# US 2023/0265404 A1 · Engineered mad7 directed endonuclease

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2023/0265404 A1
- Document ID: US20230265404A1
- Status: Published application
- Kind: A1
- Application number: 18/010,092
- Filed: 2021-06-16
- Published: 2023-08-24
- Earliest claimed: 2020-06-16
- Assignees: Bio-Techne Corporation
- Applicants: Bio-Techne Corporation
- Inventors: Bryan Jones; Neil Otto; Blake Barnes
- Web page: https://patentgrep.com/patent/US20230265404A1

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/85: for animal cells
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present disclosure provides CRISPR systems using engineered MAD7 endonucleases, as well as methods, vectors, nucleic acid compositions, and kits thereof. In particular, provided herein are MAD7 nickases, catalytically dead MAD7 enzymes, and hyperactive MAD7 enzymes.

## Claims

### Claim 1

A modified MAD7 enzyme comprising a mutation in one or more catalytic domains, wherein the modified MAD7 enzyme possesses nickase activity.

### Claim 2 (depends on 1)

The modified MAD7 enzyme of claim 1, wherein the one or more catalytic domains are selected from a RuvC endonuclease domain and a nuclease domain.

### Claim 3 (depends on 2)

The modified MAD7 enzyme of claim 2, wherein the mutation comprises a substitution mutation at one or more amino acid positions selected from 880, 881, 898, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1048, 1050, 1071, 1080, 1082, 1098, 1099, 1101, 1173, 1174, 1175, 1184, 1185, 1189, 1190, 1191, 1198, 1254, 1255, and 1258 relative to SEQ ID NO: 1.

### Claim 4 (depends on 3)

The modified MAD7 enzyme of claim 3, wherein the mutation comprises one of more of E880A, R881A, Q898A, Y1037A, T1038A, S1039A, K1040A, 11041A, D1042A, P1043A, T1045A, G1046A, F1047A, V1048A, I1050A, I1071A, F1080A, F1082A, K1098A, S1099A, W1101A, R1173A, N1174A, S1175A, Y1184A, D1185A, S1189A, P1190A, V1191A, F1198A, F1254A, D1255A, and Q1258A.

### Claim 5 (depends on 4)

The modified MAD7 enzyme of claim 4, wherein the mutation comprises R1173A.

### Claim 6

A modified MAD7 enzyme comprising a mutation in one or more catalytic domains, wherein the enzyme is catalytically inactive.

### Claim 7 (depends on 6)

The modified MAD7 enzyme of claim 6, wherein the one or more catalytic domains are selected from a RuvC endonuclease domain and a nuclease domain.

### Claim 8 (depends on 6, 7)

The modified MAD7 enzyme of claim 6 or claim 7, wherein the enzyme binds to a target DNA.

### Claim 9 (depends on 7, 8)

The modified MAD7 enzyme of claim 7 or claim 8, wherein the mutation comprises a truncation mutation in an amino acid sequence encoding the RuvC endonuclease domain and/or the nuclease domain.

### Claim 10 (depends on 9)

The modified MAD7 enzyme of claim 9, wherein the mutation comprises a deletion in one or more amino acids at positions 1023-1260 relative to SEQ ID NO: 1.

### Claim 11 (depends on 10)

The modified MAD7 enzyme of claim 10, wherein the mutation comprises a deletion of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than 90% of the amino acids at positions 1023-1260 relative to SEQ ID NO: 1.

### Claim 12 (depends on 8)

The modified MAD7 enzyme of claim 8, wherein the mutation comprises a substitution mutation at one or more amino acid positions within 6 angstroms of DNA in a homology model of the catalytic residues 962E or 877D relative to SEQ ID NO: 1.

