1086, K1088, S, 1089, N1090, R1092, T1093, I1094, C1095, A1138, Y1139, D1141, T1142, F1158, A1168, E1190, E1198, H1202, I1204, R1205, I1210, K1224, S1232, M1240, V1241, I1242, P1243, G1424, K1248, Q1254, N1257, S1258, T1262, K1263, Y1264, D1266, A1270, K1277, D1284, L1288, V1302, N1316, T1346, I1374 or a combination thereof.In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain is selected from the group consisting of D11E, D11A, E85D, A88T, T92A, E96D, Y100Q, T109D, D110N, D113N, E115D, R116S, D125E, I127D, K128A, E132K, S147T, I185L, A187T, K228N, Y229N, T232K, M255T, S271T, N273E, A294S, A327V, E355K, K357Q, N379G, T380I, S382T, A385N, D439E, R440E, S464A, H469R, Y519F, I528V, N569D, I581V, A607S, K632R, D633E , H635Q, E636Q, A647K, D648Q, T703A, P705S, K712E, S713A, A724T, V750I, D882G, S951R, D977E, E979K, S1014 P, H1027R, I1030V, E1081G, D1082E, D1086N, K1088R, S1089T, N1090D, R1092E, T1093K, I1094V, C1095R, A11 38V, Y1139L, D1141E, T1142P, F1158L, A1168T, E1190K, E1198K, H1202Q, I1204V, R1205Q, I1210M, K1224R, S In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:714.In some embodiments, the RNA-guided nuclease Streptococcus pasteurianus Cas9 domain comprises an amino acid sequence having 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 714.

[0174] In some embodiments, the RNA-guided nuclease Cas9 domain is an RNA-guided nuclease Clostridium cellulolyticum Cas9 domain. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 715. Other Clostridium cellulolyticum Cas9s can be found at www.uniprot.org / uniprot / under accession number B8I085.

[0175] In one embodiment, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises a mutation corresponding to any one of positions T4, D10, V9, D20, K21, I27, C33, K36, A47, A49, S64, Q65, E102, L103, T122, I124, K131, D137, R163, G166, I169, F170, V183, D184, I187, E193, K200, K208, L209, D221, N224, E227, F228, S234, V242, K244, L252, T256, C258, S261, V413, M415, K416, R417, K424, Y426, K427, S429, D430, A468, T470, A472, A478, Q481, K482, L485, A497, L535, W540, R541, E544, G554, P556, I570, Y574, M580, Y584, M585, T592, D593, V606, W607, I647, N650, S693, L697, E702, S704, A713, V714, I715, D776, L847, G850, G853, A854, R860, I900, H904, M905, I906, E921, Q923, S929, T930, H931, Q939, N994, I997, N1000, K1001, S1002, I1003, K1005, P1008 or a combination thereof.In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain is selected from the group consisting of T4S, D10E, V9I, D20N, K21E, I27E, C33I, K36V, A47S, A49P, S64R, Q65H, E102L, L103V, T122V, I124F, K131Q, D137E, R163Q, G166S, I169L, F170L, V183G, D184G, I187T, E193S, K200Q, K208A, L209Y, D221K, N224Q, E227S, F228S, S234T, V242I, K244N, L252K, T256K, C258T, S261F, V413K, M415 L, K416R, R417N, K424Q, Y426I, K427P, S429H, D430Q, A468S, T470S, A472V, A478G, Q481K, K482R, L48 5S, A497M, L535H, W540Y, R541K, E544Q, G554F, P556S, I570V, Y574I, M580F, Y584N, M585N, T592A, D 593A, V606W, W607F, I647R, N650H, S693K, L697F, E702Q, S704N, A713V, V714I, I715V, D776E, L847A , G850P, G853A, A854P, R860K, I900V, H904D, M905V, I906L, E921Y, Q923E, S929D, T930E, H931Y, Q939P, N994Q, I997P, N1000R, K1001M, S1002N, I1003K, K1005H, P1008K, or a combination thereof. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO:715. In some embodiments, the RNA-guided nuclease Clostridium cellulolyticum Cas9 domain comprises an amino acid sequence having 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 715.

[0176] In some embodiments, the RNA-guided nuclease Cas9 domain is a RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 716. Other Geobacillus thermodenitrificans T1 Cas9s can be found at www.uniprot.org / uniprot / under accession number A0A1W6VMQ3.

[0177]

[0155] In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain is K2, D8, I14, D35, K41, F74, V75, K91, I117, R128, T136, Q151, S152, S156, A161, V164, S17 1st place, E178th place, D179th place, V185th place, R192nd place, K195th place, A199th place, Y204th place, I207th place, V208th place, A212th place, H215th place, S219th place, F227th place, T260th place, V261st place, V271st place, G274th, I276th, A278th, L279th, D282nd, I287th, K289th, H293rd, F299th, V302nd, N307th, R313th, L317th, L318th, V331st, G337th, K34 1st, S348th, A354th, A355th, K356th, R359th, M372nd, T377th, R380th, E395th, D399th, E404th, S416th, T441st, R445th, N464th, E504th, S508th, M515th, Q516th, E520th, G521st, V534th, L545th, K559th, T578th, K603rd, T612th, L619th, S621st, N656th, N660th, L673rd, D68 5th place, I699th place, N708th place, N717th place, R737th place, V738th place, S752nd place, D756th place, Q771st place, N777th place, N792nd place, E793rd place, I811th place, I824th place, K839th place, Q845th place, K848th place, The mutations include any one of positions T849, L895, I902, T908, V929, I943, I946, M948, F990, T995, V1000, Q1014, D1017, S1019, N1020, G1021, S1024, N1030, N1031, R1035, S1036, I1037, V1067, S1071, A1075, and I1079, or a combination thereof. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of positions D8, D179, D282, D399, D685, D756, and D1071.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain is selected from the group consisting of K2R, D8E, D8A, I14V, D35E, K41Q, F74V, V75I, K91E, I117V, R128K, T136S, Q151R, S152A, S156G, A161G, V164I, S171A, E178G, D179E, V185I, R192H, K195R, A199S, Y204F, I207M, V208S, A212K, H215N, S219T, F227V, T260I, V261A, V27 1I, G274S, I276A, A278G, L279P, D282E, I287L, K289E, H293Q, F299Y, V302I, N307R, R313Y, L317I, L318V, V331I, G337D, K341Q, S348K, A354K, A355S, K356S, R359L, M372L, T377A, R380H, E395P, D399N, E404N, S416T, T441S, R445K, N464T, E504 D, S508T, M515T, Q516K, E520D, G521E, V534M, L545H, K559R, T578V, K603R, T612I, L619V, S621T, N656M, N660S, L673F, D 685E, I699V, N708E, N717D, ​​R737K, V738I, S752A, D756E, Q771R, N777H, N792D, E793Q, I811V, I824V, K839T, Q845K, K848A , T849S, L895P, I902V, T908K, V929V, I943V, I946M, M948I, F990L, T995I, V1000G, Q1014K, D1017H, S1019G, N1020T, G1021A, S1024E, N1030C, N1031S, R1035S, S1036G, I1037V, V1067L, S1071A, A1075T, I1079V, or a combination thereof. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises a mutation corresponding to any one of D8E, D179E, D282E, D399N, D685E, D756E, D1071H.In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 716. In some embodiments, the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain comprises an amino acid sequence having 85-90%, 90-95%, 95-97%, 97-98%, or 98-99% sequence identity to SEQ ID NO: 716.

