Genome editing compositions and methods for treatment of cystic fibrosis

Abstract

Provided herein are compositions and methods of using prime editing systems comprising prime editors and prime editing guide RNAs for treatment of genetic disorders such as cystic fibrosis.

Description

WSGR Docket No.59761-775.601 GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF CYSTIC FIBROSIS CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 482,785, filed February 1, 2023, and U.S. Provisional Application No.63 / 596,171, filed November 03, 2023, each of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Cystic fibrosis (CF) is one of the most common genetic disease, particularly in the Caucasian population. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR / ABCC7) gene (chr7: 117480025-117668665 (GRCh38 / hg38)). The CFTR protein encoded by the CFTR gene is a chloride channel, which is involved in fluid transport and surface hydration across epithelial cells of the body’s tubular organs (e.g., lungs and intestines). Mutations in the CFTR gene Mutations in the CFTR gene results in defective biosynthesis, trafficking, and / or activity of the CFTR protein, and may cause severe damage to the lungs, pancreas, liver, intestines, sinuses, and limited ability to breathe overtime. While technological advances have increased the life expectancy of certain CF patients, numerous mutations remain non-responsive to currently existing therapies, and there is still no effective cure for the disease. SUMMARY OF THE DISCLOSURE

[0003] This disclosure provides prime editing methods and compositions for correcting mutations in the CFTR gene associated with Cystic fibrosis.

[0004] Provided herein, in some embodiments, are methods and compositions for prime editing of alterations in a target sequence in a target gene, for example, a CFTR gene. The target CTFR gene may comprise double stranded DNA. As exemplified in FIG.1, in some embodiments, the target gene, e.g., a CFTR gene, is edited by prime editing. In some embodiments, the prime editing described herein results in efficient correction of one or more pathogenic mutations in the CFTR gene, thereby treating cystic fibrosis in a subject.

[0005] Without wishing to be bound by any particular theory, the prime editing process may search specific targets and edit endogenous sequences in a target gene, e.g., the CFTR gene. As exemplified in FIG.1, the spacer sequence of a PEgRNA recognizes and anneals with a search target sequence in a target strand of the target gene. A prime editing complex may generate a nick in the target gene on the edit strand which is the complementary strand of the target strand. The prime editing complex may then use a free 3’ end formed at the nick site of the edit strand to initiate DNA synthesis, where a primer binding site (PBS) of the PEgRNA complexes with the free 3’ end, and a single stranded DNA is synthesized using an editing template of the PEgRNA as a template. The editing template mayWSGR Docket No.59761-775.601 comprise one or more nucleotide edits compared to the endogenous target CFTR gene sequence. Accordingly, the newly-synthesized single stranded DNA also comprises the nucleotide edit(s) encoded by the editing template. Through removal of an editing target sequence on the edit strand of the target gene and DNA repair, the intended nucleotide edit(s) included in the newly synthesized single stranded DNA are incorporated into the target CFTR gene.

[0006] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.

[0007] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer comprising at its 3’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template comprising at its 3’ end sequence number 64, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1.

[0008] In some embodiments, the gRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), wherein T indicates the presence of a uridine nucleotide.

[0009] In some embodiments, the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

[0010] In some embodiments, the 3’ motif is directly connected to the PBS at its 3’ end.

[0011] In some embodiments, the 3’ motif is linked to the PBS at its 3’ end via a linker.

[0012] In some embodiments, the linker is 4 nucleotides in length.

[0013] In some embodiments, the editing template comprises at its 3’end SEQ ID NOs: 68, 76, 84, 91, or 97.

[0014] In some embodiments, the editing template has a length of 20 nucleotides or less.

[0015] In some embodiments, the editing template has a length of 10, 13, 17, or 20 nucleotides.

[0016] In some embodiments, the editing template consists of SEQ ID NO: 64.WSGR Docket No.59761-775.601

[0017] In some embodiments, the spacer is from 17-22 nucleotides in length.

[0018] In some embodiments, the spacer comprises at its 3’ end SEQ ID NO: 10.

[0019] In some embodiments, the spacer has the sequence of SEQ ID NO: 10.

[0020] In some embodiments, the PBS comprises at its 5’ end sequence number 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, or 61.

[0021] In some embodiments, the PBS comprises at its 5’ end sequence number 28, 37, 43, or 49.

[0022] In some embodiments, the PBS comprises at its 5’ end sequence number 19, 22, 25, 55, 58, or 61.

[0023] In some embodiments, the PBS has a length of 20 nucleotides or less.

[0024] In some embodiments, the PBS is 8-15 nucleotides in length.

[0025] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 306, 309, 310, 314, 317, 318, 322, 328, 335, 336, 345, 353, 363, 364, 371, 382, 390, 399, 400, 410, 425, 426, 443, and 457.

[0026] In one aspect, provided herein is a prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of the aspects and embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: an ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 473, 474, 475, 476, or 477, and an ngRNA core capable of binding a Cas9 protein.

[0027] In some embodiments, the ngRNA spacer comprises at its 3’ end SEQ ID NO: 473.

[0028] In some embodiments, the ngRNA spacer comprises at its 3’ end SEQ ID NO: 475.

[0029] In some embodiments, the ngRNA spacer comprises at its 3’ end SEQ ID NOs: 474, 476, or 477.

[0030] In some embodiments, the ngRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), wherein T indicates the presence of a uridine nucleotide.WSGR Docket No.59761-775.601

[0031] In some embodiments, the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 491, 493, 494, 496, 499, 500, 501, 504, 505, 506, 507, or 508.

[0032] In some embodiments, the ngRNA comprise SEQ ID NOs: 486, 487, 489, 491, 493, 494, 496, 500, 501, 504, 505, 506, 507, or 508.

[0033] In some embodiments, the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507, or 508.

[0034] In some embodiments, the ngRNA comprises SEQ ID NOs: 491, 493, 494, or 496.

[0035] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 2; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 2, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.

[0036] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer comprising at its 3’ end SEQ ID NO: 2; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template comprising at its 3’ end nucleotides 4-8 of sequence number 66, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 2.

[0037] In some embodiments, the editing template comprises at its 3’ end sequence number 66.

[0038] In some embodiments, the editing template comprises at its 3’ end sequence number 66, 67, 69, 71, 75, 77, 78, 83, 87, 88, 89, 92, 93, 94, 96, 98, or 100.

[0039] In some embodiments, the editing template consists of sequence number 66.

[0040] In some embodiments, the editing template has a length of 25 nucleotides or less.

[0041] In some embodiments, the editing template is 11 or 12 nucleotides in length.

[0042] In some embodiments, the editing template comprises at its 3’ end SEQ ID NOs: 829, 830, 831, 832, 833, 834, 853, 854, 855, 856, 857, 858, 877, 878, 879, 880, 881, 882, 901, 902, 903, 904, 905, 906, 925, 926, 927, 928, 929, 930, 949, 950, 951, 952, 953, 954, 973, 974, 975, 976, 977, 993, 994, 995, 996, 997, or 998.

[0043] In some embodiments, the editing template further encodes a PAM silencing edit.

[0044] In some embodiments, the editing template encodes a TGA-to-GGT PAM silencing edit.WSGR Docket No.59761-775.601

[0045] In some embodiments, the editing template comprises at its 3’ end nucleotides 7-12 of SEQ ID NO: 72.

[0046] In some embodiments, the editing template comprises at its 3’ end SEQ ID NOs: 72, 80 or 85.

[0047] In some embodiments, the editing template encodes a TGA-to-GGG PAM silencing edit.

[0048] In some embodiments, the editing template comprises at its 3’ end nucleotides 7-12 of SEQ ID NO: 73.

[0049] In some embodiments, the editing template comprises at its 3’ end SEQ ID NOs: 73, 81 or 86.

[0050] In some embodiments, the editing template encodes a TGA-to-GGC PAM silencing edit.

[0051] In some embodiments, the editing template comprises at its 3’ end nucleotides 7-12 of SEQ ID NO: 74.

[0052] In some embodiments, the editing template comprises at its 3’ end SEQ ID NO: 74 or 82.

[0053] In some embodiments, the editing template encodes a GGA-to-GGC PAM silencing.

[0054] In some embodiments, the editing template comprises at its 3’ end SEQ ID NOs: 835, 836, 837, 838, 839, 840, 859, 860, 861, 862, 863, 864, 883, 884, 885, 886, 887, 888, 907, 908, 909, 910, 911, 912, 931, 932, 933, 934, 935, 936, 955, 956, 957, 958, 959, 960, 978, 979, 980, 981, 982, 999, 1000, 1001, 1002, 1003, or 1004.

[0055] In some embodiments, the editing template encodes a GGA-to-GGG PAM silencing.

[0056] In some embodiments, the editing template comprises at its 3’ end SEQ ID NOs: 841, 842, 843, 844, 845, 846, 865, 866, 867, 868, 869, 870, 889, 890, 891, 892, 893, 894, 913, 914, 915, 916, 917, 918, 937, 938, 939, 940, 941, 942, 961, 962, 963, 964, 965, 966, 983, 984, 985, 986, 987, 1005, 1006, 1007, 1008, 1009, or 1010.

[0057] In some embodiments, the editing template encodes a GGA-to-GGT PAM silencing.

[0058] In some embodiments, the editing template comprises at its 3’ end SEQ ID NOs: 847, 848, 849, 850, 851, 852, 871, 872, 873, 874, 875, 876, 895, 896, 897, 898, 899, 900, 919, 920, 921, 922, 923, 924, 943, 944, 945, 946, 947, 948, 967, 968, 969, 970, 971, 972, 988, 989, 990, 991, 992, 1011, 1012, 1013, 1014, 1015, or 1016.

[0059] In some embodiments, the editing template has a length of 16 nucleotides or less.

[0060] In some embodiments, the editing template is 12-16 nucleotides in length.

[0061] In some embodiments, the gRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 603),WSGR Docket No.59761-775.601 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), wherein T indicates the presence of a uridine nucleotide.

[0062] In some embodiments, the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

[0063] In some embodiments, the 3’ motif is directly connected to the PBS at its 3’ end.

[0064] In some embodiments, the 3’ motif is linked to the PBS at its 3’ end via a linker.

[0065] In some embodiments, the linker is 4 nucleotides in length.

[0066] In some embodiments, the spacer is from 17-22 nucleotides in length.

[0067] In some embodiments, the spacer comprises at its 3’ end SEQ ID NO: 11.

[0068] In some embodiments, the spacer has the sequence of SEQ ID NO: 11.

[0069] In some embodiments, the PBS is 8-15 nucleotides in length.

[0070] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 129, 130, 131, 132, 133, 134, 135, 136, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 159, 160, 161, 162, 163, 164, 165, 166, 167, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 315, 319, 321, 323, 326, 327, 329, 331, 332, 333, 334, 337, 340, 341, 342, 343, 344, 346, 347, 348, 349, 350, 351, 352, 354, 358, 359, 360, 361, 362, 365, 366, 367, 368, 369, 370, 372, 373, 376, 377, 378, 379, 380, 381, 383, 385, 386, 387, 388, 389, 391, 394, 395, 396, 397, 398, 401, 402, 404, 405, 406, 407, 408, 409, 411, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 427, 428, 429, 432, 433, 434, 435, 436, 438, 439, 440, 441, 442, 444, 446, 447, 448, 449, 450, 451, 453, 454, 455, 456, 458, 460, 461, 462, 463, 464, 465, 466, 468, 469, 470, 471, 472, 1210, 1211, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 828, and 1017.WSGR Docket No.59761-775.601

[0071] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 126, 137, 156, 168, 244, and 1184.

[0072] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 127, 138, 157, 169, 245, and 1178.

[0073] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 128, 139, 158, 170, and 1172.

[0074] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1018, 1019, 1020, 1021, 1022, 1023, 1042, 1043, 1044, 1045, 1046, 1047, 1066, 1067, 1068, 1069, 1070, 1071, 1090, 1091, 1092, 1093, 1094, 1095, 1114, 1115, 1116, 1117, 1118, 1119, 1138, 1139, 1140, 1141, 1142, 1143, 1162, 1163, 1164, 1165, 1166, 1185, 1186, 1187, 1188, 1189, or 1190.

[0075] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1024, 1025, 1026, 1027, 1028, 1029, 1048, 1049, 1050, 1051, 1052, 1053, 1072, 1073, 1074, 1075, 1076, 1077, 1096, 1097, 1098, 1099, 1100, 1101, 1120, 1121, 1122, 1123, 1124, 1125, 1144, 1145, 1146, 1147, 1148, 1149, 1167, 1168, 1169, 1170, 1171, 1191, 1192, 1193, 1194, 1195, and 1196.