### Claim 13 (depends on 12)

The modified MAD7 enzyme of claim 12, wherein the mutation comprises a substitution at one or more amino acid positions selected from 858, 874, 875, 876, 877, 878, 879, 880, 881, 883, 885, 893, 895, 902, 927, 933, 934, 937, 939, 940, 942, 944, 962, 963, 964, 967, 968, 969, 972, 973, 974, 975, 976, 980, 981, 982, 983, 984, 987, 988, 990, 991, 992, 993, 994, 995, 997, 1003, 1005, 1006, 1008, 1011, 1012, 1013, 1014, 1024, 1026, 1028, 1031, 1032, 1033, 1034, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1045, 1046, 1047, 1054, 1064, 1068, 1069, 1071, 1073, 1080, 1082, 1085, 1086, 1089, 1101, 1107, 1109, 1116, 1129, 1141, 1146, 1153, 1168, 1171, 1173, 1174, 1175, 1185, 1189, 1190, 1191, 1198, 1200, 1201, 1208, 1209, 1211, 1213, 1215, 1216, 1218, 1220, 1223, 1224, 1225, 1231, 1246, 1248, 1249, 1250, 1253, 1256, 1258, 1262, and 1263 relative to SEQ ID NO: 1.

### Claim 14 (depends on 13)

The modified MAD7 enzyme of claim 13, wherein the mutation comprises one or more of N858A, I874A, G875A, I876A, D877A, R878A, G879A, E880A, R881A, L883A, Y885A, G893A, I895A, N902A, W927A, I933A, K934A, K937A, G939A, Y940A, S942A, V944A, E962A, D963A, L964A, G967A, F968A, K969A, R972A, F973A, K974A, V975A, E976A, Y980A, Q981A, K982A, F983A, E984A, L987A, I988A, K990A, L991A, N992A, Y993A, L994A, V995A, K997A, E1003A, G1005A, G1006A, L1008A, Y1011A, Q1012A, L1013A, T1014A, G1024A, Q1026A, G1028A, F1031A, Y1032A, V1033A, P1034A, Y1037A, T1038A, S1039A, K1040A, I1041A, D1042A, P1043A, T1045A, G1046A, F1047A, K1054A, F1064A, F1068A, D1069A, I1071A, Y1073A, F1080A, F1082A, D1085A, Y1086A, F1089A, W1101A, G1107A, R1109A, N1116A, T1129A, I1141A, G1146A, I1153A, L1168A, Q1171A, R1173A, N1174A, S1175A, D1185A, S1189A, P1190A, V1191A, F1198A, D1200A, 51201A, L1208A, P1209A, D1211A, D1213A, N1215A, G1216A, Y1218A, I1220A, K1223A, G1224A, L1225A, I1231A, L1246A, I1248A, S1249A, N1250A, W1253A, F1256A, Q1258A, Y1262A, and L1263A relative to SEQ ID NO: 1.

### Claim 15 (depends on 13)

The modified MAD7 enzyme of claim 13, wherein the mutation comprises one or more of N858Q, I874Q, G875Q, I876Q, D877Q, R878Q, G879Q, E880Q, R881Q, L883Q, Y885Q, S887Q, V888Q, I889Q, D890Q, G893Q, I895Q, E897Q, Q898Q, S900Q, N902Q, W927Q, I930Q, I933Q, K934Q, E935Q, K937Q, E938Q, G939Q, Y940Q, L941Q, S942Q, V944Q, H946Q, I948Q, Y955Q, N956Q, I958Q, E962Q, D963Q, L964Q, G967Q, F968Q, K969Q, G971Q, R972Q, K974Q, V975Q, E976Q, Q978Q, V979Q, Y980Q, Q981Q, K982Q, F983Q, E984Q, L987Q, I988Q, K990Q, L991Q, N992Q, Y993Q, L994Q, V995Q, K997Q, E1003Q, G1005Q, G1006Q, L1008Q, Y1011Q, Q1012Q, L1013Q, T1014Q, G1024Q, Q1026Q, G1028Q, F1031Q, Y1032Q, V1033Q, P1034Q, Y1037Q, T1038Q, S1039Q, K1040Q, I1041Q, D1042Q, P1043Q, T1045Q, G1046Q, F1047Q, K1054Q, F1064Q, F1068Q, D1069Q, I1071Q, Y1073Q, F1080Q, F1082Q, D1085Q, Y1086Q, F1089Q, W1101Q, G1107Q, R1109Q, N1116Q, T1129Q, I1141Q, G1146Q, I1153Q, L1168Q, Q1171Q, R1173Q, N1174Q, S1175Q, D1185Q, S1189Q, P1190Q, V1191Q, F1198Q, D1200Q, S1201Q, L1208Q, P1209Q, D1213Q, N1215Q, G1216Q, Y1218Q, I1220Q, K1223Q, G1224Q, L1225Q, I1231Q, L1246Q, I1248Q, S1249Q, N1250Q, W1253Q, F1256Q, Q1258Q, Y1262Q, and L1263Q relative to SEQ ID NO: 1.