[0178] [Table 2]

[0179] In some embodiments, the Cas domain is a CasX domain. In some embodiments, the CasX domain is derived from a Planctomycetes bacterium. In some embodiments, the CasX domain is from a Deltaproteobacteria. In some embodiments, the CasX domain comprises or is identical to an amino acid sequence at least about 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:721.In some embodiments, the CasX domain includes positions R11, R12, V14, K15, S17, N18, A22, G23, T25, P38, K41, E42, N46, L47, N53, I54, P57, T61, S6 2nd, R63rd, A64th, E75th, H82nd, Q89th, P104th, N106th, I113th, N199th, S124th, S125th, C133rd, Y137th, N145th, D146th, H151st, S161st, R165th, N177th, L 180th, R202nd, N205th, G215th, C219th, V236th, T241st, L248th, I254th, S269th, I290th, E291st, V297th, Q299th, I314th, E318th, Q323rd, L333rd, E359th , D360th, K362nd, Q366th, N367th, L368th, A369th, G370th, Y371st, H404th, H409th, G410th, E411th, Y417th, V428th, E429th, S432nd, K433rd, L437th, S443 A451st, I464th, A470th, I502nd, L503rd, I531st, G537th, L540th, N553rd, I559th, S563rd, V571st, N579th, H589th, S607th, L608th, L620th, R623rd, R6 24th, L644th, S646th, M652nd, I657th, R679th, L684th, N686th, H689th, S696th, T702nd, T737th, L742nd, Y744th, Q748th, M751st, I753rd, A771st, R777th, P The mutations include any one of positions 792, S818, R823, V824, E826, K827, A832, T833, M836, I839, G841, V846, N860, V862, D864, V867, V877, S883, S889, G890, S894, K908, N913, F916, T918, R936, Q938, Y940, K942, S963, R966, K967, and K968, or any combination thereof.In some embodiments, the CasX domain is selected from the group consisting of R11K, R12K, V14S, K15A, S17N, N18A, A22V, G23S, T25S, P38D, K41K, E42K, N46K, L47R, N53V, I54M, P57V, T61N, S62A, R63A, A64N, E75K, H82Q, Q89K, P104S, N106K, I113K, N199K, S124T, S125A, C133G, Y137F, N145S, D146E, H151Y, S161A, R165K, N177S, L1 80A, R202K, N205T, G215A, C219Y, V236I, T241S, L248I, I254V, S269G, I290V, E291D, V297I, Q299R, I314L, E318D, Q323L, L333V, E359D, D 360M, K362R, Q366S, N367G, L368V, A369T, G370A, Y371E, H404Y, H409Y, G410A, E411G, Y417F, V428I, E429A, S432T, K433S, L437R, S443A, A451V, I464L, A470M, I502V, L503V, I531L, G537K, L540I, N553S, I559L, S563G, V571L, N579Q, H589T, S607L, L608I, L620I, R623K, R624K , L644V, S646P, M652V, I657V, R679E, L684S, N686G, H689D, S696G, T702A, T737S, L742F, Y744H, Q748H, M751V, I753V, A771T, R777K, P792 The mutations or substitutions include any one or more of T, S818T, R823G, V824M, E826V, K827R, A832S, T833D, M836A, I839L, G841N, V846A, N860T, V862E, D864E, V867A, V877G, S883K, S889R, G890D, S894F, K908Q, N913D, F916H, T918V, R936N, Q938N, Y940F, K942S, S963A, R966K, K967R, and K968R, or a combination thereof.

[0180] In some embodiments, the Cas12 domain is derived from Acidaminococcus species BV3L6. In some embodiments, the Cas12 domain comprises or is identical to an amino acid sequence at least about 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:720. In some embodiments, the Cas12 domain includes positions T1, Q2, E4, G5, N8, L9, K28, H29, I30, Q31, E32, Q33, F35, I36, E37, E38, A41, N43, D 44th, H45th, E48th, I52nd, R55th, T59th, Y60th, A61st, D62nd, Q63rd, C64th, Q66th, L67th, Q69th, L70th, N74th, S76th, A77th, D80th, S81st, Y82nd, E85 E88th, T90th, R91st, N92nd, A93rd, I95th, E97th, A99th, T100th, Y101st, N103rd, A104th, H106th, D107th, I110th, R112th, T113th, D114th, R159th , S169th, S185th, A187th, I192nd, D195th, K201st, T212th, R218th, N223rd, I228th, S233rd, I236th, E237th, V239th, F242nd, Q249th, Y257th, V279 284th place, I284th place, F305th place, N313th place, S324th place, I329th place, S331st place, T337th place, L338th place, L345th place, E349th place, S357th place, I358th place, N386th place, I393rd place, L396th place, I400th place, S403rd place, V40 8th, Q409th, G427th, K428th, Q436th, L442nd, S468th, Q469th, S472nd, L473rd, L479th, E487th, S488th, A497th, L510th, A516th, K522nd, Q535th, M5 36th, S541st, V545th, K549th, N550th, G552nd, V557th, N559th, S586th, Y596th, A601st, I604th, A613th, S628th, E637th, A657th, K660th, G663rd, Q 665th, C673rd, L683rd, L697th, A711th, L717th, Q723rd, A733rd, E735th, Y740th, K751st, K756th, G766th, I778th, R793rd, L844th, I858th, S865th,I874th, H898th, I903rd, I916th, L931st, K941st, N945th, V951st, S958th, V959th, D965th, I938th, H984th, A1009th, C1 024th place, G1037th place, T1049th place, G1055th place, T1056th place, Y1068th place, L1075th place, V1083rd place, K1085th place, L1097th place, H1104th place, D1106th place, D1111 position, L1122, A1134, V1138, D1147, V1160, P1161, R1171, R1173, Y1176, N1205, D1207, S1220, V1221, A1230, N1237, L1243, M1259, Q1274, G1291, Q1295, A1299, or L1304. In some embodiments, the Cas12 domain is selected from the group consisting of T1S, Q2N, E4S, G5E, N8H, L9K, K28E, H29N, I30L, Q31T, E32A, Q33Y, F35M, I36V, E37N, E38D, A41L, N43S, D44E, H45N, E48K, I52V, R55K, T59Y, Y60F, A61I, D62E, Q63E, C64T, Q6 6K, L67H, Q69A, L70I, N74P, S76Y, A77K, D80T, S81A, Y82F, E85D, E88L, T90N, R91N, N92T, A93N, I95 R, E97I, A99D, T100N, Y101C, N103K, A104S, H106A, D107G, I110E, R112K, T113V, D114P, R159K, S169 V, S185A, A187S, I192L, D195E, K201I, T212K, R218N, N223T, I228T, S233G, I236L, E237D, V239I, F 242V, Q249C, Y257F, V279T, I284V, F305Y, N313S, S324N, I329L, S331A, T337E, L338K, L345I, E349Q , S357L, I358A, N386D, I393V, L396A, I400L, S403N, V408I, Q409E, G427D, K428D, Q436A, L442I, S4 68V, Q469L, S472A, L473V, L479T, E487D, S488D, A497V, L510I, A516V, K522Q, Q535S, M536N, S541D,V545E, K549Q, N550Q, G552C, V557E, N559E, S586N, Y596Q, A601S, I604L, A613D, S62 8N, E637T, A657D, K660R, G663N, Q665K, C673H, L683V, L697V, A711G, L717F, Q723E, A 733L, E735D, Y740F, K751E, K756A, G766A, I778V, R793P, L844F, I858V, S865T, I874L , H898N, I903V, I916A, L931F, K941N, N945Q, V951I, S958T, V959A, D965E, I938V, H98 4Q, A1009S, C1024Y, G1037S, T1049E, G1055R, T1056N, Y1068F, L1075A, V1083R, K10 85G, L1097I, H1104K, D1106N, D1111N, L1122K, A1134D, V1138I, D1147A, V1160E, P11 The mutations or substitutions include any one or more of 61F, R1171Q, R1173E, Y1176L, N1205T, D1207N, S1220L, V1221T, A1230E, N1237S, L1243I, M1259K, Q1274L, G1291A, Q1295N, A1299N, or L1304K.

[0181] In some embodiments, the chimeric nuclease comprises a non-naturally occurring Cas domain. In some embodiments, the chimeric nuclease may comprise a Cas domain from another Class 1 or Class 2 CRISPR-Cas protein, CRISPR-Cas3, CRISPR-Cascade, or Cas13d.

[0182] Nuclear localization signal In some embodiments, the chimeric nuclease further comprises one or more nuclear localization sequences (NLS). In some embodiments, the NLS helps promote translocation of the protein to the cell nucleus. In some embodiments, the chimeric nuclease comprises one or more NLS. In some embodiments, the chimeric nuclease is fused or linked to one or more NLS.

[0183] In some embodiments, the chimeric nuclease comprises at least one NLS. In some embodiments, the chimeric nuclease comprises at least two NLSs. In embodiments comprising at least two NLSs, the NLSs may be the same or different.

[0184]

[0161] In some embodiments, the chimeric nuclease may further comprise at least one nuclear localization sequence (NLS). In some embodiments, the chimeric nuclease may further comprise one NLS. In some cases, the chimeric nuclease may further comprise two NLSs. In some embodiments, the chimeric nuclease may further comprise three NLSs. In some embodiments, the chimeric nuclease may further comprise at least four NLSs. In some embodiments, the chimeric nuclease may further comprise at least five NLSs. In some embodiments, the chimeric nuclease may further comprise at least six NLSs. In some embodiments, the chimeric nuclease may further comprise at least seven NLSs. In some embodiments, the chimeric nuclease may further comprise at least eight NLSs. In some embodiments, the chimeric nuclease may further comprise at least nine NLSs. In some embodiments, the chimeric nuclease may further comprise no more than 10 NLSs.