[0076] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1030, 1031, 1032, 1033, 1034, 1035, 1054, 1055, 1056, 1057, 1058, 1059, 1078, 1079, 1080, 1081, 1082, 1083, 1102, 1103, 1104, 1105, 1106, 1107, 1126, 1127, 1128, 1129, 1130, 1131, 1150, 1151, 1152, 1153, 1154, 1155, 1173, 1174, 1175, 1176, 1177, 1197, 1198, 1199, 1200, 1201, and 1202.

[0077] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1036, 1037, 1038, 1039, 1040, 1041, 1060, 1061, 1062, 1063, 1064, 1065, 1084, 1085, 1086, 1087, 1088, 1089, 1108, 1109, 1110, 1111, 1112, 1113, 1132, 1133, 1134, 1135, 1136, 1137, 1156, 1157, 1158, 1159, 1160, 1161, 1179, 1180, 1181, 1182, 1183, 1203, 1204, 1205, 1206, 1207, and 1208.

[0078] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,WSGR Docket No.59761-775.601 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 315, 319, 321, 323, 326, 327, 329, 331, 332, 333, 334, 337, 340, 341, 342, 343, 344, 346, 347, 348, 349, 350, 351, 352, 354, 358, 359, 360, 361, 362, 365, 366, 367, 368, 369, 370, 372, 373, 376, 377, 378, 379, 380, 381, 383, 385, 386, 387, 388, 389, 391, 394, 395, 396, 397, 398, 401, 402, 404, 405, 406, 407, 408, 409, 411, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 427, 428, 429, 432, 433, 434, 435, 436, 438, 439, 440, 441, 442, 444, 446, 447, 448, 449, 450, 451, 453, 454, 455, 456, 458, 460, 461, 462, 463, 464, 465, 466, 468, 469, 470, 471, 472, 1210, 1211, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 828, and 1017.

[0079] In one aspect, provided herein is a prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of the aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 473, 474, 476 or 477; and (ii) an ngRNA core capable of binding a Cas9 protein.

[0080] In some embodiments, the ngRNA spacer comprises SEQ ID NO: 473.

[0081] In some embodiments, the ngRNA comprises SEQ ID NOs: 491, 493, 494, or 496.

[0082] In some embodiments, the ngRNA comprises SEQ ID NO: 496.

[0083] In one aspect, provided herein is a prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of the aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 475; and (ii) an ngRNA core capable of binding a Cas9 protein.

[0084] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NO: 475.

[0085] In some embodiments, the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507, or 508.

[0086] In one aspect, provided herein is a prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 478; and an ngRNA core capable of binding a Cas9 protein.

[0087] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NO: 478.

[0088] In some embodiments, the ngRNA comprises SEQ ID NO: 502.

[0089] In one aspect, provided herein is a prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of aspects or embodiments herein, and (b) a ngRNA, or oneWSGR Docket No.59761-775.601 or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 479; and an ngRNA core capable of binding a Cas9 protein.

[0090] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NO: 479.

[0091] In some embodiments, the ngRNA comprises SEQ ID NO: 503.

[0092] In some embodiments, the ngRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), wherein T indicates the presence of a uridine nucleotide.

[0093] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 3; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.

[0094] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer comprising at its 3’ end SEQ ID NO: 3; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template comprising at its end sequence number 65, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3.

[0095] In some embodiments, the gRNA core comprises nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAAWSGR Docket No.59761-775.601 CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.

[0096] In some embodiments, the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

[0097] In some embodiments, the 3’ motif is directly connected to the PBS at its 3’ end.

[0098] In some embodiments, the 3’ motif is linked to the PBS at its 3’ end via a linker.

[0099] In some embodiments, the linker is 4 nucleotides in length.

[0100] In some embodiments, the editing template comprises at its 3’end SEQ ID NOs: 70, 79, 90, 95, or 99.

[0101] In some embodiments, the editing template has a length of 24 nucleotides or less.

[0102] In some embodiments, the editing template is a length of 10, 14, 18, 21, or 24 nucleotides.

[0103] In some embodiments, the editing template consists of sequence number 65.

[0104] In some embodiments, the spacer is from 17-22 nucleotides in length.

[0105] In some embodiments, the spacer comprises at its 3’ end SEQ ID NO: 12.

[0106] In some embodiments, the spacer has the sequence of SEQ ID NO: 12.

[0107] In some embodiments, the PBS comprises at its 5’ end sequence number 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, or 63.

[0108] In some embodiments, the PBS comprises at its 5’ end sequence number 27, 54, 57, 60, or 63.

[0109] In some embodiments, the PBS comprises at its 5’ end sequence number 27, 33, 39, 45, or 51.

[0110] In some embodiments, the PBS consists of SEQ ID NO: 27.

[0111] In some embodiments, the PBS has a length of 15 nucleotides or less.

[0112] In some embodiments, the PBS is 7-15 nucleotides in length.

[0113] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 307, 308, 312, 313, 316, 320, 324, 325, 330, 338, 339, 355, 356, 357, 374, 375, 384, 392, 393, 403, 412, 413, 419, 430, 431, 437, 445, 452, 459, and 467.

[0114] In one aspect, provided herein is a prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 480, 11, 481, 482, 483, or 484, and (ii) an ngRNA core capable of binding a Cas9 protein.

[0115] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NOs: 480, 11, 481, 482, 483, or 484.

[0116] In some embodiments, the gRNA core comprises nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAAWSGR Docket No.59761-775.601 CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.

[0117] In some embodiments, the ngRNA comprises SEQ ID NOs: 488, 490, 492, 495, 497, or 498.

[0118] In some embodiments, the PEgRNA comprises from 5’ to 3’, the spacer, the gRNA core, the RTT, and the PBS.

[0119] In some embodiments, the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule.

[0120] In some embodiments, the PEgRNA further comprises 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

[0121] In some embodiments, the PEgRNA comprises s 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.

[0122] In some embodiments, the PEgRNA and / or the ngRNA further comprises 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

[0123] In some embodiments, the PEgRNA and / or the ngRNA comprises s 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.

[0124] In one aspect, provided herein is a prime editing system comprising (a) the PEgRNA of any one of the aspects or embodiments herein, or one or more polynucleotides encoding the PEgRNA, and (b) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.

[0125] In some embodiments, the prime editing system further comprises a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.

[0126] In some embodiments, the prime editor is a fusion protein.

[0127] In one aspect, provided herein is a prime editing system comprising (a) the PEgRNA of any one of the aspects or embodiments herein or one or more polynucleotides encoding the PEgRNA, (b) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N- intein or a polynucleotide encoding the N-terminal extein; and (c) a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding theWSGR Docket No.59761-775.601 C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.

[0128] In some embodiments, the prime editing system further comprises: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (d) a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C- terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.

[0129] In one aspect, provided herein is a population of viral particles collectively comprising the one or more polynucleotides encoding the prime editing system of any one of the aspects or embodiments herein.

[0130] In some embodiments, the viral particles are AAV particles.

[0131] In one aspect, provided herein is an LNP comprising the prime editing system of any one of the aspects or embodiments herein.

[0132] In some embodiments, the PEgRNA, the polynucleotide encoding the Cas9 nickase, and the polynucleotide encoding the reverse transcriptase.

[0133] In some embodiments, the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are mRNA.

[0134] In some embodiments, the polynucleotide encoding the Cas9 nickase and the polynucleotide d encoding the reverse transcriptase are the same molecule.

[0135] In one aspect, provided herein is a method of editing a CFTR gene, the method comprising contacting the CFTR gene with: (a) the PEgRNA of any one of the aspects or embodiments herein, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase or (b) the prime editing system of any one of the aspects or embodiments herein.

[0136] In some embodiments, the CFTR gene is in a cell.

[0137] In some embodiments, the cell is a mammalian cell.

[0138] In some embodiments, the cell is a human cell.

[0139] In some embodiments, the cell is a primary cell.

[0140] In some embodiments, the cell is an epithelial cell.

[0141] In some embodiments, the cell is in a subject or obtained from a subject or a cell bank.

[0142] In some embodiments, the subject is a human.WSGR Docket No.59761-775.601

[0143] In some embodiments, contacting the CFTR gene comprises contacting the cell with (i) the population of viral particles of claim 113 or 114 or (ii) the LNP of any one of the aspects or embodiments herein.

[0144] In one aspect, provided herein is a method of treating cystic fibrosis in a subject in need thereof, the method comprising administering to the subject (i) the PEgRNA of any one of the aspects or embodiments herein, , and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (ii) the prime editing system of any one of the aspects or embodiments herein, (iii) the population of viral particles of any one of the aspects or embodiments herein, or (iv) the LNP of any one of the aspects or embodiments herein.

[0145] In one aspect, provided herein is a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a spacer that comprises at its 3’ end a PEgRNA spacer sequence selected from any one of Tables 18-20; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template that comprises at its 3’ end an RTT sequence selected from same Table as the PEgRNA spacer sequence, and a primer binding site (PBS) that comprises at its 5’ end a PBS sequence selected from same Table as the PEgRNA spacer sequence.

[0146] In some embodiments, the spacer of the PEgRNA is from 17 to 22 nucleotides in length.

[0147] In some embodiments, the spacer of the PEgRNA is 20 nucleotides in length.

[0148] In some embodiments, the PEgRNA comprised from 5’ to 3’, the spacer, the gRNA core, the editing template, and the PBS.

[0149] In some embodiments, the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule.

[0150] In some embodiments, the gRNA core comprises a gRNA core sequence selected from Table 10.

[0151] In one aspect, provided herein is a prime editing system comprising: (a) the prime editing guide RNA (PEgRNA) of any one of the aspects or embodiments herein, or one or more polynucleotides encoding the PEgRNA; and optionally (b) a nick guide RNA (ngRNA), or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises a spacer comprising at its 3’ end nucleotides 4-20 of any ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence, and a ngRNA core capable of binding to a Cas9 protein.

[0152] In some embodiments, the ngRNA spacer is from 17 to 22 nucleotides in length.

[0153] In some embodiments, the spacer of the ngRNA comprises at its 3’ end nucleotides 3-20, 2- 20, or 1-20 of the ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence.

[0154] In some embodiments, the ngRNA spacer is 20 nucleotides in length.WSGR Docket No.59761-775.601

[0155] In some embodiments, the ngRNA core comprises a gRNA core sequence selected from Table 10.

[0156] In some embodiments, the prime editing system further comprises: (c) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or a nucleic acid encoding the Cas9 nickase, and a reverse transcriptase, or a nucleic acid encoding the reverse transcriptase.

[0157] In some embodiments, the prime editing system further comprises: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (d) a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C- terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase and a reverse transcriptase (RT) domain. INCORPORATION BY REFERENCE

[0158] All publications, patents, and patent applications mentioned in this specification 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0160] FIG.1 depicts a schematic of a prime editing guide RNA (PEgRNA) binding to a double stranded target DNA sequence.

[0161] FIG.2 depicts a PEgRNA architectural overview in an exemplary schematic of PEgRNA designed for a prime editor.

[0162] FIG.3 is a schematic showing the spacer and gRNA core part of an exemplary guide RNA, in two separate molecules. The rest of the PEgRNA structure is not shown.

[0163] FIG.4 shows restoration of swelling and CFTR function in prime edited patient-derived intestinal organoids after incubation with 10 µM of forskolin. DETAILED DESCRIPTION OF THE DISCLOSURE

[0164] Provided herein, in some embodiments, are compositions and methods to edit the target gene cystic fibrosis transmembrane conductance regulator (CFTR / ABCC7) with prime editing. In certainWSGR Docket No.59761-775.601 embodiments, provided herein are compositions and methods for correction of mutations in the CFTR gene associated with cystic fibrosis. Compositions provided herein can comprise prime editors (Pes) that may use engineered guide polynucleotides, e.g., prime editing guide RNAs (PEgRNAs), that can direct Pes to specific DNA targets and can encode DNA edits on the target gene CFTR that serve a variety of functions, including direct correction of disease-causing mutations associated with cystic fibrosis.

[0165] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. Definitions

[0166] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

[0167] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof as used herein mean “comprising”.

[0168] Unless otherwise specified, the words “comprising”, “comprise”, “comprises”, “having”, “have”, “has”, “including”, “includes”, “include”, “containing”, “contains” and “contain” are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0169] Reference to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure.

[0170] The term “about” or “approximately” in relation to a numerical means a range of values that fall within 10% greater than or less than the value. For example, about x means x±(10% * x).

[0171] The term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.WSGR Docket No.59761-775.601

[0172] The terms “protein” and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three- dimensional conformation. In some embodiments, a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined 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, enzyme precursor proteins, regulatory protein, structural protein, receptor, 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 may be a full-length protein (e.g., a fully processed protein having certain biological function). In some embodiments, a protein may be a variant or a fragment of a full-length protein. A variant of a protein or enzyme comprises 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 a reference protein.