### Claim 16 (depends on 15)

The mutation of claim 15, wherein the mutation comprises E962Q.

### Claim 17

A modified MAD7 enzyme, wherein the enzyme comprises a mutation in a domain selected from a PAM binding domain, a RuvC endonuclease domain, and a nuclease domain, wherein the enzyme possesses increased nuclease activity compared to wild-type MAD7 enzyme.

### Claim 18 (depends on 17)

The modified MAD7 enzyme of claim 17, wherein the enzyme further possesses increased nickase activity compared to wild-type MAD7.

### Claim 19 (depends on 17, 18)

The modified MAD7 enzyme of claim 17 or 18, wherein the enzyme comprises a substitution at one or more amino acid positions selected from 121, 124, 125, 158, 168, 172, 180, 272, 275, 280, 290, 363, 406, 409, 443, 503, 510, 537, 557, 561, 583, 599, 601, 604, 618, 621, 622, 624, 652, 675, 852, 855, 916, 918, 922, 907, 977, 985, 1022, 1025, 1029, 1114, 1115, 1118, 1157, 1160, 1167, 1241, and 1242 relative to SEQ ID NO: 1.

### Claim 20 (depends on 19)

The modified MAD7 enzyme of claim 19, wherein the mutation comprises one or more of N121K, S124K, A125K, S158K, F168H, A172K, I180K, N190H, E272K, N275K, Q280K, A290R, N363R, N406K, L409K, H443K, L503K, Q510K, Y537K, A557K, P561K, N583K, S599K, T601K, E604K, Q618K, H621K, I622K, S624K, N652K, L675K, N852K, G855K, Q916R, G918K, I922K, K970R, R977K, T985K, N1022K, H1025K, Q1092K, F1114R, V1115K, R1118K, E1157K, Q1160K, R1167K, F1241K, and S1242K relative to SEQ ID NO: 1.

### Claim 21 (depends on 17, 18)

The modified MAD7 enzyme of claim 17 or 18, wherein the enzyme comprises one or more substitution mutations selected from I12T, S15Y, Q185, A24E, E29G, T30K, Q33E, F34N, V36E, G48A, R51Y, D56K, G64D, S67E, T69A, K84Y, Q88Y, G92D, D96K, T97E, 199E, Y105L, A108E, H110V, A114K, M122L, N141E, Q152E, A161T, S163Y, D166G, Y167F, A172K, C174M, S182T, S184I, C185A, H186Y, A193L, E194P, F197L, S198D, A200I, R204E, V207K, N212P, S219E, S225E, M229K, Y235F, Y237L, K239Y, G241N, I244L, S250D, C256I, K258G, S261E, M263I, N275K, Y277P, Q280K, C288S, I289D, A290R, Y294S, E295F, Y298E, Y307L, G312E, L314Y, H321N, V323L, G330F, Y333L, V344K, S345N, F347A, Y348L, E349T, T355L, R357G, E360S, I368E, H369Y, N377K, N391K, L393K, Q394S, K395F, T398A, C410E, T419N, H422K, H426E, Q434L, E435L, H443K, L449E, A451V, V457F, V460S, A464L, W467F, C468L, S469K, V470P, M472L, L476E, K516E, I524N, S538D, M545R, F555M, A557K, K563F, N583K, T601K, T631E, I646K, D656K, D689Y, L692E, Q694V, D717P, N755K, R768K, A772N, Q782K, D802G, A813K, N817D, G820K, H822S, T826Y, N827D, Y832K, Y836E, M843V, F856N, E868N, T891Q, C892K, Y907T, I911E, K914D, Q916R, A919E, Q921D, I922K, E926N, I936L, L943Q, A960V, S965N, K970R, T985K, N989D, I999K, I1001P, T1002D, I1016P, P1017F, K1019S, L1020F, N1022K, V1023L, H1025K, C1029I, I1050L, T1057K, V1058N, R1062K, C1081E, I1090T, Q1092K, V1095E, M1096G, S1100K, S1102T, V1108E, R1113F, F1114R, V1115K, F1119W, S1120D, D1124E, D1131E, M1132L, E1133K, T1135L, M1138K, T1139Y, W1143Y, Y1156K, I1158F, V1159F, Q1160K, H1161S, I1162L, L1176D, L1179K, R1186Y, N1196G, A1202R, A1207S, C1219N, T1232K, and S1242K relative to SEQ ID NO: 1.