[0185] Additionally, an NLS can be expressed as part of a chimeric nuclease. In some embodiments, the NLS can be located almost anywhere in the amino acid sequence of the protein, generally comprising a short sequence of three or more or four or more amino acids. The location of the NLS fusion can be at the N-terminus, C-terminus, or anywhere in the sequence of the chimeric nuclease or its components (e.g., between the I-TevI ​​domain and the Cas domain of the chimeric nuclease, between the I-TevI ​​domain and the linker domain, between the Cas domain polymerase and the linker domain, or inserted at the N-terminus or C-terminus of the chimeric nuclease). In some embodiments, the chimeric nuclease comprises an NLS at the N-terminus. In some embodiments, the chimeric nuclease comprises an NLS at the C-terminus. In some embodiments, the chimeric nuclease comprises at least one NLS at both the N-terminus and the C-terminus. In some embodiments, the chimeric nuclease comprises two NLSs at the N-terminus and / or the C-terminus.

[0186] Any NLS known in the art is contemplated herein. The NLS may be any naturally occurring NLS or any NLS that does not occur in nature (e.g., an NLS having one or more mutations relative to the wild-type NLS). In some embodiments, one or more NLSs of the chimeric nuclease comprise a bipartite NLS. In some embodiments, the nuclear localization signal (NLS) is predominantly basic. In some embodiments, one or more NLSs of the chimeric nuclease are rich in lysine and arginine residues. In some embodiments, one or more NLSs of the chimeric nuclease comprise a proline residue. In some embodiments, the nuclear localization signal (NLS) comprises the sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 742), KRTADGSEFESPKKKRKV (SEQ ID NO: 743), KRTADGSEFEPKKKRKV (SEQ ID NO: 744), NLSKRPAAIKKAGQAKKKK (SEQ ID NO: 745), RQRRNELKRSF (SEQ ID NO: 746), or NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 747).

[0187] In some embodiments, the NLS is a single-part NLS. For example, in some embodiments, the NLS is the SV40 large T antigen NLS PKKKRKV (SEQ ID NO: 740). In some embodiments, the NLS is a bipartite NLS. In some embodiments, the bipartite NLS comprises two basic domains separated by a spacer sequence comprising a varying number of amino acids. In some embodiments, the NLS is a bipartite NLS. In some embodiments, the bipartite NLS consists of two basic domains separated by a spacer sequence comprising a varying number of amino acids. In some embodiments, the spacer amino acid sequence comprises the sequence KRXXXXXXXXXXKKKL (Xenopus nucleoplasmin NLS) (SEQ ID NO: 748), where X is any amino acid. In some embodiments, the NLS comprises the nucleoplasmin NLS sequence KRPAATKKAGQAKKKK (SEQ ID NO: 741). In some embodiments, the NLS is a non-canonical sequence, such as M9 of the hnRNP A1 protein, influenza virus nucleoprotein NLS, and yeast Gal4 protein NLS. In some embodiments, the NLS is a non-canonical sequence, such as M9 of the hnRNP A1 protein, influenza virus nucleoprotein NLS, and yeast Gal4 protein NLS.

[0188] In certain embodiments, the chimeric nuclease comprises a nuclear localization signal. In certain embodiments, the nuclear localization signal comprises an SV40 nuclear localization signal comprising the amino acid sequence of SEQ ID NO: 740 (PKKKRKV). In certain embodiments, the nuclear localization signal comprises a nucleoplasmin nuclear localization signal comprising the amino acid sequence of SEQ ID NO: 741 (KRPAATKKAGQAKKKK).

[0166] Non-limiting examples of NLS sequences are provided in Table 3 below.

[0189] [Table 3]

[0190] Donor polynucleotide The chimeric nucleases of the present disclosure may be used or administered in conjunction with a donor nucleic acid sequence. The nucleic acid sequence is a sequence that can be used as a template to replace a sequence excised by the chimeric nuclease. In some embodiments, the donor nucleic acid restores non-oncogenic function (e.g., restores wild-type function) of a gene that contains an oncogenic mutation. In some embodiments, the donor nucleic acid is DNA.

[0191] The methods and techniques described herein are useful for genetic modification of cells harboring oncogenes within a cell population. The donor nucleic acid may be configured for integration by homologous recombination. Such donor DNA for integration by homologous recombination may include a first flanking region of homology, an exogenous polynucleotide sequence of interest, and a second flanking region of homology. Alternatively, the exogenous donor DNA may be inserted into a genomic location by non-homologous end joining and integration into the genomic location at a single double-strand break or a double double-strand break.

[0192] When a chimeric nuclease (e.g., TevSaCas9) cleaves double-stranded DNA, the Cas (e.g., Cas9) may leave a blunt end and I-TevI ​​may leave a 3' overhang. The provided donor DNA may include a blunt end and a 1-nucleotide 3' overhang configured to bind to the 3' overhang created at the site cleaved by the chimeric nuclease (TevCas9). In some embodiments, the donor nucleic acid includes a blunt end and a 3' overhang. In some embodiments, the 3' overhang is at least 1, 2, 3, 4, or 5 nucleotides in length. It will be understood that the length of the nucleotide overhang may be modified to accommodate the use of Cas domains that generate overhangs other than 2 nucleotides.

[0193] In some embodiments, the donor DNA comprises a DNA sequence intended to be inserted into a site of a gene, such as an oncogene. In certain embodiments, the donor DNA comprises double-stranded DNA of the same length cleaved by the nuclease and comprises DNA ends complementary to the DNA ends cleaved by the chimeric nuclease. In certain embodiments, the donor DNA comprises the 5' end of the DNA to be phosphorylated. In certain embodiments, the donor DNA comprises circular double-stranded DNA comprising an I-TevI ​​target site and a Cas target site, and the product cleaved from the double-stranded DNA comprises ends complementary to the ends of the product cleaved by the chimeric nuclease.

[0194]

[0171] The donor nucleic acid can include homology arms flanking either end of the DNA sequence to be inserted into the gene. In some embodiments, the homology arms include 5' and 3' homology arms flanking both ends of the DNA sequence to be inserted. In some embodiments, the 5' and 3' homology arms are the same or different lengths.

[0195]

[0172] Double-stranded donor nucleic acids may contain various 5'-end chemical modifications, such as biotin. Other versions of donor DNA may include stabilizing modifications to the 2' position of the ribose, including, but not limited to, 2'-fluoro, 2'-amino, and 2'-O-methyl. In some embodiments, donor nucleic acids may contain 3'-end modifications, such as inverted dT or biotin. Other versions of donor nucleic acids may include locked nucleic acids (LNAs), in which the 2'-O and 4'-C atoms of the ribose sugar are joined by a methylene bridge. Other versions of double-stranded donor nucleic acids may include circular plasmid DNA containing a chimeric nuclease target site, where cleavage by the chimeric nuclease creates DNA ends complementary to those in the genomic target. Double-stranded donor nucleic acids may include synthetic or amplified linear double-stranded DNA. In certain embodiments, the donor nucleic acid is delivered using a viral vector, such as an adeno-associated virus or lentivirus.

[0196] guide RNA

[0173] The chimeric nuclease of the present disclosure may further comprise a guide RNA. The guide RNAs may target the same region of DNA in an oncogene but contain different sequences accounting for genetic polymorphism in the population. Other versions of guide RNAs may target different oncogenes. Other versions may contain a mixture of guide RNAs targeting multiple sequences in the same gene. The guide RNA may comprise a single strand containing all elements necessary for activity (e.g., target binding and nuclease binding). Alternatively, the guide RNA may comprise two or more non-covalently linked nucleic acids that form a single moiety through base pairing between the two or more nucleic acids.

[0197]

[0174] A gRNA is generally supported by a scaffold, where a scaffold refers to a portion of a gRNA or crRNA molecule that contains a sequence that is substantially identical or highly conserved (e.g., does not confer target specificity) across naturally occurring biological species. A scaffold includes a tracrRNA segment and a portion of a crRNA segment other than a polynucleotide target guide sequence at or near the 5' end of the crRNA segment, and does not include any non-naturally occurring portions that contain sequences that are not conserved in naturally occurring crRNAs and tracrRNAs. In some embodiments, a gRNA comprises a CRISPR RNA (crRNA):trans-activating cRNA (tracrRNA) duplex. In some embodiments, a gRNA comprises a stem-loop that mimics the naturally occurring duplex between the crRNA and tracrRNA. In some embodiments, the stem-loop comprises a nucleotide sequence that includes a non-naturally occurring sequence. For example, in some embodiments, a composition comprises a synthetic or chimeric guide RNA comprising a crRNA, a stem, and a tracrRNA.