[0173] In some embodiments, a protein comprises one or more protein domains or subdomains. As used herein, the term “polypeptide domain”, “protein domain”, or “domain” when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function. In some embodiments, a protein comprises multiple protein domains. In some embodiments, a protein comprises multiple protein domains that are naturally occurring. In some embodiments, a protein comprises multiple protein domains from different naturally occurring proteins. For example, in some embodiments, a prime editor may be a fusion protein comprising a Cas9 protein domain of S. pyogenes and a reverse transcriptase protein domain of a retrovirus (e.g., a Moloney murine leukemia virus) or a variant of the retrovirus. A protein that comprises amino acid sequences from different origins or naturally occurring proteins may be referred to as a fusion, or chimeric protein.

[0174] In some embodiments, a protein comprises a functional variant or functional fragment of a full-length wild type protein. A “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 of the functions, e.g., catalytic or binding functions. For example, a functional fragment of a reverse transcriptase may encompass less than the entire amino acid sequence of a wild type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide. When the reference protein is a fusion of multiple functional domains, a functional fragment thereof may retain one or more of the functions of at least one of the functional domains. For example, aWSGR Docket No.59761-775.601 functional fragment of a Cas9 may encompass less than the entire amino acid sequence of a wild type Cas9 but retains its DNA binding ability and lacks its nuclease activity partially or completely.

[0175] A “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 encompasses one or more alterations to the amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions. In some embodiments, the one or more alterations to the amino acid sequence comprises amino acid substitutions, insertions or deletions, or any combination thereof. In some embodiments, the one or more alterations to the amino acid sequence comprises amino acid substitutions. For example, a functional variant of a reverse transcriptase may comprise one or more amino acid substitutions compared to the amino acid sequence of a wild type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide. When the reference protein is a fusion of multiple functional domains, a functional variant thereof may retain one or more of the functions of at least one of the functional domains. For example, in some embodiments, a functional fragment of a Cas9 may comprise one or more amino acid substitutions in a nuclease domain, e.g., an H840A amino acid substitution, compared to the amino acid sequence of a wild type Cas9, but retains the DNA binding ability and lacks the nuclease activity partially or completely.

[0176] The term “function” and its grammatical equivalents as used herein may refer to a capability of operating, having, or serving an intended purpose. Functional may comprise any percent from baseline to 100% of an intended purpose. For example, functional may comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose. In some embodiments, the term functional may mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.

[0177] In some embodiments, a protein or polypeptides includes naturally occurring amino acids (e.g., one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V). In some embodiments, a protein or polypeptides includes non-naturally occurring amino acids (e.g., amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics). In some embodiments, a protein or polypeptide is modified.

[0178] In some embodiments, a protein comprises an isolated polypeptide. The term “isolated” means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.WSGR Docket No.59761-775.601

[0179] In some embodiments, a protein is present within a cell, a tissue, an organ, or a virus particle. In some embodiments, a protein is present within a cell or a part of a cell (e.g., a bacteria cell, a plant cell, or an animal cell). In some embodiments, a protein is present in a mixture of analytes (e.g., a lysate). In some embodiments, the protein is present in a lysate from a plurality of cells or from a lysate of a single cell.

[0180] The terms “homologous,” “homology,” or “percent homology” as used herein refer to the degree of sequence identity between an amino acid and a corresponding reference amino acid sequence or a polynucleotide sequence and a corresponding reference polynucleotide sequence. “Homology” can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar. Homology can mean, for example, nucleic acid sequences with 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 nucleic acid sequences may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid sequence. For example, a “region of homology to a genomic region” can be a region of DNA that has a similar sequence to a given genomic region in the genome. A region of homology can be of any length that is sufficient to promote binding of a spacer, a primer binding site, or protospacer sequence to the genomic region. For example, the region of homology can comprise 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, 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 such that the region of homology has sufficient homology to undergo binding with the corresponding genomic region.

[0181] When a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or specified portion of the length.

[0182] Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is 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. Additional known algorithms include those published in: Smith & Waterman, “Comparison of Biosequences”, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch,WSGR Docket No.59761-775.601 “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 implementation of these or similar algorithms. Global alignment programs may also be used to align similar sequences of roughly 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 optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al (“Current Protocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998). In some embodiments, alignment between a query sequence and a reference sequence is performed with Needleman-Wunsch alignment with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment, as further described in Altschul et al.(“Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res.25:3389-3402, 1997) and Altschul et al, (“Protein database searches using compositionally adjusted substitution matrices”, FEBS J.272:5101-5109, 2005).

[0183] A skilled person understands that amino acid (or nucleotide) positions may be determined in homologous sequences based on alignment, for example, “H840” in a reference Cas9 sequence may correspond to H839, or another position in a Cas9 homolog.

[0184] The term “polynucleotide” or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules. In some embodiments, a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA. In some embodiments, a polynucleotide is double stranded, e.g., a double-stranded DNA in a gene. In some embodiments, a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.

[0185] Polynucleotides can have any three-dimensional structure. The following are nonlimiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolatedWSGR Docket No.59761-775.601 RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a tRNA-derived small RNA (tsRNA), an antisense RNA, an shRNA, or a small rDNA-derived RNA (srRNA).

[0186] In some embodiments, a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof. In some embodiments, a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

[0187] In some embodiments, a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. In some embodiments, the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).

[0188] In some embodiments, a polynucleotide may be modified. As used herein, the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides. In some embodiments, modifications may be on the nucleoside base and / or sugar portion of the nucleosides that comprise the polynucleotide. In some embodiments, the modification may be on the internucleoside linkage (e.g., phosphate backbone). In some embodiments, multiple modifications are included in the modified nucleic acid molecule. In some embodiments, a single modification is included in the modified nucleic acid molecule.

[0189] The term “complement”, “complementary”, or “complementarity” as used herein, refers to the ability of two polynucleotide molecules to base pair with each other. Complementary polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, an adenine on one polynucleotide molecule will base pair to a thymine or an uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to a guanine on a second polynucleotide molecule. Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence. For instance, the two DNA molecules 5’-ATGC-3’ and 5’- GCAT-3’ are complementary, and the complement of the DNA molecule 5’-ATGC-3’ is 5’-GCAT- 3’. A percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule. “Substantially complementary” as used herein refers to a degree of complementarity that can be 70%, 75%, 80%,WSGR Docket No.59761-775.601 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. “Substantially complementary” can also refer to a 100% complementarity over a portion“or a region of two polynucl”otide molecules. In some embodiments, the portion or the region of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.

[0190] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which polynucleotides, e.g., the transcribed mRNA, translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of a functional form of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a gene is determined by the amount of the mRNA, or transcript, that is encoded by the gene after transcription the gene. In some embodiments, expression of a polynucleotide, e.g., an mRNA, is determined by the amount of the protein encoded by the mRNA after translation of the mRNA. In some embodiments, expression of a polynucleotide, e.g., a mRNA or coding RNA, is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide.

[0191] The term “sequencing” as used herein, may comprise capillary sequencing, bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high- throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, or any combination thereof.

[0192] The terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular molecule, or biological or cellular material, and means a molecule having minimal homology to another molecule while still maintaining a desired structure or functionality.

[0193] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and / or translated to produce an amino acid, or a polypeptide or fragment thereof. In some embodiments, aWSGR Docket No.59761-775.601 polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid. In some embodiments, a polynucleotide comprises one or more codons that encode a polypeptide. In some embodiments, a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide. In some embodiments, the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide.

[0194] The term “mutation” as used herein refers to a change and / or alteration in an amino acid sequence of a protein or nucleic acid sequence of a polynucleotide. Such changes and / or alterations may comprise the 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 nucleic acid sequence, compared to a reference amino acid or a reference nucleic acid sequence. In some embodiments, the reference sequence is a wild-type sequence. In some embodiments, a mutation in a nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide. In some embodiments, the mutation in the amino acid sequence of the polypeptide or the mutation in the nucleic acid sequence of the polynucleotide is a mutation associated with a disease state.

[0195] The term “subject” and its grammatical equivalents as used herein may refer to a human or a non-human. A subject may be a mammal. In some embodiments, a subject is human. A human subject may be male or female. A human subject may be of any age. A subject may be a human embryo. A human subject may be a newborn, an infant, a child, an adolescent, or an adult. A human subject may be in need of treatment for a genetic disease or disorder.

[0196] The terms “treatment” or “treating” and their grammatical equivalents may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder. Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder. Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder. In addition, this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder. Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder. In some embodiments, a condition may be pathological. In some embodiments, a treatment may not completely cure or prevent a disease, condition, or disorder. In some embodiments, a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder. In some embodiments, a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.WSGR Docket No.59761-775.601

[0197] The term “ameliorate” and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

[0198] The terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder. In some embodiments, a composition, e.g., a pharmaceutical composition, prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject.

[0199] The term “effective amount” or “therapeutically effective amount” refers to a quantity of a composition, for example a prime editing composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein. An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo. An effective amount can be the amount to induce, for example, at least about a 2-fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g., expression of a target CFTR gene to produce functional CFTR protein) observed relative to a negative control. An effective amount or dose can induce, for example, about 2-fold increase, about 3-fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25-fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500-fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation (e.g., expression of a target CFTR gene to produce functional CFTR protein). The amount of target gene modulation may be measured by any suitable method known in the art. In some embodiments, the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo).

[0200] In some embodiments, an effective amount can be an amount to induce, when administered to a population of cells, a certain percentage of the population of cells to have a correction of a mutation. For example, in some embodiments, an effective amount can be the amount to induce, when administered to or introduced to a population of cells, installation of one or more intended nucleotide edits that correct a mutation in the target CFTR gene, in at least about 1%, 2%, 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the population of cells.WSGR Docket No.59761-775.601 Prime Editing

[0201] The term “prime editing” refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit (also referred to herein as a nucleotide change) into the target DNA through target-primed DNA synthesis. A target gene of prime editing may comprise a double stranded DNA molecule having two complementary strands: a first strand that may be referred to as a “target strand” or a “non-edit strand”, and a second strand that may be referred to as a “non-target strand,” or an “edit strand.” In some embodiments, in a prime editing guide RNA (PEgRNA), a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a “search target sequence”. In some embodiments, the spacer sequence anneals with the target strand at the search target sequence. The target strand may also be referred to as the “non-Protospacer Adjacent Motif (non-PAM strand).” In some embodiments, the non-target strand may also be referred to as the “PAM strand”. In some embodiments, the PAM strand comprises a protospacer sequence and optionally protospacer adjacent motif (PAM) sequence. In prime editing using a Cas-protein-based prime editor, a PAM sequence refers to a short DNA sequence immediately adjacent to the protospacer sequence on the PAM strand of the target gene. A PAM sequence may be specifically recognized by a programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease. In some embodiments, a specific PAM is characteristic of a specific programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease. A protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence. In a PEgRNA, a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T).

[0202] In some embodiments, the double stranded target DNA comprises a nick site on the PAM strand (or non-target strand). As used herein, a “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target DNA. In some embodiments, the position of a nick site is determined relative to the position of a specific PAM sequence. In some embodiments, the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence. In some embodiments, the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is upstream of a PAM sequence recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. In some embodiments, the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P. lavamentivorans Cas9 nickase, a C. diphtheriae Cas9 nickase, a N. cinerea Cas9, a S. aureus Cas9, or a N. lari Cas9 nickase. In someWSGR Docket No.59761-775.601 embodiments, the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. In some embodiments, the nick site is 2 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a S. thermophilus Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. By “upstream” and “downstream” it is intended to define relevant positions at least two regions or upstream of a second sequence in a DNA molecule where the first sequence is positioned 5’ to the second sequence. Accordingly, the second sequence is downstream of the first sequence.

[0203] A “primer binding site” (also referred to as PBS or primer binding site sequence) is a single- stranded portion of the PEgRNA that comprises a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand). The PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site. In some embodiments, in the process of prime editing, the PEgRNA complexes with and directs a prime editor to bind the search target sequence on the target strand of the double stranded target DNA and generates a nick at the nick site on the non-target strand of the double stranded target DNA. In some embodiments, the PBS is complementary to or substantially complementary to, and primed DNA synthesis.