### Claim 22 (depends on 17, 18)

The modified MAD7 enzyme of claim 17 or 18, wherein the enzyme comprises one or more substitution mutations selected from N91K, N121K, S124K, A125K, L156K, S158K, R159K, D166K, F168H, A172K, I180K, N190H, D254R, D254K, F262H, C267R, E272K, N275R, N275K, Q280R, Q280K, A290R, A290K, T292K, Y298K, S345K, F347K, R357K, E360R, E360H, N363R, N363K, S405K, N406K, L409K, C410K, C410H, H443R, H443K, S499K, L503K, Q510K, I524K, Y537K, A557K, P561K, I565K, N583K, S599K, T601K, E604K, T605K, Q618K, N619K, H621K, I622K, I622H, S624K, D627K, I630K, N652K, L675R, L675K, N852K, G855K, F856R, F856K, Q916R, Q916K, G918K, A919K, Q921K, I922R, I922K, K970R, R977K, T985K, I1016K, N1022K, H1025R, H1025K, I1050H, D1055K, I1090K, Q1092R, Q1092K, Q1092H, N1093K, V1095K, M1096K, S1097K, R1112K, R1113K, F1114R, F1114K, V1115K, R1118K, S1120K, E1157K, V1159H, Q1160R, Q1160K, Q1160H, H1161R, H1161K, E1164R, E1164K, R1167K, F1241K, S1242K, and R1243K relative to SEQ ID NO: 1.