[0198]

[0175] Generally, a protospacer adjacent motif (PAM) is also a critical sequence element mediating the enzymatic activity of Cas nucleases. A PAM sequence or element also refers to and includes a DNA sequence of approximately 2-6 base pairs that is a critical target component for Cas nucleases. In certain cases, the PAM sequence further includes a DNA sequence required for the Cas / sgRNA to form an R-loop and probe specific DNA through Watson-Crick pairing of its guide RNA with the genome. In certain cases, the specificity of the PAM can be a function of the DNA-binding specificity of the Cas protein (e.g., the Cas PAM recognition domain), and a protospacer adjacent motif recognition domain refers to a Cas amino acid sequence that contains the binding site for the DNA target PAM sequence.

[0199] Typically, the PAM sequence is located on either strand, downstream in the 5' to 3' direction from the Cas9 cleavage site. The canonical PAM sequence (i.e., the PAM sequence associated with the Streptococcus pyogenes Cas9 nuclease, or SpCas9) is 5'-NGG-3' (SEQ ID NO: 760), where "N" is any nucleobase followed by two guanine ("G") nucleobases. Different Cas9 nucleases or equivalent proteins from different organisms may be associated with different PAM sequences. Furthermore, any given Cas9 nuclease, e.g., SpCas9, may be engineered to alter the PAM specificity of the nuclease, thereby allowing the nuclease to recognize alternative PAM sequences. In the CRISPR-Cas system derived from S. pyogenes (spCas9), the protospacer region DNA is typically immediately preceded by a 5'-NGG (SEQ ID NO: 761) or NAG (SEQ ID NO: 762) protospacer adjacent motif (PAM). Other Cas9 orthologs may have different PAM specificities. For example, Cas9 from S. thermophilus (stCas9) requires 5'-NNAGAA (SEQ ID NO: 763) for CRISPR1 and 5'-NGGNG (SEQ ID NO: 764) for CRISPR3, while Neiseria menigiditis (nmCas9) requires 5'-NNNNGATT (SEQ ID NO: 765). Cas9 from Staphylococcus aureus subsp. aureus (saCas9) requires 5'-NNGRRT (SEQ ID NO: 766) (R = A or G). In some embodiments, Cas9 enzymes from different bacterial species (i.e., Cas9 orthologs) may have varying PAM specificities. For example, Cas9 from Staphylococcus aureus (SaCas9) recognizes NGRRT (SEQ ID NO: 767) or NGRRN (SEQ ID NO: 768). Furthermore, Cas9 from Neisseria meningitis (NmCas) recognizes NNNNGATT (SEQ ID NO: 769). In another example, Cas9 from Streptococcus thermophilis (StCas9) recognizes NNAGAAW (SEQ ID NO: 770).In yet another example, Cas9 from Treponema denticola (TdCas) recognizes NAAAAC (SEQ ID NO: 771). These examples are not intended to be limiting. It will be further recognized that non-SpCas9s bind to a variety of PAM sequences, thereby making them useful when a suitable SpCas9 PAM sequence does not exist for the desired target cleavage site. Additionally, non-SpCas9s may have other characteristics that make them more useful than SpCas9.

[0200] In some embodiments, the gRNA spacer sequence comprises from about 15 nucleotides to about 28 nucleotides. In some embodiments, the gRNA comprises at least about 15 nucleotides. In some embodiments, the gRNA spacer sequence comprises at most about 28 nucleotides. In some embodiments, the gRNA spacer sequence is from about 15 nucleotides to about 16 nucleotides, from about 15 nucleotides to about 17 nucleotides, from about 15 nucleotides to about 18 nucleotides, from about 15 nucleotides to about 19 nucleotides, from about 15 nucleotides to about 20 nucleotides, from about 15 nucleotides to about 21 nucleotides, from about 15 nucleotides to about 22 nucleotides, from about 15 nucleotides to about 23 nucleotides, from about 15 nucleotides to about 24 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 15 nucleotides to about 28 nucleotides, from about 16 nucleotides to about 17 nucleotides, from about 16 nucleotides to about 18 nucleotides, from about 16 nucleotides to about 19 nucleotides, from about 16 nucleotides to about 20 nucleotides, from about 16 nucleotides to about 21 nucleotides, from about 16 nucleotides to about 22 nucleotides, from about 16 nucleotides to about 23 nucleotides, from about 16 nucleotides to about 24 nucleotides, from about 16 nucleotides to about 25 nucleotides, or from about 16 nucleotides to about 17 nucleotides. to about 28 nucleotides, about 17 nucleotides to about 18 nucleotides, about 17 nucleotides to about 19 nucleotides, about 17 nucleotides to about 20 nucleotides, about 17 nucleotides to about 21 nucleotides, about 17 nucleotides to about 22 nucleotides, about 17 nucleotides to about 23 nucleotides, about 17 nucleotides to about 24 nucleotides, about 17 nucleotides to about 25 nucleotides, about 17 nucleotides to about 28 nucleotides, about 18 nucleotides to about 19 nucleotides, about 18 nucleotides to about 20 nucleotides, about 18 nucleotides to about 21 nucleotides, about 18 nucleotides to about 22 nucleotides, about 18 nucleotides to about 23 nucleotides, about 18 nucleotides to about 24 nucleotides, about 18 nucleotides to about 25 nucleotides, about 18 nucleotides to about 28 nucleotides, about 19 nucleotides to about 20 nucleotides, about 19 nucleotides to about 21 nucleotides, about 19 nucleotides to about 22 nucleotides, about 19 nucleotides to about 23 nucleotides,The amino acid sequence may be from about 19 nucleotides to about 24 nucleotides, from about 19 nucleotides to about 25 nucleotides, from about 19 nucleotides to about 28 nucleotides, from about 20 nucleotides to about 21 nucleotides, from about 20 nucleotides to about 22 nucleotides, from about 20 nucleotides to about 23 nucleotides, from about 20 nucleotides to about 24 nucleotides, from about 20 nucleotides to about 25 nucleotides, from about 20 nucleotides to about 28 nucleotides, from about 21 nucleotides to about 22 nucleotides, from about 21 nucleotides to about 23 nucleotides, from about 21 nucleotides to about 24 nucleotides, from about 21 nucleotides to about 25 nucleotides, from about 21 nucleotides to about 28 nucleotides, from about 22 nucleotides to about 23 nucleotides, from about 22 nucleotides to about 24 nucleotides, from about 22 nucleotides to about 25 nucleotides, from about 22 nucleotides to about 28 nucleotides, from about 23 nucleotides to about 24 nucleotides, from about 23 nucleotides to about 25 nucleotides, from about 23 nucleotides to about 28 nucleotides, from about 24 nucleotides to about 25 nucleotides, from about 24 nucleotides to about 28 nucleotides, or from about 25 nucleotides to about 28 nucleotides. In some embodiments, the gRNA spacer sequence comprises about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, or about 28 nucleotides.

[0201] In some embodiments, the guide RNA may be selected from the group consisting of 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives of adenine, 6-methyl derivatives of guanine, 2-propyl derivatives of adenine, 2-propyl derivatives of guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-propynyluracil, 5-propyl nylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-halouracil, 5-bromouracil, 5 -Trifluoromethyluracil, 5-halocytosine, 5-bromocytosine, 5-trifluoromethylcytosine, 5-substituted uracil, 5-substituted cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytosine

[00130] Guide RNAs contain a variety of nucleobases, including, but not limited to, phenothiazine cytidine, substituted phenoxazine cytidine, carbazole cytidine, pyridoindole cytidine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, 5-substituted pyrimidine, 6-azapyrimidine, N-2, N-6, or O-6 substituted purines, 2-aminopropyl adenine, 5-propynyl uracil, and 5-propynyl cytosine. Other versions of guide RNAs can include other nucleic acids, such as bridged or locked nucleic acids.