[0204] the PBS and encodes a single strand of DNA. The editing template may comprise a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand), and comprises one or more intended nucleotide edits compared to the endogenous sequence of the double stranded target DNA. In some embodiments, the editing template and the PBS are immediately adjacent to each other. Accordingly, in some embodiments, a PEgRNA in prime editing comprises a single-stranded portion that comprises the PBS and the editing template immediately adjacent to each other. In some embodiments, the single stranded portion of the PEgRNA comprising both the PBS and the editing template is complementary or substantially complementary to an endogenous sequence on the PAM strand (i.e., the non-target strand or the edit strand) of the double stranded target DNA except for one or more non-complementary nucleotides at the intended nucleotide edit position(s). As used herein, the editing template, and the relative positions as among elements of a PEgRNA, are determined by double stranded target DNA that may have complementarity or identity to elements of the PEgRNA. In some embodiments, the editing template is complementary or substantially complementary to aWSGR Docket No.59761-775.601 sequence on the PAM strand that is immediately downstream of the nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions. The endogenous, e.g., genomic, sequence that is complementary or substantially complementary to the editing template, except for the one or more non-complementary nucleotides at the position corresponding to the intended nucleotide edit, may be referred to as an “editing target sequence”. In some embodiments, the editing template has identity or substantial identity to a sequence on the target strand that is complementary to, or having the same position in the genome as, the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions. In some embodiments, the editing template encodes a single stranded DNA, wherein the single stranded DNA has identity or substantial identity to the editing target sequence except for one or more insertions, deletions, or substitutions at the positions of the one or more intended nucleotide edits. In some embodiments, the editing template may encode the wild-type or non-disease associated gene sequence (or its complement if the edit strand is the antisense strand of a gene). In some embodiments, the editing template may encode the wild-type or non-disease associated protein, but contain one or more synonymous mutations relative to the wild-type or non-disease associated protein coding region. Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence once the desired edit is installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer).

[0205] In some embodiments, a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene. In some embodiments, the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene at the nick site. In some embodiments, a primer binding site (PBS) of the PEgRNA anneals with a free 3’ end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3’ end as a primer. Subsequently, a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized. In some embodiments, the newly synthesized single-stranded DNA comprises one or more intended nucleotide edits compared to the endogenous target gene sequence. Accordingly, in some embodiments, the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template. The endogenous, e.g., genomic, sequence that is partially complementary to the editing template may be referred to as an “editing target sequence”. Accordingly, in some embodiments, the newly synthesized single stranded DNA has identity or substantial identity to a sequence in the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions. In some embodiments, the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 nucleotides contiguous are located upstream of the 5’ most edit in the editing template.WSGR Docket No.59761-775.601

[0206] In some embodiments, the newly synthesized single-stranded DNA equilibrates with the editing target on the edit strand of the target gene for pairing with the target strand of the target gene. In some embodiments, the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FEN1. In some embodiments, the FEN is an endogenous FEN, for example, in a cell comprising the target gene. In some embodiments, the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided in trans. In some embodiments, the newly synthesized single stranded DNA, which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the edit strand of the target gene. In some embodiments, the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene. In some embodiments, the newly synthesized single-stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the target strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch between the two otherwise complementary strands. In some embodiments, the mismatch is recognized by DNA repair machinery, e.g., an endogenous DNA repair machinery. In some embodiments, through DNA repair, the intended nucleotide edit is incorporated into the target gene. Prime Editor

[0207] The term “prime editor (PE)” refers to the polypeptide or polypeptide components involved in prime editing. In various embodiments, a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity. In some embodiments, the prime editor further comprises a polypeptide domain having nuclease activity. In some embodiments, the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity. In some embodiments, the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease. As used herein, the term “nickase” refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target. In some embodiments, the prime editor comprises a polypeptide domain that is an inactive nuclease. In some embodiments, the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease. In some embodiments, the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5’ endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation. In some embodiments, the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.WSGR Docket No.59761-775.601

[0208] A prime editor may be engineered. In some embodiments, the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment. In some embodiments, the polypeptide components of a prime editor may be of different origins or from different organisms. In some embodiments, a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species. In some embodiments, a prime editor comprises a Cas polypeptide (DNA binding domain) and a reverse transcriptase polypeptide (DNA polymerase) that are derived from different species. For example, a prime editor may comprise a S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.

[0209] In some embodiments, polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein. In other embodiments, a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences. For example, a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA. Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part. In some embodiments, a single polynucleotide, construct, or vector encodes the prime editor fusion protein. In some embodiments, multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein. For example, a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector. Prime Editor Nucleotide Polymerase Domain

[0210] In some embodiments, a prime editor comprises a nucleotide polymerase domain, e.g., a DNA polymerase domain. The DNA polymerase domain may be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, or may be a functional mutant, a functional variant, or a functional fragment thereof. In some embodiments, the polymerase domain is a template dependent polymerase domain. For example, the DNA polymerase may rely on a template polynucleotide strand, e.g., the editing template sequence, for new strand DNA synthesis. In some embodiments, the prime editor comprises a DNA-dependent DNA polymerase. For example, a prime editor having a DNA-dependent DNA polymerase can synthesize a new single stranded DNA using a PEgRNA editing template that comprises a DNA sequence as a template. In such cases, the PEgRNA is a chimeric or hybrid PEgRNA, and comprising an extension arm comprising a DNA strand. The chimeric or hybrid PEgRNA may comprise an RNA portion (including the spacer and the gRNA core) and a DNA portion (the extension arm comprising the editing template that includes a strand of DNA).WSGR Docket No.59761-775.601

[0211] In some embodiments, the DNA polymerases can be wild type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and / or the polymerases may be modified by genetic engineering, mutagenesis, or directed evolution-based processes. The polymerases can be a T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like. The polymerases can be thermostable, and can include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants and derivatives thereof.

[0212] In some embodiments, the DNA polymerase is a bacteriophage polymerase, for example, a T4, T7, or phi29 DNA polymerase. In some embodiments, the DNA polymerase is an archaeal polymerase, for example, pol I type archaeal polymerase or a pol II type archaeal polymerase. In some embodiments, the DNA polymerase comprises a thermostable archaeal DNA polymerase. In some embodiments, the DNA polymerase comprises a eubacterial DNA polymerase, for example, Pol I, Pol II, or Pol III polymerase. In some embodiments, the DNA polymerase is a Pol I family DNA polymerase. In some embodiments, the DNA polymerase is a E.coli Pol I DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is a E.coli Pol IV DNA polymerase. In some embodiments, the DNA polymerase comprises a eukaryotic DNA polymerase. In some embodiments, the DNA polymerase is a Pol-beta DNA polymerase, a Pol- lambda DNA polymerase, a Pol-sigma DNA polymerase, or a Pol-mu DNA polymerase. In some embodiments, the DNA polymerase is a Pol-alpha DNA polymerase. In some embodiments, the DNA polymerase is a POLA1 DNA polymerase. In some embodiments, the DNA polymerase is a POLA2 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-delta DNA polymerase. In some embodiments, the DNA polymerase is a POLD1 DNA polymerase. In some embodiments, the DNA polymerase is a POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a human POLD1 DNA polymerase. In some embodiments, the DNA polymerase is a human POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a POLD3 DNA polymerase. In some embodiments, the DNA polymerase is a POLD4 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-epsilon DNA polymerase. In some embodiments, the DNA polymerase is a POLE1 DNA polymerase. In some embodiments, the DNA polymerase is a POLE2 DNA polymerase. In some embodiments, the DNA polymerase is a POLE3 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-eta (POLH) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-iota (POLI) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-kappa (POLK) DNA polymerase. In some embodiments, the DNA polymerase is a Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a human Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a viral DNA-dependent DNA polymerase. In someWSGR Docket No.59761-775.601 embodiments, the DNA polymerase is a B family DNA polymerases. In some embodiments, the DNA polymerase is a herpes simplex virus (HSV) UL30 DNA polymerase. In some embodiments, the DNA polymerase is a cytomegalovirus (CMV) UL54 DNA polymerase.

[0213] In some embodiments, the DNA polymerase is an archaeal polymerase. In some embodiments, the DNA polymerase is a Family B / pol I type DNA polymerase. For example, in some embodiments, the DNA polymerase is a homolog of Pfu from Pyrococcus furiosus. In some embodiments, the DNA polymerase is a pol II type DNA polymerase. For example, in some embodiments, the DNA polymerase is a homolog of P. furiosus DP1 / DP22-subunit polymerase. In some embodiments, the derived from archaea with optimal growth temperatures that are similar to the desired assay temperatures.

[0214] In some embodiments, the DNA polymerase comprises a thermostable archaeal DNA polymerase. In some embodiments, the thermostable DNA polymerase is isolated or derived from Pyrococcus species (furiosus, species GB-D, woesii, abysii, horikoshii), Thermococcus species (kodakaraensis KOD1, litoralis, species 9 degrees North-7, species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus.

[0215] Polymerases may also be from eubacterial species. In some embodiments, the DNA polymerase is a Pol I family DNA polymerase. In some embodiments, the DNA polymerase is an E.coli Pol I DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase. In some embodiments, the DNA Polymerase is a Pol III family DNA polymerase. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is an E.coli Pol IV DNA polymerase. In some embodiments, the Pol I DNA activity.

[0216] Suitable thermostable pol I DNA polymerases can be isolated from a variety of thermophilic eubacteria, including Thermus species and Thermotoga maritima such as Thermus aquaticus (Taq), Thermus thermophilus (Tth) and Thermotoga maritima (Tma UlTma).

[0217] In some embodiments, a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT). A RT or an RT domain may be a wild type RT domain, a full-length RT domain, or may be a functional mutant, a functional variant, or a functional fragment thereof. An RT or an RT domain of a prime editor may comprise a wild-type RT, or may be engineered or evolved to contain specific amino acid substitutions, truncations, or variants. An engineered RT may comprise sequences or amino acid changes different from a naturally occurring RT. In some embodiments, the engineered RT may have improved reverse transcription activity over a naturally occurring RT or RT domain. In some embodiments, the engineered RT may haveWSGR Docket No.59761-775.601 improved features over a naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity. In some embodiments, a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a reference naturally occurring RT.

[0218] In some embodiments, a prime editor comprises a virus RT, for example, a retrovirus RT. Non-limiting examples of virus RT include Moloney murine leukemia virus (M-MLV MMLVRT or M-MLV RT); human T-cell leukemia virus type 1 (HTLV-1) RT; bovine leukemia virus (BLV) RT; Rous Sarcoma Virus (RSV) RT; human immunodeficiency virus (HIV) RT, M-MFV RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus (UR2AV) RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT, all of which may be suitably used in the methods and composition described herein.

[0219] A prime editor can comprise a wild-type M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof. Table 1 provides sequences of illustrative M-MLV RTs suitable for use with compositions and methods of the disclosure.

[0220] In some embodiments, a prime editor comprises a wild-type M-MLV RT as set forth in SEQ ID NO: 518. In some embodiments, a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 519. In some embodiments, a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 520. In some embodiments, a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 1209.

[0221] Table 1. Illustrative M-MLV SequencesWSGR Docket No.59761-775.601

[0222] In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions: H8X, P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X as compared to a reference M-MLV RT where X is any amino acid other than the original amino acid in the reference M-MLV RT. In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions: H8Y, P51L, S67K, E69K, L139P,WSGR Docket No.59761-775.601 T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N as compared to a reference M- MLV RT. In some embodiments, the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 518.

[0223] In some embodiments, a prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions D200N, T330P, L603W, T306K, or W313F as compared to a reference M- MLV RT. In some embodiments, the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 518.

[0224] In some embodiments, a prime editor comprises a M-MLV RT comprising amino acid substitutions H8Y, D200N, T330P, L603W, T306K, and W313F as compared to a reference M- MMLV RT. In some embodiments, the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 518.

[0225] In some embodiments, a prime editor comprises a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in in Table 1. In some embodiments, the prime editor comprises a M- MLV RT that comprises an amino acid sequence that is selected from the group consisting of: amino acid sequences provided in Table 1 or a variant or fragment thereof. In some embodiments, the prime editor comprises a variant M-MLV RT that comprises an amino acid sequence set forth in SEQ ID NO: 520.

[0226] In some embodiments, an RT variant may be a functional fragment of a reference RT that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, up to 100, up to 200, up to 300, up to 400, or up to 500 or more amino acid changes compared to a reference RT. In some embodiments, the RT variant comprises a fragment of a reference RT, such that the fragment is 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 corresponding fragment of the reference RT. In some embodiments, the fragment is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical, 96%, 97%, 98%, 99%, or 99.5% of the amino acid length of a corresponding reference RT (M-MLV reverse transcriptase). A reference RT can be any one of the RTs shown in Table 1.WSGR Docket No.59761-775.601

[0227] In some embodiments, a functional RT fragment or variant is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600 or more amino acids in length.

[0228] In still other embodiments, the functional RT variant is truncated at the N-terminus or the C- terminus, or both, by a certain number of amino acids which results in a truncated variant that retains sufficient DNA polymerase function. In some embodiments, the functional RT variant, e.g., a functional MMLV RT variant, is truncated at the C-terminus to abolish or reduce RNAase H activity and still retain DNA polymerase activity.