### Claim 23 (depends on 17, 18)

The modified MAD7 enzyme of claim 17 or 18, herein the enzyme comprises one or more substitution mutations selected from N91R, N91K, N121R, N121K, S124K, A125K, L156K, L156H, S158R, S158K, R159K, D166K, F168H, A172R, A172K, S176K, D178K, D179K, I180K, S181H, N190H, L210K, L210H, D213R, D213K, F251R, F251K, D254R, D254K, S261K, F262K, F262H, N264K, L265K, Y266H, C267R, C267K, N270K, N270H, E272R, E272K, K274R, N275R, N275K, L276R, L276K, K278R, Q280R, Q280K, K281R, I289K, A290R, A290K, D291K, T292K, S293K, V296K, Y298K, S345R, S345K, S345H, K346R, F347K, Y348K, S350K, Q353R, Q353K, Q353H, K354R, R357K, D358R, D358K, E360R, E360H, T361K, N363R, N363K, S405K, N406K, N406H, Y407K, L409K, C410K, C410H, H443R, H443K, S499K, L503R, L503K, Q510R, Q510K, S514K, G523K, I524K, T526K, D529K, K533R, Y537R, Y537K, Y537H, S538K, N539K, N540R, N556K, A557R, A557K, K558R, N559K, N559H, K560R, P561R, P561K, P561H, D562R, D562K, K564R, I565K, N583R, N583K, P586K, G587K, N589R, N589K, K590R, P593R, K594R, V595K, S598R, S598K, S599K, K600R, T601K, G602R, G602K, V603K, E604K, T605R, T605K, Y606K, L613K, G615K, Y616R, Y616K, K617R, Q618R, Q618K, N619K, K620R, K620H, H621R, H621K, I622K, I622H, S624K, S625K, D627K, F628K, I630R, I630K, H647R, P648K, E649K, K651R, N652K, N652H, E664K, I666K, S667K, G668K, R671K, E674K, L675R, L675K, L675H, K679R, E743K, T846K, F849R, F849K, A851K, N852K, T854R, T854K, G855R, G855K, F856R, F856K, D859K, K914R, Q916R, Q916K, G918K, A919K, Q921K, I922R, I922K, K925R, E929K, E938R, E938K, Y966K, G967R, K970R, G971K, F973K, R977K, Q981K, T985R, T985K, M986K, I1016K, D1018K, K1021R, N1022K, G1024R, G1024K, H1025R, H1025K, P1034R, V1048R, N1049K, 11050R, I1050H, K1052R, K1052H, K1054R, D1055R, D1055K, I1090K, T1091K, Q1092R, Q1092K, Q1092H, N1093K, T1094K, V1095K, M1096K, S1097K, I1110R, I1110K, K1111R, R1112K, R1113K, F1114R, F1114K, V1115R, V1115K, V1115H, N1116K, G1117R, G1117K, R1118K, R1118H, F1119R, F1119K, S1120K, E1157K, V1159H, Q1160R, Q1160K, Q1160H, H1161R, H1161K, F1163R, E1164R, E1164K, E1164H, R1167K, G1239K, F1241K, S1242K, R1243K, D1244K, L1246K, K1247R, S1249R, S1249K, N1250H, and K1251R relative to SEQ ID NO: 1.

### Claim 24 (depends on 17, 18)

The modified MAD7 enzyme of claim 17 or 18, wherein the mutation comprises a substitution selected from K169R, D529R, and K535R.

### Claim 25

A fusion protein comprising the modified MAD7 enzyme of any of the preceding claims.

### Claim 26 (depends on 25)

The fusion protein of claim 25, further comprising one or more moieties selected from a base editor, an inhibitor of base repair, a homology directed repair enhancer, a chromatin remodeling peptide, a transposase, a photoregulatory protein, an epigenetic modifier, a transcriptional repressor, a transcriptional activator, and a nuclear colocalization signal protein.

### Claim 27 (depends on 26)

The fusion protein of claim 26, wherein the modified MAD7 is conjugated to the one or more additional moieties by a linker.

### Claim 28 (depends on 25, 26, 27)

A system comprising:
i. a modified MAD7 enzyme of any of the preceding claims or a fusion protein of any one of claims 25-27; and
ii. a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence.

### Claim 29 (depends on 28)

The system of claim 28, further comprising donor nucleic acid.

### Claim 30

The system of any of the preceding claims, wherein the target DNA sequence is a genomic DNA sequence in a host cell.

### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

A vector comprising a nucleic acid sequence encoding the modified MAD7 enzyme of any of claims 1-24 or the fusion protein of any one of claims 25-27, and a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence.

### Claim 32 (depends on 28, 29, 30, 31)

A host cell comprising the system of any one of claims 28-30 or the vector of claim 31.

### Claim 33 (depends on 28, 29, 30, 31)

A method of altering a target genomic DNA sequence in a host cell, comprising introducing the system of any one of claims 28-30 or the vector claim 31 into a host cell comprising a target genomic DNA sequence.

### Claim 34 (depends on 33)

The method of claim 33, wherein the host cell is a mammalian cell.

### Claim 35 (depends on 34)

The method of claim 34, wherein the host cell is a human cell.

### Claim 36 (depends on 33, 34, 35)

The method of any one of claims 33-35, wherein the target genomic DNA sequence encodes a gene product.