[0202]

[0179] The guide RNAs described herein may further comprise one or more of an unnatural internucleoside linkage, a nucleic acid mimic, a modified sugar moiety, and a modified nucleobase. In certain embodiments, the unnatural internucleoside linkage comprises one or more of a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3'-alkylenephosphonate, a 5'-alkylenephosphonate, a chiral phosphonate, a phosphinate, a 3'-aminophosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, and a boranophosphate. In certain embodiments, the nucleic acid mimic comprises one or more of peptide nucleic acid (PNA), morpholino nucleic acid, cyclohexenyl nucleic acid (CeNA), or locked nucleic acid (LNA). In certain embodiments, the modified sugar moiety comprises one or more of 2'-O-(2-methoxyethyl), 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-O-methyl, and 2'-fluoro.In certain embodiments, the modified nucleobase is 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivative of adenine, 6-methyl derivative of guanine, 2-propyl derivative of adenine, 2-propyl derivative of guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 5-halo, 5-bromo, 5-trifluoromethyl, 5-substituted uracil,

[00130] The guide RNAs may include one or more of: cytosine, 5-substituted cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidine, phenoxazine cytidine, phenothiazine cytidine, substituted phenoxazine cytidine, carbazole cytidine, pyridoindole cytidine, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, 5-substituted pyrimidine, 6-azapyrimidine, N-2, N-6, or O-6 substituted purine, 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. Exemplary guide RNAs of the present disclosure can be found in Table 4.

[0203] Production of chimeric nucleases The chimeric nucleases described herein may be produced by a number of methods, including using an E. coli expression system such as that described in WO 2020 / 225719 A1. Alternatively, the chimeric nuclease may be produced in the target cell to be modified by providing one or more genetic vectors that direct the expression and production of the nuclease in the target cell. Additionally, the vector may provide sequences that direct the expression of a guide RNA that targets the chimeric nuclease to a specific genomic region.

[0181] Exemplary methods for producing the genetically engineered cells described herein are described below.

[0204] Populations of cells are grown in T-flasks to 70-90% confluence. Cells are harvested by centrifugation and diluted to 1.0 x 10 in Buffer T (Invitrogen, Carlsbad, CA). 7 cells / ml (practical range is 0.2–2 × 10 7The cells are electroporated with a chimeric nuclease described herein, including a chimeric nuclease selected from SEQ ID NOs: 750-756, formulated in tris(hydroxymethyl)aminomethane or phosphate buffered saline, using a Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) at 2000 V (working range 1100-2500), 20 ms (working range 10-30 ms), and 1 pulse (working range 1-4). Cells were cultured in RPMI 1640 containing 0.3 g / L glutamine and 2 g / L glucose (Sigma-Aldrich, Irvine, UK), 10% fetal bovine serum (Sigma-Aldrich, Oakville, ON, Canada), 2 mM L-glutamine, and 100 units penicillin and 0.1 mg / mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) for 24 hours before recovery. Dead cells were removed using a Dead Cell Removal Kit (Miltenyi Biotec, Somerville, MA, USA). Knockout efficiency was determined by amplifying the target gene by polymerase chain reaction and measuring the percentage of edited cells by targeted amplicon sequencing (GENEWIZ, South Plainfield, NJ, USA). Amplicon sequencing is a targeted next-generation sequencing method that allows for the analysis of genetic alterations in specific genomic regions. This method uses PCR to create sequences of DNA called amplicons. Amplicons from different samples can be multiplexed (also called indexing or pooling), which involves adding barcodes (indexes) to the samples so they can be identified. Prior to multiplexing, the individual samples used for amplicon sequencing must be transformed into a library by adding adapters and enriching the target regions through PCR amplification. The adapters enable the formation of indexed amplicons and allow the amplicons to attach to a flow cell for sequencing.Amplicon sequencing is typically used to detect variants in a population of cells.

[0205] Other methods for delivering nucleases to cells may also be used, such as lipid nanoparticles, polymers, viral vectors, or cell-penetrating peptides. The chimeric nuclease or guide RNA may be delivered individually or in combination, as DNA or RNA, in single-stranded or double-stranded form. Additionally, the chimeric nuclease may be delivered as RNA containing one or more of the following elements: a 5' cap, a 5' untranslated region, a coding sequence, a 3' untranslated region, and a polyadenine (polyA) tail. For stability, RNA is modified with various amino acids such as 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives of adenine, 6-methyl derivatives of guanine, 2-propyl derivatives of adenine, 2-propyl derivatives of guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, pseudouracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thioalkyladenine, 8-hydroxyladenine, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine, 5-halouracil, 5-bromouracil, 5-trifluoromethyluracil, uracil, 5-halocytosine, 5-bromocytosine, 5-trifluoromethylcytosine, 5-substituted uracil, 5-substituted cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidine, phenoxazine cytidine, phenothiazine The nucleic acid may comprise a variety of nucleobases, including, but not limited to, cytosine, substituted phenoxazine cytidine, carbazole cytidine, pyridoindole cytidine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, 5-substituted pyrimidine, 6-azapyrimidine, N-2, N-6, or O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0206] In another embodiment, the chimeric nuclease may be delivered as an integrating vector, including but not limited to, retroviral vectors, lentiviral vectors, transposon vectors, and adeno-associated viral vectors. The chimeric nuclease may be delivered by other electroporation systems, including but not limited to, Nucleofector™ (Lonza, Basel, Switzerland), MaxCyte (Gaithersburg, MD), or CliniMACS® (Bergisch Gladbach, Germany).

[0207]

[0185] The chimeric nuclease may further be included in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents, or excipients. The term "pharmaceutically acceptable excipient," as used herein, refers to carriers and vehicles that are compatible with (and preferably capable of stabilizing) the active ingredients of the pharmaceutical composition of the present invention (e.g., the compounds of the present invention) and are not harmful to the individual being treated. For example, solubilizing agents that form specific, more soluble complexes with the compounds of the present invention can be utilized as pharmaceutical excipients for delivery of the compounds. Suitable carriers and vehicles are known to those skilled in the art. The term "excipient," as used herein, encompasses all such carriers, adjuvants, diluents, solvents, or other inert additives. Pharmaceutical formulations may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, glycerol, tetrahydrofuryl alcohol, and fatty acid esters of sorbitan, cyclodextrin, albumin, hyaluronic acid, chitosan, and mixtures thereof. Polyethylene glycol (PEG) may be used to achieve desirable solubility, stability, half-life, and other beneficial pharmaceutical properties. Representative examples of stabilizing ingredients include polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof.Other excipients, such as solution binders or antioxidants, that can be employed include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha tocopherol), vitamin C, and xylitol.

[0208]

[0186] The guide RNA of the present disclosure hybridizes to a target sequence. In some embodiments, the target sequence is an oncogenic mutation. In some embodiments, the oncogenic mutation is selected from any one of KRAS, PIK3CA, EGFR, Muc4, or a combination thereof.

[0209] Methods for targeting oncogenic mutations

[0187] In some embodiments, provided herein are methods for targeting oncogenic mutations in cells, the methods comprising contacting a cell with a chimeric nuclease of the present disclosure. In some embodiments, provided herein are methods for targeting oncogenic mutations. In some embodiments, the methods comprise administering a chimeric nuclease composition of the present disclosure to an individual having a disease or disorder, thereby treating the disease or disorder. In some embodiments, the disease or disorder is cancer.

[0210] In some embodiments, the methods include administering a chimeric nuclease composition of the present disclosure to an individual having cancer, thereby treating the cancer. The methods include administering to the individual a therapeutically effective amount of a chimeric nuclease composition or a pharmaceutical composition comprising a chimeric nuclease composition described herein. In some embodiments, administration of the chimeric nuclease composition results in the incorporation of one or more intended nucleotide edits into a target gene in the individual or cell. In some embodiments, administration of the chimeric nuclease results in the correction of one or more oncogenic mutations, such as a point mutation, insertion, or deletion, associated with an oncogene in the individual or cell.

[0211]

[0189] The present disclosure provides methods for targeting oncogenic mutations in cells. The present disclosure also provides methods for targeting oncogenic mutations in an individual. In some embodiments, the cells are cells of an individual afflicted with cancer. In some embodiments, the individual has cancer.

[0212]

[0190] The present disclosure also provides the use of a chimeric nuclease composition to target oncogenic mutations in a cell. The present disclosure provides a method of editing a genome in a cell. The present disclosure also provides a method of editing the genome of an individual. The present disclosure also provides the use of a chimeric nuclease composition to target oncogenic mutations in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer. In some embodiments, the individual has undergone a treatment that may induce metastasis. In some embodiments, the treatment includes surgery, radiation treatment, and chemotherapy. In some embodiments, the individual is human. In some embodiments, the cancer is a carcinoma or sarcoma. In some embodiments, the carcinoma includes breast cancer, lung cancer, colon cancer, or prostate cancer. In some embodiments, the sarcoma includes osteosarcoma or soft tissue sarcoma. In some embodiments, the cancer is a glioblastoma.