[0229] In some embodiments, a prime editing composition or a prime editing system disclosed herein comprises a polynucleotide (e.g., a DNA, a RNA, e.g., a mRNA) that encodes a M-MLV RT. In some embodiments, the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in Table 1. In some embodiments, the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NOs: 518, 519, or 520. In some embodiments, the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is selected from the group consisting of: the amino acid sequences provided in Table 1. In some embodiments, the polynucleotide encodes a variant M-MLV RT that comprises an amino acid sequence that is set forth in SEQ ID NO: 520.

[0230] In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a Geobacillus stearothermophilus Group II Intron (GsI-IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT. In some embodiments, the prime editor comprises a retron RT. In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (GsI-IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT. In some embodiments, the prime editor comprises a retron RT. Programmable DNA Binding Domain

[0231] In some embodiments, the DNA-binding domain of a prime editor is a programmable DNA binding domain.

[0232] A programmable DNA binding domain refers to a protein domain that is designed to bind a specific nucleic acid sequence, e.g., a target DNA or a target RNA. In some embodiments, the DNA- binding domain is a polynucleotide programmable DNA-binding domain that can associate with a guide polynucleotide (e.g., a PEgRNA) that guides the DNA-binding domain to a specific DNAWSGR Docket No.59761-775.601 sequence, e.g., a search target sequence in a target gene. In some embodiments, the DNA-binding domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated (Cas) protein. A Cas protein may comprise any Cas protein described herein or a functional fragment or functional variant thereof. In some embodiments, a DNA-binding domain may also comprise a zinc-finger protein domain. In other cases, a DNA-binding domain comprises a transcription activator-like effector domain (TALE). In some embodiments, the DNA-binding domain comprises a DNA nuclease. For example, the DNA-binding domain of a prime editor may comprise an RNA-guided DNA endonuclease, e.g., a Cas protein. In some embodiments, the DNA-binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator like effector domain nuclease (TALEN), where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, e.g., a Fok I nuclease domain.

[0233] In some embodiments, the DNA-binding domain comprises a nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises an endonuclease domain having single strand DNA cleavage activity. For example, the endonuclease domain may comprise a FokI nuclease domain. In some embodiments, the DNA-binding domain of a prime editor comprises a nuclease having full nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises a nuclease having modified or reduced nuclease activity as compared to a wild type endonuclease domain. For example, the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild type endonuclease domain. In some embodiments, the DNA- binding domain of a prime editor has nickase activity. In some embodiments, the DNA-binding domain of a prime editor comprises a Cas protein domain that is a nickase. In some embodiments, compared to a wild type Cas protein, the Cas nickase comprises one or more amino acid substitutions in a nuclease domain that reduces or abolishes its double strand nuclease activity but retains DNA binding activity. In some embodiments, the Cas nickase comprises an amino acid substitution in a HNH domain. In some embodiments, the Cas nickase comprises an amino acid substitution in a RuvC domain.

[0234] In some embodiments, the DNA-binding domain comprises a CRISPR associated protein (Cas protein) domain. A Cas protein may be a Class 1 or a Class 2 Cas protein. A Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or a type VI Cas protein. Non-limiting examples of Cas proteins include , Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a / Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx1S, Csx11, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI CasWSGR Docket No.59761-775.601 effector proteins, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, Cas12c / C2c3, homologues, modified or engineered variants, mutants, and / or functional fragments thereof. A Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms.

[0235] A Cas protein, e.g., Cas9, can be from any suitable organism. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis.

[0236] Non-limiting examples of suitable organism include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus 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, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia 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., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some embodiments, the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis (S. lugdunensis).

[0237] In some embodiments, a Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae,WSGR Docket No.59761-775.601 Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0238] In some embodiments, a Cas protein, e.g., Cas9, can be a wild type or a modified form of a Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can be a wild type or a modified form of a Cas protein. A Cas protein, e.g., Cas9, can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can comprise an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof relative to a corresponding wild- type version of the Cas protein. In some embodiments, a Cas protein can be a polypeptide with at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein.

[0239] A Cas protein, e.g., Cas9, may comprise one or more domains. Non-limiting examples of Cas domains include, guide nucleic acid recognition and / or binding domain, nuclease domains (e.g., Dnase or Rnase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains. In various embodiments, a Cas protein comprises a guide nucleic acid recognition and / or binding domain can interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage.

[0240] In some embodiments, a Cas protein, e.g., Cas9, comprises one or more nuclease domains. A Cas protein can comprise an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein. In some embodiments, a Cas protein comprises a single nuclease domain. For example, a Cpf1 may comprise a RuvC domain but lacksWSGR Docket No.59761-775.601 HNH domain. In some embodiments, a Cas protein comprises two nuclease domains, e.g., a Cas9 protein can comprise an HNH nuclease domain and a RuvC nuclease domain.

[0241] In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, wherein all nuclease domains of the Cas protein are active. In some embodiments, a prime editor comprises a Cas protein having one or more inactive nuclease domains. One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity. In some embodiments, a Cas protein, e.g., Cas9, comprising mutations in a nuclease domain has reduced (e.g., nickase) or abolished nuclease activity while maintaining its ability to target a nucleic acid locus at a search target sequence when complexed with a guide nucleic acid, e.g., a PEgRNA.

[0242] In some embodiments, a prime editor comprises a Cas nickase that can bind to the target gene in a sequence-specific manner and generate a single-strand break at a protospacer within double- stranded DNA in the target gene, but not a double-strand break. For example, the Cas nickase can cleave the edit strand or the non-edit strand of the target gene but may not cleave both. In some embodiments, a prime editor comprises a Cas nickase comprising two nuclease domains (e.g., Cas9), with one of the two nuclease domains modified to lack catalytic activity or deleted. In some embodiments, the Cas nickase of a prime editor comprises a nuclease inactive RuvC domain and a nuclease active HNH domain. In some embodiments, the Cas nickase of a prime editor comprises a nuclease inactive HNH domain and a nuclease active RuvC domain. In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the RuvC domain e.g., an amino acid substitution that reduces or abolishes nuclease activity of the RuvC domain. In some embodiments, the Cas9 nickase comprises a D10X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than D. In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the HNH domain e.g., an amino acid substitution that reduces or abolishes nuclease activity of the HNH domain. In some embodiments, the Cas9 nickase comprises a H840X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than H.

[0243] In some embodiments, a prime editor comprises a Cas protein that can bind to the target gene in a sequence-specific manner but lacks or has abolished nuclease activity and may not cleave either strand of a double stranded DNA in a target gene. Abolished activity or lacking activity can refer to an enzymatic activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type exemplary activity (e.g., wild-type Cas9 nuclease activity). In some embodiments, a Cas protein of a prime editor completely lacks nuclease activity. A nuclease, e.g., Cas9, that lacks nuclease activity may be referred to as nuclease inactive or “nuclease dead” (abbreviated by “d”). A nuclease dead Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. InWSGR Docket No.59761-775.601 some embodiments, a dead Cas protein is a dead Cas9 protein. In some embodiments, a prime editor comprises a nuclease dead Cas protein wherein all of the nuclease domains (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpf1 protein) are mutated to lack catalytic activity or are deleted.

[0244] A Cas protein can be modified. A Cas protein, e.g., Cas9, can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and / or enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of the Cas protein.

[0245] A Cas protein can be a fusion protein. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional regulation domain, or a polymerase domain. A Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.

[0246] In some embodiments, the Cas protein of a prime editor is a Class 2 Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, a Cas9 protein homolog, mutant, variant, or a functional fragment thereof. As used herein, a Cas9, Cas9 protein, Cas9 polypeptide or a Cas9 nuclease refers to an RNA guided nuclease comprising one or more Cas9 nuclease domains and a Cas9 gRNA binding domain having the ability to bind a guide polynucleotide, e.g., a PEgRNA. A Cas9 protein may refer to a wild type Cas9 protein from any organism or a homolog, ortholog, or paralog from any organisms; any functional mutants or functional variants thereof; or any functional fragments or domains thereof. In some embodiments, a prime editor comprises a full-length Cas9 protein. In some embodiments, the Cas9 protein can generally comprises at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity to a wild type reference Cas9 protein (e.g., Cas9 from S. pyogenes). In some embodiments, the Cas9 comprises an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof as compared to a wild type reference Cas9 protein.

[0247] In some embodiments, a Cas9 protein may comprise a Cas9 protein from Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), Streptococcus canis (Sc), Streptococcus thermophilus (St), Staphylococcus lugdunensis (Slu), Neisseria meningitidis (Nm), Campylobacter jejuni (Cj), Francisella novicida (Fn), or Treponema denticola (Td), or any Cas9 homolog or ortholog from an organism known in the art. In some embodiments, a Cas9 polypeptide is a SpCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP_038431314 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a SaCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. J7RUA5 or a fragment orWSGR Docket No.59761-775.601 variant thereof. In some embodiments, a Cas9 polypeptide is a ScCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. A0A3P5YA78 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a StCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP_007896501.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a SluCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_230580236.1 or WP_250638315.1 or WP_242234150.1, WP_241435384.1, WP_002460848.1, KAK58371.1, or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a NmCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_002238326.1 or WP_061704949.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a CjCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_100612036.1, WP_116882154.1, WP_116560509.1, WP_116484194.1, WP_116479303.1, WP_115794652.1, WP_100624872.1, or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a FnCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in Uniprot Accession No. A0Q5Y3 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a TdCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in NCBI Accession No. WP_147625065.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a chimera comprising domains from two or more of the organisms described herein or those known in the art. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide from Streptococcus macacae, e.g., comprising the amino acid sequence as set forth in NCBI Accession No. WP_003079701.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide generated by replacing a PAM interaction domain of a SpCas9 with that of a Streptococcus macacae Cas9 (Spy-mac Cas9).

[0248] Exemplary Cas9 and Cas9 nickase variants are provided in Table 2.

[0249] Table 2: Exemplary Cas protein sequencesWSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601

[0250] In some embodiments, a prime editor comprises a DNA binding domain that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in Table 2. In some embodiments, the DNA binding domain comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 differences e.g., mutations e.g., deletions, substitutions and / or insertions compared to any one of the amino acid sequences set forth in Table 2.

[0251] In some embodiments, a prime editor comprises a Cas9 protein that is a Cas9 nickase that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the nickase sequences set forth in Table 2. In some embodiments, a prime editor comprises a Cas9 protein that comprises an amino acid sequence that is selected from the group consisting of the sequences set forth in Table 2. In some embodiments, a prime editor comprises a Cas9 protein that comprises an amino acid sequence that lacks a N-terminus methionine relative to an amino acid sequence set forth in Table 2. In some embodiments, a prime editing compositions or prime editing systems disclosed herein comprises a polynucleotide (e.g., a DNA, or an RNA, e.g., an mRNA) that encodes a Cas9 protein that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in Table 2.

[0252] In some embodiments, a Cas9 protein comprises a Cas9 protein from Streptococcus pyogenes (Sp), e.g., as according to NC_002737.2:854751-858857 or the protein encoded by UniProt Q99ZW2, e.g., as according to SEQ ID NO: 521. In some embodiments, a prime editor comprises a Cas9 protein (e.g., a SpCas9) as according to any one of the sequences set forth in SEQ ID NOs: 521-524 or a variant thereof. In some embodiments, the Cas9 protein is a SpCas9. In some embodiments, a SpCas9 can be a wild type SpCas9, a SpCas9 variant, or a nickase SpCas9. In some embodiments, the SpCas9 lacks the N-terminus methionine relative to a corresponding SpCas9 (e.g., a wild type SpCas9, a SpCas9 variant or a nickase SpCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 521, not including the N-terminus methionine. In some embodiments, a wild type SpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 521. In some embodiments, a prime editor comprises a Cas9 protein comprising one orWSGR Docket No.59761-775.601 more mutations (e.g., amino acid substitutions, insertions and / or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SpCas9). In some embodiments, the Cas9 protein comprising one or more mutations relative to a wild type Cas9 (e.g., a wild type SpCas9) protein comprises an amino acid sequence set forth in SEQ ID NOs: 522, 523, or 524. Exemplary Streptococcus pyogenes Cas9 (SpCas9) amino acid sequence useful in the prime editors disclosed herein are provided in Table 2.

[0253] In some embodiments, a prime editor comprises a Cas9 protein (e.g., a SluCas9) as according to any one of the SEQ ID NOs: 525-527 or a variant thereof. In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus lugdunensis (SluCas9) e.g., as according to any one of the SEQ ID NO: 525, or a variant thereof. In some embodiments, the Cas9 protein is a SluCas9. In some embodiments, a SluCas9 can be a wild type SluCas9, a SluCas9 variant, or a nickase SluCas9. In some embodiments, the SluCas9 lacks the N-terminus methionine relative to a corresponding SluCas9 (e.g., a wild type SluCas9, a SluCas9 variant or a nickase SluCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 525, not including the N-terminus methionine. In some embodiments, a wild type SluCas9 comprises an amino acid sequence set forth in SEQ ID NO: 525. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and / or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SluCas9). In some embodiments, the Cas9 protein comprising one or mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NOs: 526 or 527. Exemplary Staphylococcus lugdunensis Cas9 (SluCas9) amino acid sequence useful in the prime editors disclosed herein are provided in Table 2.

[0254] In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as according to any of the SEQ ID NOs: 528-530, or a variant thereof. In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as set forth in Table 2, or a variant thereof. In some embodiments, the Cas9 protein is a SaCas9. In some embodiments, a SaCas9 can be a wild type SaCas9, a SaCas9 variant, or a nickase SaCas9. In some embodiments, the SaCas9 lacks the N-terminus methionine relative to a corresponding SaCas9 (e.g., a wild type SaCas9, a SaCas9 variant or a nickase SaCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 528, not including the N-terminus methionine. In some embodiments, a wild type SaCas9 comprises an amino acid sequence set forth in SEQ ID NO: 528. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and / or deletions relative to a corresponding wild type Cas9 protein (e.g., a wild type SaCas9). In some embodiments, the Cas9 protein comprising one or more mutations relative to a wild type Cas9 protein comprises anWSGR Docket No.59761-775.601 amino acid sequence set forth in SEQ ID NOs: 529 or 530. Exemplary Staphylococcus aureus Cas9 (SaCas9) amino acid sequence useful in the prime editors disclosed herein are provided in Table 2.

[0255] In some embodiments, a prime editor comprises a Cas9 protein as according to any one of the sequences set forth in SEQ ID NOs: 531-539, 546-548 or a variant thereof. In some embodiments, the Cas9 protein is a Cas9 variant, for example, a SpCas9 variant (e.g., SpCas9-NG, SpCas9-NGA, SpRY, or SpG). In some embodiments, a prime editor comprises a Cas9 protein that lacks the N- terminus methionine relative to a corresponding Cas9 protein (e.g., a Cas9 variant set forth in any one of SEQ ID NOs: 531-539, 546-548). In some embodiments, a prime editor comprises a Cas9 protein (e.g., a Cas9 variant), having an amino acid sequence as according to any one of SEQ ID NOs: 531- 539, 546-548 not including the N-terminus methionine. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and / or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in any one of SEQ ID NOs: 531-539, 546-548). In some embodiments, the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 532, 533, 535, 536, 538, 539, 547, or 548.

[0256] In some embodiments, a Cas9 protein is a chimeric Cas9, e.g., modified Cas9, e.g., synthetic RNA-guided nucleases (sRGNs), e.g., modified by DNA family shuffling, e.g., sRGN3.1, sRGN3.3. In some embodiments, the DNA family shuffling comprises, fragmentation and reassembly of parental Cas9 genes, e.g., one or more of Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa). In some embodiments, a modified sluCas9 shows increased editing efficiency and / or specificity relative to a sluCas9 that is not modified. In some embodiments, a modified Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in editing efficiency compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in specificity compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in cleavage activity compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a editor comprises a Cas9 protein (e.g., a chimeric Cas9), e.g., as according any one of the sequencesWSGR Docket No.59761-775.601 set forth in SEQ ID NOs: 540-545, or a variant thereof. Exemplary amino acid sequences of Cas9 protein (e.g., sRGN) useful in the prime editors disclosed herein are provided in Table 2. In some embodiments, a prime editor comprises a Cas9 protein, that lacks a N-terminus methionine relative to SEQ ID NO: 540 or SEQ ID NO: 543. In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and / or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in any one of SEQ ID NOs: 540, or 543). In some embodiments, the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 541, 542, 544, or 545.

[0257] In some embodiments, a Cas9 protein comprises a variant Cas9 protein containing one or more amino acid substitutions. In some embodiments, a wildtype Cas9 protein comprises a RuvC domain and an HNH domain. In some embodiments, a prime editor comprises a nuclease active Cas9 protein that may cleave both strands of a double stranded target DNA sequence. In some embodiments, the nuclease active Cas9 protein comprises a functional RuvC domain and a functional HNH domain. In some embodiments, a prime editor comprises a Cas9 nickase that can bind to a guide polynucleotide and recognize a target DNA, but can cleave only one strand of a double stranded target DNA. In some embodiments, the Cas9 nickase comprises only one functional RuvC domain or one functional HNH domain. In some embodiments, a prime editor comprises a Cas9 that has a non- functional HNH domain and a functional RuvC domain. In some embodiments, the prime editor can cleave the edit strand (i.e., the PAM strand), but not the non-edit strand of a double stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 having a non-functional RuvC domain that can cleave the target strand (i.e., the non-PAM strand), but not the edit strand of a double stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 that has neither a functional RuvC domain nor a functional HNH domain, which may not cleave any strand of a double stranded target DNA sequence.

[0258] In some embodiments, a prime editor comprises a Cas9 having a mutation in the RuvC domain that reduces or abolishes the nuclease activity of the RuvC domain. In some embodiments, the Cas9 comprises a mutation at amino acid D10 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 comprises a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid D10, G12, and / or G17 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a D10A mutation, a G12A mutation, and / or a G17A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.WSGR Docket No.59761-775.601

[0259] In some embodiments, a prime editor comprises a Cas9 polypeptide having a mutation in the HNH domain that reduces or abolishes the nuclease activity of the HNH domain. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid H840 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid E762, D839, H840, N854, N856, N863, H982, H983, A984, D986, and / or a A987 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a E762A, D839A, H840A, N854A, N856A, N863A, H982A, H983A, A984A, and / or a D986A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid residue R221, N394, and / or H840 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 521). In some embodiments, the Cas9 polypeptide comprises a R221K, N394L, and / or H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation at amino acid residue R220, N393, and / or H839 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 521) lacking a N-terminal methionine, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a R220K, N393K, and / or H839A mutation as compared to a wild type SpCas9 (as set forth in SEQ ID NO: 521) lacking a N-terminal methionine, or a corresponding mutation thereof.

[0260] In some embodiments, a prime editor comprises a Cas9 having one or more amino acid substitutions in both the HNH domain and the RuvC domain that reduce or abolish the nuclease activity of both the HNH domain and the RuvC domain. In some embodiments, the prime editor comprises a nuclease inactive Cas9, or a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 comprises a H840X substitution and a D10X mutation compared to a wild type SpCas9 as set forth in SEQ ID NO: 521 or corresponding mutations thereof, wherein X is any amino acid other than H for the H840X substitution and any amino acid other than D for the D10X substitution. In some embodiments, the dead Cas9 comprises a H840A and a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or corresponding mutations thereof.

[0261] In some embodiments, the N-terminal methionine is removed from the amino acid sequence of a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus (Met (-)) Cas9 nickases include any one of the sequences set forth in SEQ ID NOs: 523, 524, 527, 530, 533, 536, 539, 542, 545, 548, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.WSGR Docket No.59761-775.601

[0262] Besides dead Cas9 and Cas9 nickase variants, the Cas9 proteins used herein may also include other Cas9 variants having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9, e.g., a wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of a reference Cas9, e.g., a wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9.

[0263] In some embodiments, a Cas9 fragment is a functional fragment that retains one or more Cas9 activities. In some embodiments, the Cas9 fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, a prime editor comprises a Cas protein, e.g., a Cas9 variant, comprising modifications that allow altered PAM recognition. Exemplary Cas9 protein amino acid sequence (e.g., Cas9 variant with altered PAM recognition specificities) that are useful in the Prime editors of the disclosure are provided in Table 2. In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow altered PAM recognition. In prime editing using a Cas- protein-based prime editor, a “protospacer adjacent motif (PAM)”, PAM sequence, or PAM-like motif, may be used to refer to a short DNA sequence immediately following the protospacer sequence on the PAM strand of the target gene. In some embodiments, the PAM is recognized by the Cas nuclease in the prime editor during prime editing. In certain embodiments, the PAM is required for target binding of the Cas protein. The specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length. In some embodiments, the PAM can be a 5’ PAM (i.e., located upstream of the 5’ end of the protospacer). InWSGR Docket No.59761-775.601 other embodiments, the PAM can be a 3’ PAM (i.e., located downstream of the 5’ end of the protospacer). In some embodiments, the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5’-NGG-3’ PAM. In some embodiments, the Cas protein of a prime editor has altered or non-canonical PAM specificities. Exemplary PAM sequences and corresponding Cas variants are described in Table 3. It should be appreciated that for each of the variants provided, the Cas protein comprises one or more of the amino acid substitutions as indicated compared to a wild type Cas protein sequence, for example, the Cas9 as set forth in SEQ ID NO: 521. The PAM motifs as shown in Table 3 are in the order of 5’ to 3’.

[0264] In some embodiments, the Cas proteins of the disclosure can also be used to direct transcriptional control of target sequences, for example silencing transcription by sequence-specific binding to target sequences. In some embodiments, a Cas protein described herein may have one or mutations in a PAM recognition motif. In some embodiments, a Cas protein described herein may have altered PAM specificity.

[0265] As used in PAM sequences in Table 3, “N” refers to any one of nucleotides A, G, C, and T, “R” refers to nucleotide A or G, “W” refers to A or T; “V” refers to A, C, or G; and “Y” refers to nucleotide C or T.

[0266] Table 3: Cas protein variants and corresponding PAM sequencesWSGR Docket No.59761-775.601

[0267] In some embodiments, a prime editor comprises a Cas9 polypeptide comprising one or mutations selected from the group consisting of: A61R, L111R, D1135V, R221K, A262T, R324L, N394K, S409I, S409I, E427G, E480K, M495V, N497A, Y515N, K526E, F539S, E543D, R654L, R661A, R661L, R691A, N692A, M694A, M694I, Q695A, H698A, R753G, M763I, K848A, K890N, Q926A, K1003A, R1060A, L1111R, R1114G, D1135E, D1135L, D1135N, S1136W, V1139A, D1180G, G1218K, G1218R, G1218S, E1219Q, E1219V, E1219V, Q1221H, P1249S, E1253K, N1317R, A1320V, P1321S, A1322R, I1322V, D1332G, R1332N, A1332R, R1333K, R1333P, R1335L, R1335Q, R1335V, T1337N, T1337R, S1338T, H1349R, and any combinations thereof as compared to a wildtype SpCas9 polypeptide as set forth in SEQ ID NO: 521.

[0268] In some embodiments, a prime editor comprises a SaCas9 polypeptide. In some embodiments, the SaCas9 polypeptide comprises one or more of mutations E782K, N968K, and R1015H as compared to a wild type SaCas9. In some embodiments, a prime editor comprises a FnCas9 polypeptide, for example, a wildtype FnCas9 polypeptide or a FnCas9 polypeptide comprising one or more of mutations E1369R, E1449H, or R1556A as compared to the wild type FnCas9. In some embodiments, a prime editor comprises a Sc Cas9, for example, a wild type ScCas9 or a ScCas9 polypeptide comprises one or more of mutations I367K, G368D, I369K, H371L, T375S, T376G, and T1227K as compared to the wild type ScCas9. In some embodiments, a prime editor comprises a St1 Cas9 polypeptide, a St3 Cas9 polypeptide, or a SluCas9 polypeptide.

[0269] In some embodiments, a prime editor comprises a Cas polypeptide that comprises a circular permutant Cas variant. For example, a Cas9 polypeptide of a prime editor may be engineered such that the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein, or a Cas9 nickase) are topically rearranged to retain the ability to bind DNA when complexed with a guide RNA (gRNA). An exemplary circular permutant configuration may be N-terminus-[original C-terminus]- [original N-terminus]-C-terminus. Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.

[0270] In various embodiments, the circular permutants of a Cas protein, e.g., a Cas9, may have the following structure: N-terminus–[original C-terminus]–[optional linker]–[original N-terminus]–C- terminus. In some embodiments, a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 521):

[0271] N-terminus–[1268-1368]–[optional linker]–[1-1267]–C-terminus;WSGR Docket No.59761-775.601

[0272] N-terminus–[1168-1368]–[optional linker]–[1-1167]–C-terminus;

[0273] N-terminus–[1068-1368]–[optional linker]–[1-1067]–C-terminus;

[0274] N-terminus–[968-1368]–[optional linker]–[1-967]–C-terminus;

[0275] N-terminus–[868-1368]–[optional linker]–[1-867]–C-terminus;

[0276] N-terminus–[768-1368]–[optional linker]–[1-767]–C-terminus;

[0277] N-terminus–[668-1368]–[optional linker]–[1-667]–C-terminus;

[0278] N-terminus–[568-1368]–[optional linker]–[1-567]–C-terminus;

[0279] N-terminus–[468-1368]–[optional linker]–[1-467]–C-terminus;

[0280] N-terminus–[368-1368]–[optional linker]–[1-367]–C-terminus;

[0281] N-terminus–[268-1368]–[optional linker]–[1-267]–C-terminus;

[0282] N-terminus–[168-1368]–[optional linker]–[1-167]–C-terminus;

[0283] N-terminus–[68-1368]–[optional linker]–[1-67]–C-terminus;

[0284] N-terminus–[10-1368]–[optional linker]–[1-9]–C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).