[0213]

[0191] The present disclosure also provides for the use of the chimeric nuclease composition for editing a genome in a cell. The present disclosure also provides for the use of the chimeric nuclease composition for editing the genome of an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0214]

[0192] The present disclosure provides methods for deleting at least a portion of an oncogene in a cell. The present disclosure also provides methods for deleting at least a portion of an oncogene in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0215]

[0193] The present disclosure also provides for the use of the chimeric nuclease composition for deleting at least a portion of an oncogene in a cell. The present disclosure also provides for the use of the chimeric nuclease composition for deleting at least a portion of an oncogene in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0216]

[0194] The present disclosure provides methods for silencing or disrupting at least a portion of an oncogene in a cell. The present disclosure also provides methods for silencing or disrupting at least a portion of an oncogene in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0217]

[0195] The present disclosure also provides for the use of the chimeric nuclease composition to silence or disrupt at least a portion of an oncogene in a cell. The present disclosure also provides for the use of the chimeric nuclease composition to silence or disrupt at least a portion of an oncogene in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0218]

[0196] The present disclosure provides methods for replacing at least a portion of an oncogene in a cell. The present disclosure also provides methods for replacing at least a portion of an oncogene in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0219]

[0197] The present disclosure also provides for the use of a chimeric nuclease composition to replace at least a portion of an oncogene in a cell. The present disclosure also provides for the use of a chimeric nuclease composition to replace at least a portion of an oncogene in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0220]

[0198] The present disclosure provides methods of restoring non-oncogenic function in a cell. The present disclosure also provides methods of restoring non-oncogenic function in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0221]

[0199] The present disclosure also provides for the use of the chimeric nuclease composition to restore a non-oncogenic function in a cell. The present disclosure also provides for the use of the chimeric nuclease composition to restore a non-oncogenic function in an individual. In some embodiments, the cell is a cell of an individual afflicted with cancer. In some embodiments, the individual is an individual with cancer.

[0222]

[0200] The chimeric nucleases of the present disclosure may be formulated to treat individuals (e.g., humans) having disorders involving pathological angiogenesis (e.g., cancer, such as breast cancer, ovarian cancer, renal cancer, colorectal cancer, liver cancer, gastric cancer, and lung cancer, obesity, macular degeneration, diabetic retinopathy, psoriasis, rheumatoid arthritis, cell-mediated immunity, and rosacea).

[0223] In some embodiments, the cancer treated by the chimeric nuclease is selected from any one of prostate cancer, liver cancer, colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, gastric (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell carcinoma, non-small cell carcinoma), sarcoma (soft tissue sarcoma and osteosarcoma), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioblastoma, renal cancer, lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS). In some embodiments of this method, the cancer is selected from any one of triple-negative breast cancer, gastric (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, myelodysplastic syndrome (MDS), or a combination thereof. In some embodiments of this method, the cancer is selected from the group consisting of triple-negative breast cancer, gastric (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel cell carcinoma, MSI-high cancer, or a combination thereof. In certain embodiments, the cancer comprises a BRAF mutation (e.g., a BRAF V600E mutation), BRAF wild-type, KRAS wild-type, or an activating KRAS mutation. The cancer may be early-stage, intermediate-stage, or late-stage.

[0224] In some embodiments, the methods provided herein comprise administering to an individual an effective amount of a chimeric nuclease composition. In some embodiments, the methods comprise administering to an individual an effective amount of a chimeric nuclease composition described herein, e.g., a polynucleotide, vector, or construct encoding a chimeric nuclease component, an LNP, and / or a polypeptide comprising a chimeric nuclease component. The chimeric nuclease composition can be administered to target an oncogene in the individual. Identification of an individual in need of such treatment can be by the judgment of the individual or a healthcare professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).

[0225]

[0203] In some embodiments, the methods comprise administering a chimeric nuclease composition provided herein directly to an individual. The chimeric nuclease compositions described herein can be delivered in any form described herein, e.g., LNP, RNP, polynucleotide vector, e.g., viral vector, or mRNA. The chimeric nuclease composition can be formulated with any pharmaceutically acceptable carrier described herein or known in the art for direct administration to an individual. Components of the chimeric nuclease composition or pharmaceutical composition thereof may be administered to an individual simultaneously or sequentially. For example, in some embodiments, the methods comprise administering to an individual a chimeric nuclease composition or pharmaceutical composition thereof comprising a chimeric nuclease. In some embodiments, the methods comprise administering to an individual a polynucleotide or vector encoding the chimeric nuclease together with a donor nucleic acid.

[0226] Suitable routes of administering the chimeric nuclease to an individual include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intra-articular, intraperitoneal, intracapsular, transmuscular, intragingival, intradental, intracochlear, transtympanic, intravisceral, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration. In some embodiments, the described compositions are administered intraperitoneally, intravenously, or by direct injection or infusion. In some embodiments, the compositions described herein are administered to an individual by injection, by means of a catheter, by means of a suppository, or by means of an implant.

[0227]

[0205] In some embodiments, the method includes administering to an individual cells edited with a chimeric nuclease composition described herein.

[0206] The specific dose administered may be a uniform dose for each individual. Alternatively, an individual's dose may be adjusted according to the individual's approximate body weight. Other factors that determine the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and medical condition of the patient.

[0228]

[0207] In embodiments in which the components of the chimeric nuclease composition are administered sequentially, the time between sequential administrations may be at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days.

[0229]

[0208] In some embodiments, methods of monitoring the progress of treatment are provided, hi some embodiments, the methods comprise determining the level of correction of a diagnostic marker, e.g., a mutation in a proto-oncogene. [Example]

[0230] Example 1: Generating a dataset of disease-causing mutations

[0209] Provided herein is a study that utilizes a method for systematically searching large datasets of mutant genes to identify putative allele-specific targets of chimeric nucleases using a computer algorithm. To increase the accuracy of Cas9 endonuclease, a computer algorithm was developed to optimize the design of guide RNAs and predict possible target sites for the nuclease. This algorithm incorporated a machine learning model to determine the thermodynamic properties of target sites to better predict editing outcomes. None of these algorithms identified allele-specific targets for chimeric nucleases.

[0231]

[0210] This disclosure provides a method for systematically identifying allele-specific targets by first generating a curated dataset of mutant and non-mutant alleles from a database of disease-causing mutations, e.g., the NCBI database, the Cancer Genome Atlas database. Here, using a custom Python script, the genomic locations of disease-causing mutations were extracted from the dataset and used as queries against the GRCh38.p13 human genome. The 50 nucleotides surrounding the mutation were called and printed in a tabular format, with the location of the mutation annotated in the sequence. The result was a pair of sequences: one wild-type sequence, the second the annotated mutation. The process was repeated for subsequent mutations in the dataset.

[0232] Identifying allele-specific targets using predictive algorithms This method was used computationally to predict allele-specific chimeric nuclease sites. Specifically, as shown in Figure 2A, a specially coded Python script was created that uses the pairwise mutant and wild-type sequences generated above as input. The user selects from a set of chimeric nuclease parameters accompanied by a position-weighted matrix of binding and cleavage preferences for the I-TevI ​​site, linker, and Cas9 site. The algorithm then inputs pairs of wild-type and mutant sequences from the dataset and identifies and scores chimeric nuclease sites. Sites found in the mutant sequence compared to the wild-type sequence, as well as sites restricted to the mutant sequence or that are allele-specific, are printed in lines in a table. The script then proceeds to the next pair of wild-type and mutant sequences in the dataset until all paired sequences have been compared. The output consists of a table of predicted chimeric nuclease sites where the nuclease targets the mutant sequence but not the wild-type.

[0233] The development of predictive algorithms allows for the systematic identification of allele-specific chimeric nuclease targets for potential therapeutic gene editing, replacing traditional manual processes. In this example, 682 potential TevSaCas9 sequences, SEQ ID NOS: 1-682, were identified from the top 1,000 most common cancer-causing mutations in the Genomic Data Commons (GDC) Data Portal. Single nucleotide polymorphisms, deletions, or insertions are oncogenic mutations. Each of these variants can be targeted by therapeutic gene editing for the treatment of cancer. Such insertion variants were computationally predicted to contain allele-specific chimeric nuclease sites in large in-frame insertion mutations in the mucin-4 (Muc4) gene (Figures 2B and 2C).