[0285] In some embodiments, a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 521– 1368 amino acids of UniProtKB – Q99ZW2:

[0286] N-terminus–[102-1368]–[optional linker]–[1-101]–C-terminus;

[0287] N-terminus–[1028-1368]–[optional linker]–[1-1027]–C-terminus;

[0288] N-terminus–[1041-1368]–[optional linker]–[1-1043]–C-terminus;

[0289] N-terminus–[1249-1368]–[optional linker]–[1-1248]–C-terminus; or

[0290] N-terminus–[1300-1368]–[optional linker]–[1-1299]–C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).

[0291] In some embodiments, a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 521)

[0292] N-terminus–[103-1368]–[optional linker]–[1-102]–C-terminus:

[0293] N-terminus–[1029-1368]–[optional linker]–[1-1028]–C-terminus;

[0294] N-terminus–[1042-1368]–[optional linker]–[1-1041]–C-terminus;

[0295] N-terminus–[1250-1368]–[optional linker]–[1-1249]–C-terminus; or

[0296] N-terminus–[1301-1368]–[optional linker]–[1-1300]–C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).

[0297] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, thee C-terminal fragment may correspond to the 95% or more of the C-terminal amino acids of a Cas9 (e.g., amino acids about 1300-1368 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof), or the 90%, 85%, 80%, 75%, 70%, 65%, 60%,WSGR Docket No.59761-775.601 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the C-terminal amino acids of a Cas9 (e.g., SEQ ID NO: 521 or a ortholog or a variant thereof). The N-terminal portion may correspond to 95% or more of the N-terminal amino acids of a Cas9 (e.g., amino acids about 1-1300 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof), or 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the N terminal amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).

[0298] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the –terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or 1% of the amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 ( e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof). In some embodiments, the C- terminal portion that is rearranged to the N-terminus includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).

[0299] In other embodiments, circular permutant Cas9 variants may be a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 521: (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N- terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to precede the original N-terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue. The CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain. For example, the CP site may be located (as set forth in SEQ ID NO: 521 or corresponding amino acidWSGR Docket No.59761-775.601 positions thereof) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to the N-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 would become the new N-terminal amino acid. Nomenclature of these CP-Cas9 proteins may be referred to as Cas9-CP181, Cas9-CP199, Cas9- CP230, Cas9-CP270, Cas9-CP310, Cas9-CP1010, Cas9-CP1016, Cas9-CP1023, Cas9-CP1029, Cas9-CP1041, Cas9-CP1247, Cas9-CP1249, and Cas9-CP1282, respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 521, but may be implemented to make CP variants in any Cas9 sequence, either at CP sites that correspond to these positions, or at other CP sites entirely. This description is not meant to limit the specific CP sites in any way. Virtually any CP site may be used to form a CP-Cas9 variant.

[0300] In some embodiments, a prime editor comprises a Cas9 functional variant that is of smaller molecular weight than a wild type SpCas9 protein. In some embodiments, a smaller-sized Cas9 functional variant may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type II Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type V Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type VI Cas protein.

[0301] In some embodiments, a prime editor comprises a SpCas9 that is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. In some embodiments, a prime editor comprises a Cas9 functional variant or functional fragment that is less than 1300 amino acids, less than 1290 amino acids, than less than 1280 amino acids, less than 1270 amino acids, less than 1260 amino acid, less than 1250 amino acids, less than 1240 amino acids, less than 1230 amino acids, less than 1220 amino acids, less than 1210 amino acids, less than 1200 amino acids, less than 1190 amino acids, less than 1180 amino acids, less than 1170 amino acids, less than 1160 amino acids, less than 1150 amino acids, less than 1140 amino acids, less than 1130 amino acids, less than 1120 amino acids, less than 1110 amino acids, less than 1100 amino acids, less than 1050 amino acids, less than 1000 amino acids, less than 950 amino acids, less than 900 amino acids, less than 850 amino acids, less than 800 amino acids, less than 750 amino acids, less than 700 amino acids, less than 650 amino acids, less than 600 amino acids, less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the one or more functions, e.g., DNA binding function, of the Cas9 protein.

[0302] In some embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, orWSGR Docket No.59761-775.601 modified versions thereof, and preferably comprising a nickase mutation (e.g., a mutation corresponding to the D10A mutation of the wild type Cas9 polypeptide of SEQ ID NO: 521). In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a functional variant or fragment thereof.

[0303] Exemplary Cas proteins and nomenclature are shown in Table 4: Table 4: Exemplary Cas proteins and nomenclature

[0304] In some embodiments, prime editors described herein may also comprise Cas proteins other than Cas9. For example, in some embodiments, a prime editor as described herein may comprise a Cas12a (Cpf1) polypeptide or functional variants thereof. In some embodiments, the Cas12a polypeptide comprises a mutation that reduces or abolishes the endonuclease domain of the Cas12a polypeptide. In some embodiments, the Cas12a polypeptide is a Cas12a nickase. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12a polypeptide.

[0305] In some embodiments, a prime editor comprises a Cas protein that is a Cas12b (C2c1) or a Cas12c (C2c3) polypeptide. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12b (C2c1) or Cas12c (C2c3)WSGR Docket No.59761-775.601 protein. In some embodiments, the Cas protein is a Cas12b nickase or a Cas12c nickase. In some embodiments, the Cas protein is a Cas12e, a Cas12d, a Cas13, Cas14a, Cas14b, Cas14c, Cas14d, protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally- occurring Cas12e, Cas12d, Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Nuclear Localization Sequences

[0306] In some embodiments, a prime editor further comprises one or more nuclear localization sequence (NLS). In some embodiments, the NLS helps promote translocation of a protein into the cell nucleus. In some embodiments, a prime editor comprises a fusion protein, e.g., a fusion protein comprising a DNA binding domain and a DNA polymerase, that comprises one or more NLSs. In some embodiments, one or more polypeptides of the prime editor are fused to or linked to one or more NLSs. In some embodiments, the prime editor comprises a DNA binding domain and a DNA polymerase domain that are provided in trans, wherein the DNA binding domain and / or the DNA polymerase domain is fused or linked to one or more NLSs.

[0307] In certain embodiments, a prime editor or prime editing complex comprises at least one NLS. In some embodiments, a prime editor or prime editing complex comprises at least two NLSs. In some embodiments, a prime editor or prime editing complex comprises at least three NLSs. In some embodiments, a prime editor or prime editing complex comprises more than 4, 5, 6, 7, 8, 9 or 10 NLSs. In embodiments with two or more NLSs, the NLSs can be the same NLS, or they can be different NLSs. In some embodiments, the one or more NLSs of a prime editor comprise bipartite NLSs.

[0308] An NLS can be expressed as part of a prime editor or prime editing complex. In some embodiments, a NLS can be positioned anywhere in a protein’s amino acid sequence, and comprise a short sequence of three, four, or more amino acids. The location of the NLS fusion can be at the N- terminus, the C-terminus, or positioned within a sequence of a prime editor or a component thereof (e.g., inserted between the DNA-binding domain and the DNA polymerase domain of a prime editor fusion protein, between the DNA binding domain and a linker sequence, between a DNA polymerase and a linker sequence, between two linker sequences of a prime editor fusion protein or a component thereof, in either N-terminus to C-terminus or C-terminus to N-terminus order). In some embodiments, a prime editor is a fusion protein that comprises an NLS at the N terminus. In some embodiments, a prime editor is a fusion protein that comprises an NLS at the C terminus. In some embodiments, a prime editor is a fusion protein that comprises at least one NLS at both the NWSGR Docket No.59761-775.601 terminus and the C terminus. In some embodiments, the prime editor is a fusion protein that comprises two NLSs at the N terminus and / or the C terminus.

[0309] Any NLSs that are known in the art are contemplated herein. The NLSs may be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more mutations relative to a wild-type NLS).

[0310] In some embodiments, a nuclear localization signal (NLS) is predominantly basic. In some embodiments, the one or more NLSs of a prime editor are rich in lysine and arginine residues. In some embodiments, the one or more NLSs of a prime editor comprise proline residues.

[0311] Non-limiting examples of NLS sequences suitable for use with methods and compositions of the disclosure are provided in Table 5. In some embodiments, a NLS comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence provided in Table 5. In some embodiments, a NLS comprises an amino acid sequence selected from the group consisting of: the amino acid sequences provided in Table 5. In some embodiments, a prime editing composition comprises a polynucleotide that encodes a NLS that comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence provided in Table 5. In some embodiments, a prime editing composition comprises a polynucleotide that encodes a NLS that comprises any one of the amino acid sequences provided in Table 5.

[0312] Table 5: Exemplary nuclear localization sequences

[0313] In some embodiments, a nuclear localization signal (NLS) comprises SEQ ID NO: 551.

[0314] In some embodiments, a NLS is a monopartite NLS. For example, in some embodiments, a NLS is a SV40 large T antigen NLS comprising the sequence SEQ ID NO: 549. In someWSGR Docket No.59761-775.601 embodiments, a NLS is a bipartite NLS. In some embodiments, a bipartite NLS comprises two basic domains separated by a spacer sequence comprising a variable number of amino acids. In some embodiments, a NLS is a bipartite NLS. In some embodiments, a bipartite NLS consists of two basic domains separated by a linker sequence comprising a variable number of amino acids. In some embodiments, the linker amino acid sequence comprises a Xenopus nucleoplasmin NLS sequence SEQ ID NO: 566. In some embodiments, the NLS comprises a nucleoplasmin NLS sequence SEQ ID NO: 565. In some embodiments, a NLS is a noncanonical sequence such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS.

[0315] Components of a prime editor may be connected to each other in any order. In some embodiments, the DNA binding domain and the DNA polymerase domain of a prime editor may be fused to form a fusion protein, or may be joined by a peptide or protein linker, in any order from the N terminus to the C terminus. In some embodiments, a prime editor comprises a DNA binding domain fused or linked to the C-terminal end of a DNA polymerase domain. In some embodiments, a prime editor comprises a DNA binding domain fused or linked to the N-terminal end of a DNA polymerase domain. In some embodiments, the prime editor comprises a fusion protein comprising the structure NH2-[DNA binding domain]-[polymerase]-COOH; or NH2-[polymerase]-[DNA binding domain]- COOH, wherein each instance ofindicates the presence of an optional linker sequence. In some embodiments, a prime editor comprises a fusion protein and a DNA polymerase domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA binding domain]-[RNA-protein recruitment polypeptide]-COOH. In some embodiments, a prime editor comprises a fusion protein and a DNA binding domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA polymerase domain]-[RNA-protein recruitment polypeptide]-COOH.

[0316] In some embodiments, a prime editor fusion protein, a polypeptide component of a prime editor, or a polynucleotide encoding the prime editor fusion protein or polypeptide component, may be split into an N-terminal half and a C-terminal half or polypeptides that encode the N-terminal half and the C terminal half, and provided to a target DNA in a cell separately. For example, in certain embodiments, a prime editor fusion protein may be split into a N-terminal and a C-terminal half for separate delivery in AAV vectors, and subsequently translated and colocalized in a target cell to reform the complete polypeptide or prime editor protein. In such cases, separate halves of a protein or a fusion protein may each comprise a split-intein to facilitate colocalization and reformation of the complete protein or fusion protein by the mechanism of intein facilitated trans splicing. In some embodiments, a prime editor comprises a N-terminal half fused to an intein-N, and a C-terminal half fused to an intein-C, or polynucleotides or vectors (e.g., AAV vectors) encoding each thereof. When delivered and / or expressed in a target cell, the intein-N and the intein-C can be excised via protein trans-splicing, resulting in a complete prime editor fusion protein in the target cell. In someWSGR Docket No.59761-775.601 embodiments, an exemplary protein described herein may lack a methionine residue at the N- terminus.

[0317] In some embodiments, a prime editor fusion protein comprises a Cas9(H840A) nickase and a wild type M-MLV RT. In some embodiments, a prime editor fusion protein comprises a Cas9(H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT. The amino acid sequence of an exemplary prime editor fusion protein and its individual components is shown in Table 6. In some embodiments, a prime editor fusion protein comprises a Cas9 (R221K N394K H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT. The amino acid sequence of an exemplary Prime editor fusion protein and its individual components in shown in Table 7. In some embodiments an exemplary prime editor protein may comprise an amino acid sequence as set forth in any of the SEQ ID NOs: 567 or 568.