[0234] To generate Figure 2B, wild-type (WT) and mutant (MUT) sequence pairs containing 50 bases upstream and downstream of the mutation site were called from the GRCh38.p13 human genome sequence and input into the algorithm along with selected chimeric nuclease parameters, such as the preferred I-TevI ​​domain, linker, and Cas9 domain. Sequence preferences were matched against a position-weighted matrix of each domain's known binding and cleavage preferences relative to their natural preferences from in vitro data. The algorithm identified chimeric nuclease sites present in the mutant sequence but absent from the wild-type sequence, and each site found was output as a line in a table along with its position-weighted matrix score. Once all sites have been identified for a given sequence pair, the algorithm cycles through the next pair of mutant and wild-type sequences until all pairs in the input dataset have been analyzed.

[0235] Example 2: Activity of chimeric nucleases that selectively target inserted oncogenes In this study, a chimeric nuclease was designed to target a large in-frame oncogenic insertion in the designated mucin-4 (Muc4) gene, i.e., chr3:g.195781031_195781032insACCGGTGGATGCCGAGGAAGCGTCGGTGACAGGAAGAGGGGTGGTGTCACCTGTGGATACTGAGGAAAAGCTGGTGACAGGAAGAGGGGTGGCGTGACCTGTGGATACTGAGGAAGTGTCGGTGACAGGAAGAGTCGTGGTGTC (SEQ ID NO: 780) mutation. Mucin-4 is thought to be involved in various cancers, particularly colon cancer. Selective disruption of insertion mutations in Muc4 using a chimeric nuclease that generates an out-of-frame deletion can provide therapeutic benefit to patients. As shown in Figure 3A, the chimeric nuclease was targeted to Muc4 using a guide RNA of SEQ ID NO: 1685, which targets the TevSaCas9 chimeric nuclease to the insertion mutation but not wild-type Muc4. The TevSaCas9 chimeric nuclease was purified from E. coli and complexed with the guide RNA of SEQ ID NO: 1685. The complex was mixed with DNA substrate in an in vitro cleavage assay containing the Muc4 insertion mutation in one reaction and the wild-type Muc4 sequence in the second reaction. Figure 3B shows the results of the in vitro cleavage assay visualized using agarose gel electrophoresis. This demonstrates that TevSaCas9 programmed with the Muc4 insertion preferentially cleaves the oncogenic mutation sequence (presence of cleavage product) but does not cleave the wild-type Muc4 sequence (absence of cleavage product).

[0236] Example 3: Activity of chimeric nucleases to selectively eliminate cancer cells In another embodiment of the present invention, a chimeric nuclease is designed to target the oncogenic activating mutation Egfr L858R to selectively eliminate cancer cells. The Egfr L858R mutation is known to drive tumorigenesis and malignancy in non-small cell lung cancer (NSCLC). Targeting and eliminating this activating mutation can provide therapeutic benefits for treating patients with cancer. As shown in Figure 4A, a guide RNA of SEQ ID NO: 1686 is used to target the chimeric nuclease to the Egfr L858R mutation. The Egfr L858R mutation is located in the guide RNA binding site of the Egfr gene. Because TevSaCas9 activity requires accurate guide RNA binding, the mutant Egfr L858R gene can be distinguished from wild-type Egfr genes using the described nuclease. The TevSaCas9 chimeric nuclease is purified from E. coli and complexed with the guide RNA of SEQ ID NO: 1686. In an in vitro cleavage assay, the complex was mixed with a DNA substrate amplified from cells containing the Egfr L858R mutation and a separate reaction from cells wild-type for Egfr. Figure 4B shows the results of a time-course in vitro cleavage assay visualized using agarose gel electrophoresis. TevSaCas9 programmed to Egfr L858R preferentially cleaves the Egfr L858R mutation over wild-type Egfr substrates. The described chimeric nuclease selectively eliminates cancer cells. CRL-5908 cells (American Type Culture Collection, Manassas, VA, USA) are an immortalized lung epithelial cell line containing the Egfr L858R activating mutation. The control lung epithelial cell line, NuLi-1, is wild-type for the Egfr L858 amino acid. Figure 4C demonstrates that CRL-5908 cells lipofected with TevSaCas9 targeted to the Egfr L858R mutation with a guide RNA of SEQ ID NO: 1686 ("TevSaCas9-EGFRL858R") have reduced viability as measured by CellTiter-Blue viability dye (Promega, Madison, Wis., USA) compared to mock-treated cells.The viability of NuLi-1 cells was not reduced by the same TevSaCas9-EGFR L858R complex, nor by NuLi-1 cells or CRL-5908 cells treated with TevSaCas9 ("TevSaCas9") without guide RNA. Together, these data demonstrate allele-specific reduction of cell viability in human cells containing the Egfr L858R activating mutation by a chimeric nuclease that specifically targets the mutation. Identifying the location

[0237]

[0217] Other embodiments of the present methods for identifying sites include the use of coding languages ​​other than Python to encode algorithms for identifying allele-specific targets. In further embodiments, the chimeric nuclease parameters can include binding and cleavage preferences for orthologs of I-TevI ​​and / or Cas9 domains.

[0238] Table 4 shows the output from a predictive algorithm of a set of putative TevSaCas9 sites at oncogenic mutations where a nucleotide deletion is the driver mutation. Any of the sequences disclosed as target sites in Table 4 (SEQ ID NOS: 1-683) can be targeted using the chimeric nucleases and methods described herein. Exemplary guide strands that can be used to target these target sites are also disclosed (SEQ ID NOS: 1001-1686).

[0239]

[0219] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0240]

Table 4-1

[0241]

Table 4-2

[0242]

Table 4-3

[0243]

Table 4-4

[0244]

Table 4-5

[0245]

Table 4-6

[0246]

Table 4-7

[0247]

Table 4-8

[0248]

Table 4-9

[0249]

Table 4-10

[0250]

Table 4-11

[0251]

Table 4-12

[0252]

Table 4-13

[0253]

Table 4-14

[0254]

Table 4-15

[0255]

Table 4-16

[0256]

Table 4-17

[0257]

Table 4-18

[0258]

Table 4-19

[0259]

Table 4-20

[0260]

Table 4-21

[0261]

Table 4-22

[0262]

Table 4-23

[0263]

Table 4-24

[0264]

Table 4-25

[0265]

Table 4-26

[0266]

Table 4-27

[0267]

Table 4-28

[0268]

Table 4-29

[0269]

Table 4-30

[0270]

Table 4-31

[0271]

Table 4-32

[0272]

Table 4-33

[0273]

Table 4-34

[0274]

Table 4-35

[0275]

Table 4-36

[0276]

Table 4-37

[0277]

Table 4-38

[0278]

Table 4-39

[0279]

Table 4-40

[0280]

Table 4-41

[0281]

Table 4-42

[0282]

Table 4-43

[0283]

Table 4-44

[0284]

Table 4-45

[0285]

Table 4-46

[0286]

Table 4-47

[0287]

Table 4-48

[0288]

Table 4-49

[0289]

Table 4-50

[0290]

Table 4-51

[0291] [Table 4-52]

[0292] [Table 4-53]

[0293] [Table 4-54]

[0294] [Table 4-55]

[0295] [Table 4-56]

[0296] [Table 4-57]

[0297] [Table 4-58]

[0298] [Table 4-59]

[0299] SEQ ID NO: 683 (EGFR V769_D770insASV target sequence) CcacgctggcCACGCTGGCCATCACGTAGGCTTCCTGGAGGGGAGGGAGAGG SEQ ID NO: 1683 (EGFR guide RNA sequence) CGTAGGCTTCCTGGAGGGAGG SEQ ID NO: 1684 (EGFR guide RNA sequence) TCACGTAGGCTTCCTGGAGGG SEQ ID NO: 1685 (Muc4 guide RNA sequence) GAAGAGTCGTGGTGTCACCG SEQ ID NO: 1686 (EGFR L858R guide RNA sequence) GATTTTGGGCgGGCCAAACTG

[0300] SEQ ID NO: 700 MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSI LEEIPYEKDLIIERENFWIKELNSKINGYNIA Length:93 Type: Amino acid Feature: I-TEVI domain

[0301] SEQ ID NO: 701 DATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRN Length:77 Type: Amino acid Feature: Linker domain

[0302] SEQ ID NO: 702 [I-TEVI WT nuclease domain and linker domain] MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRN

[0303] SEQ ID NO: 703 DATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGS Length:83 Type: Amino acid Characteristics: Linker domain with GGSGGS

[0304] SEQ ID NO: 704 [I-TEVI WT nuclease domain and linker domain with GGSGGS] MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIADATFGDTCSTHPLKEEIIKKRSETVKAKMLKLGPDGRKALYSKPGSKNGRWNPETHKFCKCGVRIQTSAYTCSKCRNGGSGGS

[0305] SEQ ID NO:710 Length: 1,053 Type: Amino acid Characteristics: Staphylococcus aureus Cas9

[0306] SEQ ID NO:711 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA Length: 1,368 Type: Amino acid Characteristics: Streptococcus pyogenes Cas9

[0307] SEQ ID NO:712 Length: 1082 Type: Amino acid Characteristics: Neisseria meningitidis Cas9

[0308] SEQ ID NO:713 MARILAFDIGISSIWAFSENDELKDCGVRIFTKAENPKTGESLAPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDLIFKKQREGFSFSKKFEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNTLNEVLKNGTLTYKQTKKLLGLSDDYEFKREKGTYFIEFKKYKEFIKALGEHNLSQDDLNNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLITPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRA KIEKEQNENYKAKKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNKTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKDFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHEAKSGMLTSALRHTWGFSTKDRNNHLHHAIDAVIIAAYANSIVKAFSDFKKEQESNSAE LYAKKISELDYKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEFYQSYGGKEGVLALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK Length: 984 Type: Amino acid Characteristics: Campylobacter jejuni Cas9

[0309] SEQ ID NO:714 Length: 1377 Type: Amino acid Characteristics: Streptococcus pasteurianus Cas9

[0310] SEQ ID NO:715 Length: 1021 Type: Amino acid Characteristics: Clostridium cellulolyticum Cas9

[0311] SEQ ID NO:716 Length: 1082 Type: Amino acid Feature: Geobacillus thermodenitrificans T1 Cas9

[0312] SEQ ID NO: 720 [Cas12a]

[0313] SEQ ID NO: 721 [CasX] QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVAQPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYFGRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQRALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQDIILEHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNVVAQIVIWVNLNLWQKLKIGRDEAKPLQRLKGFPSFPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLSSEEDRKKKKKFARYQFGDLLLLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGD LRGKPFAIEAENSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYTVINKKSGEIVPMEVNMFNFDDPNLIFDPALFVALTFERREVLDSSNIKPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRTIQAAKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTCSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETHADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWKPAV

[0314] SEQ ID NO: 730 [I-TEVI WT nuclease domain, linker domain, and Staphylococcus aureus Cas9]

[0315] SEQ ID NO:750 [I-TEVI WT nuclease domain, linker domain with GGSGGS, and Staphylococcus aureus Cas9]

[0316] SEQ ID NO: 731 [I-TEVI WT nuclease domain, linker domain, and Streptococcus pyogenes Cas9]

[0317] SEQ ID NO:751 [I-TEVI WT nuclease domain, a linker domain containing GGSGGS, and Streptococcus pyogenes Cas9]

[0318] SEQ ID NO: 732 [I-TEVI WT nuclease domain, linker domain, and Neisseria meningitidis Cas9]

[0319] SEQ ID NO:752 [I-TEVI WT nuclease domain, a linker domain containing GGSGGS, and Neisseria meningitidis Cas9]

[0320] SEQ ID NO: 733 [I-TEVI WT nuclease domain, linker domain, and Campylobacter jejuni Cas9]

[0321] SEQ ID NO:753 [I-TEVI WT nuclease domain, a linker domain containing GGSGGS, and Campylobacter jejuni Cas9]

[0322] SEQ ID NO:734 [I-TEVI WT nuclease domain, linker domain, and Streptococcus pasteurianus Cas9]

[0323] SEQ ID NO:754 [I-TEVI WT nuclease domain, a linker domain containing GGSGGS, and Streptococcus pasteurianus Cas9]

[0324] SEQ ID NO: 735 [I-TEVI WT nuclease domain, linker domain, and Clostridium cellulolyticum Cas9]

[0325] SEQ ID NO:755 [I-TEVI WT nuclease domain, a linker domain containing GGSGGS, and Clostridium cellulolyticum Cas9]

[0326] SEQ ID NO:736 [I-TEVI WT nuclease domain, linker domain, and Geobacillus thermodenitrificans T1 Cas9]

[0327] SEQ ID NO:756 [I-TEVI WT nuclease domain, a linker domain containing GGSGGS, and Geobacillus thermodenitrificans T1 Cas9]

[0328] SEQ ID NO: 740 [SV40 nuclear localization sequence] PKKKRKV SEQ ID NO: 741 [nucleoplasmin nuclear localization sequence] KRPAATKKAGQAKKKK

Claims

1. 1. A composition comprising a chimeric nuclease, the chimeric nuclease comprising: (a) a I-TEVI nuclease domain comprising a mutation at any one of positions corresponding to T11, V16, N14, E25, K26, R27, E36, K37, G38, C39, S41, L45, F49, I60, and E81 of SEQ ID NO: 700, or a combination thereof; (b) an RNA-guided nuclease Cas domain; and (c) a guide RNA comprising a nucleic acid sequence that targets an oncogenic mutation; and the oncogenic mutation is: (i) the insertion of one or more nucleotides; (ii) a substitution or deletion of 10 or fewer nucleotides; or (iii) single nucleotide polymorphisms, The composition,

2. 2. The composition of claim 1, wherein the I-TEVI nuclease domain comprises a mutation selected from the group consisting of mutations corresponding to any one of T11V, V16I, N14G, E25D, K26R, R27A, E36S, K37N, G38N, C39V, S41H, L45F, F49Y, I60V, E81I, or a combination thereof.

3. 2. The composition of claim 1, wherein the oncogenic mutation is an oncogenic mutation in a gene selected from any one of EGFR, Muc4, PIK3CA, KRAS, or a combination thereof.

4. The composition of claim 1 , wherein the oncogenic mutation is not a deletion in exon 19 of EGFR.

5. The composition of claim 1, wherein the sequence containing an oncogenic mutation is selected from the mutations set forth in any one of SEQ ID NOs: 1 to 683.

6. 2. The composition of claim 1, wherein the oncogenic mutation comprises a mutation corresponding to an EGFR L858R mutation or an EGFR V769_D770insASV mutation.

7. 2. The composition of claim 1, wherein the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 45, 130, or 141, or comprises a nucleotide sequence set forth in SEQ ID NO: 1045, 1130, 1141, or 1686.

8. 2. The composition of claim 1, wherein the guide RNA hybridizes to a target nucleotide sequence set forth in SEQ ID NO: 683 or comprises a nucleotide sequence set forth in SEQ ID NO: 1683 or 1684.

9. 2. The composition of claim 1, further comprising a linker operably linked to the I-TEVI nuclease domain and the RNA-guided nuclease Cas domain.

10. The composition of claim 1, wherein the RNA-guided nuclease Cas domain is an RNA-guided nuclease Cas9 domain.

11. The RNA-guided nuclease Cas9 domain is selected from the group consisting of RNA-guided nuclease Staphylococcus aureus Cas9 domain, RNA-guided nuclease Streptococcus pyogenes Cas9 domain, RNA-guided nuclease Neisseria meningitidis Cas9 domain, RNA-guided nuclease Campylobacter jejuni Cas9 domain, RNA-guided nuclease Streptococcus pasteurianus Cas9 domain, RNA-guided nuclease Streptococcus pasteurianus Cas9 domain, RNA-guided nuclease Clostridium 11. The composition of claim 10, wherein the Cas9 domain is one of the following: C. cellulolyticum Cas9 domain, or the RNA-guided nuclease Geobacillus thermodenitrificans T1 Cas9 domain.

12. 12. The composition of claim 11, wherein the RNA-guided nuclease Staphylococcus aureus Cas9 domain comprises a mutation corresponding to the D10E mutation.

13. The composition of claim 1, wherein the I-TEVI nuclease domain comprises an amino acid sequence that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

700.

14. The composition of claim 1 , further comprising a donor nucleic acid.

15. The composition of claim 1 , wherein the donor nucleic acid restores a non-oncogenic function of a gene that contains an oncogenic mutation.

16. A nucleic acid or a plurality of nucleic acids encoding the chimeric nuclease or guide RNA of claim 1, said nucleic acid or a plurality of nucleic acids optionally further comprising a donor nucleic acid portion.

17. 17. The nucleic acid or nucleic acids according to claim 16, which is an expression vector selected from a plasmid, a lentiviral vector, an adeno-associated viral vector, or an adenoviral vector.

18. 10. The composition of claim 1 for use in a method of silencing or disrupting at least a portion of an oncogenic mutation in a cell comprising contacting the composition of claim 1 with the cell.

19. 15. The composition of claim 1 for use in a method of replacing at least a portion of an oncogenic mutation in a cell, comprising contacting the cell with the composition of claim 14.

20. 10. The composition of claim 1 for use in a method of treating cancer in an individual comprising administering the composition of claim 1 to an individual having cancer, thereby treating the individual's cancer.

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