[0318] In some embodiments, a prime editor fusion protein comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any of the prime editor fusion sequences described herein (e.g., PE2 or PE3; Table 6, Table 7) or known in the art.

[0319] In some embodiments, a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA. In some embodiments, the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence SEQ ID NO: 567 (Table 6). Sequence of an exemplary prime editor fusion protein comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 6.

[0320] In some embodiments, a prime editor comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the exemplary prime editor sequence in Table 6.

[0321] Table 6 lists an exemplary prime editor and its componentsWSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601

[0322] In some embodiments, a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g., Cas9((R221K N394K H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9((R221K N394K H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA. In some embodiments, the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence SEQ ID NO: 568 . Sequence of an exemplary prime editor fusion protein comprising a DNA binding domain (e.g., Cas9((R221K N394K H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9 (R221K N394K H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 7.

[0323] In some embodiments, a prime editor comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the exemplary prime editor sequence in Table 7.

[0324] Table 7 lists an exemplary prime editor and its componentsWSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601

[0325] Polypeptides comprising components of a prime editor may be fused via peptide linkers or may be provided in trans relevant to each other. For example, a reverse transcriptase may be expressed, delivered, or otherwise provided as an individual component rather than as a part of a fusion protein with the DNA binding domain. In such cases, components of the prime editor may be associated through non-peptide linkages or co-localization functions. In some embodiments, a prime editor further comprises additional components capable of interacting with, associating with, or capable of recruiting other components of the prime editor or the prime editing system. For example, a prime editor may comprise an RNA-protein recruitment polypeptide that can associate with an RNA-protein recruitment RNA aptamer. In some embodiments, an RNA-protein recruitment polypeptide can recruit, or be recruited by, a specific RNA sequence. Non limiting examples of RNA- protein recruitment polypeptide and RNA aptamer pairs include a MS2 coat protein and a MS2 RNA hairpin, a PCP polypeptide and a PP7 RNA hairpin, a Com polypeptide and a Com RNA hairpin, a Ku protein and a telomerase Ku binding RNA motif, and a Sm7 protein and a telomerase Sm7 binding RNA motif. In some embodiments, the prime editor comprises a DNA binding domain fused or linked to an RNA-protein recruitment polypeptide. In some embodiments, the prime editor comprises a DNA polymerase domain fused or linked to an RNA-protein recruitment polypeptide. In some embodiments, the DNA binding domain and the DNA polymerase domain fused to the RNA-protein recruitment polypeptide, or the DNA binding domain fused to the RNA-protein recruitment polypeptide and the DNA polymerase domain are co-localized by the corresponding RNA-protein recruitment RNA aptamer of the RNA-protein recruitment polypeptide. In some embodiments, the corresponding RNA-protein recruitment RNA aptamer fused or linked to a portion of the PEgRNA or ngRNA. For example, an MS2 coat protein fused or linked to the DNA polymerase and a MS2 hairpin installed on the PEgRNA for co-localization of the DNA polymerase and the RNA-guided DNA binding domain (e.g., a Cas9 nickase).WSGR Docket No.59761-775.601

[0326] In certain embodiments, components of a prime editor are directly fused to each other. In certain embodiments, components of a prime editor are associated to each other via a linker.

[0327] In some embodiments, a prime editor comprises a polypeptide domain, an MS2 coat protein (MCP), that recognizes an MS2 hairpin. In some embodiments, the nucleotide sequence of the MS2 hairpin (or equivalently referred to as the “MS2 aptamer”) is a sequence provided in Table 8. In some embodiments, the amino acid sequence of the MCP is a sequence provided in Table 8.

[0328] Table 8: Exemplary MS2 hairpin and MCP sequences

[0329] As used herein, a linker can be any chemical group or a molecule linking two molecules or moieties, e.g., a DNA binding domain and a polymerase domain of a prime editor. In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker comprises a non-peptide moiety. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length, for example, a polynucleotide sequence. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).

[0330] In certain embodiments, two or more components of a prime editor are linked to each other by a peptide linker. In some embodiments, a peptide linker is 5-100 amino acids in length, for example, 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, 28, 29, 30, 30- 35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some embodiments, the peptide linker is 16 amino acids in length, 24 amino acids in length, 64 amino acids in length, or 96 amino acids in length.

[0331] In some embodiments, a linker comprises 1-100 amino acids.

[0332] Non-limiting examples of linkers are provided in Table 9. In some embodiments, a linker comprises any one of the amino acid sequences set forth in Table 9, or any combination thereof.

[0333] Table 9. Illustrative Peptide Linker SequencesWSGR Docket No.59761-775.601

[0334] In certain embodiments, two or more components of a prime editor are linked to each other by a non-peptide linker. In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3- aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. PEgRNA for editing of CFTR gene

[0335] The term “prime editing guide RNA”, or “PEgRNA”, refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA. In some embodiments, the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing. “Nucleotide edit” or “intended nucleotide edit” refers to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene. Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene, or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA that replaces the editing target sequence, as compared to the editing target sequence. In some embodiments, a PEgRNA comprises a spacer sequence that is complementary or substantially complementary to a search target sequence on a target strand of the target gene. In some embodiments, the PEgRNA comprises a gRNA core that associates with a DNAWSGR Docket No.59761-775.601 binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor. In some embodiments, the PEgRNA further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence may be referred to as an extension arm.

[0336] In certain embodiments, the extension arm comprises a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis. In some embodiments, the PBS is complementary or substantially complementary to a free 3’ end on the edit strand of the target gene at a nick site generated by the prime editor. In some embodiments, the extension arm further comprises an editing template that comprises one or more intended nucleotide edits to be incorporated in the target gene by prime editing. In some embodiments, the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the prime editor, for example, a reverse transcriptase domain. The reverse transcriptase editing template may also be referred to herein as an RT template, or RTT. In some embodiments, the editing template comprises partial complementarity to an editing target sequence in the target gene, e.g., an CFTR gene. In some embodiments, the editing template comprises substantial or partial complementarity to the editing target sequence except at the position of the intended nucleotide edits to be incorporated into the target gene. An exemplary architecture of a PEgRNA including its components is as demonstrated in Fig.2.

[0337] In some embodiments, a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide. In some embodiments, a PEgRNA is a chimeric polynucleotide that includes both RNA and DNA nucleotides. For example, a PEgRNA can include DNA in the spacer sequence, the gRNA core, or the extension arm. In some embodiments, a PEgRNA comprises DNA in the spacer sequence. In some embodiments, the entire spacer sequence of a PEgRNA is a DNA sequence. In some embodiments, the PEgRNA comprises DNA in the gRNA core, for example, in a stem region of the gRNA core. In some embodiments, the PEgRNA comprises DNA in the extension arm, for example, in the editing template. An editing template that comprises a DNA sequence may serve as a DNA synthesis template for a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase. Accordingly, the PEgRNA may be a chimeric polynucleotide that comprises RNA in the spacer, gRNA core, and / or the PBS sequences and DNA in the editing template.

[0338] Components of a PEgRNA may be arranged in a modular fashion. In some embodiments, the spacer and the extension arm comprising a primer binding site sequence (PBS) and an editing template, e.g., a reverse transcriptase template (RTT), can be interchangeably located in the 5’ portion of the PEgRNA, the 3’ portion of the PEgRNA, or in the middle of the gRNA core. In some embodiments, a PEgRNA comprises a PBS and an editing template sequence in 5’ to 3’ order. In some embodiments, the gRNA core of a PEgRNA of this disclosure may be located in between a spacer and an extension arm of the PEgRNA. In some embodiments, the gRNA core of a PEgRNA may be located at the 3’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may beWSGR Docket No.59761-775.601 located at the 5’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 3’ end of an extension arm. In some embodiments, the gRNA core of a PEgRNA may be located at the 5’ end of an extension arm. In some embodiments, the PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, and an extension arm. In some embodiments, the PEgRNA comprises, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, the PEgRNA comprises, from 5’ to 3’: an extension arm, a spacer, and a gRNA core. In some embodiments, the PEgRNA comprises, from 5’ to 3’: an editing template, a PBS, a spacer, and a gRNA core.

[0339] In some embodiments, a PEgRNA comprises a single polynucleotide molecule that comprises the spacer sequence, the gRNA core, and the extension arm. In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules. In some embodiments, a PEgRNA comprise a first polynucleotide molecule that comprises the spacer and a portion of the gRNA core, and a second polynucleotide molecule that comprises the rest of the gRNA core and the extension arm. In some embodiments, the gRNA core portion in the first polynucleotide molecule and the gRNA core portion in the second polynucleotide molecule are at least partly complementary to each other. In some embodiments, the PEgRNA may comprise a first polynucleotide comprising the spacer and a first portion of a gRNA core comprising, which may also be referred to as a crRNA. In some embodiments, the PEgRNA comprise a second polynucleotide comprising a second portion of the gRNA core and the extension arm, wherein the second portion of the gRNA core may also be referred to as a trans-activating crRNA, or tracr RNA. In some embodiments, the crRNA portion and the tracr RNA portion of the gRNA core are at least partially complementary to each other. In some embodiments, the partially complementary portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem, as exemplified in FIG.3.

[0340] In some embodiments, a spacer sequence comprises a region that has substantial complementarity to a search target sequence on the target strand of a double stranded target DNA, e.g., an CFTR gene. In some embodiments, the spacer sequence of a PEgRNA is identical or substantially identical to a protospacer sequence on the edit strand of the target gene (except that the protospacer sequence comprises thymine and the spacer sequence may comprise uracil). In some embodiments, the spacer sequence is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a search target sequence in the target gene. In some embodiments, the spacer comprises is substantially complementary to the search target sequence.

[0341] In some embodiments, the length of the spacer varies from about 10 nucleotides to about 100 nucleotides. In some embodiments, the spacer is 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length. In some embodiments, the spacer is from 15 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, orWSGR Docket No.59761-775.601 20 to 30 nucleotides in length. In some embodiments, the spacer is 16 to 22 nucleotides in length, e.g., about 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, the spacer is 17 to 22 nucleotides in length, e.g., about 17, 18, 19, 20, 21, or 22 nucleotides in length.

[0342] As used herein in a PEgRNA or a nick guide RNA sequence, or fragments thereof such as a spacer, PBS, or RTT sequence, unless indicated otherwise, it should be appreciated that the letter “T” or “thymine” indicates a nucleobase in a DNA sequence that encodes the PEgRNA or guide RNA sequence, and is intended to refer to a uracil (U) nucleobase of the PEgRNA or guide RNA or any chemically modified uracil nucleobase known in the art, such as 5-methoxyuracil.

[0343] The extension arm of a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g., an RTT). The extension arm may be partially complementary to the spacer. In some embodiments, the editing template (e.g., RTT) is partially complementary to the spacer. In some embodiments, the editing template (e.g., RTT) and the primer binding site (PBS) are each partially complementary to the spacer.

[0344] An extension arm of a PEgRNA may comprise a primer binding site sequence (PBS, or PBS sequence) that comprises complementarity to and can hybridize with a free 3’ end of a single stranded DNA in the target gene (e.g., the CFTR gene) generated by nicking with a prime editor at the nick site on the PAM strand.

[0345] The length of the PBS sequence may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA.

[0346] In some embodiments, the PBS is about 3 to 19 nucleotides in length. In some embodiments, the PBS is about 3 to 17 nucleotides in length. In some embodiments, the PBS is about 4 to 16 nucleotides, about 6 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleotides in length. In some embodiments, the PBS is 8 to 17 nucleotides in length. In some embodiments, the PBS is 8 to 16 nucleotides in length. In some embodiments, the PBS is 8 to 15 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length. In some embodiments, the PBS is 8 to 13 nucleotides in length. In some embodiments, the PBS is 8 to 12 nucleotides in length. In some embodiments, the PBS is 8 to 11 nucleotides in length. In some embodiments, the PBS is 8 to 10 nucleotides in length. In some embodiments, the PBS is 8 or 9 nucleotides in length. In some embodiments, the PBS is 16 or 17 nucleotides in length. In some embodiments, the PBS is 15 to 17 nucleotides in length. In some embodiments, the PBS is 14 to 17 nucleotides in length. In some embodiments, the PBS is 13 to 17 nucleotides in length. In some embodiments, the PBS is 12 to 17 nucleotides in length. In some embodiments, the PBS is 11 to 17 nucleotides in length. In some embodiments, the PBS is 10 to 17 nucleotides in length. In some embodiments, the PBS is 9 to 17 nucleotides in length. In some embodiments, the PBS is about 7 to 15 nucleotides in length. In some embodiments, the PBS is 6, 7,WSGR Docket No.59761-775.601 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length. In some embodiments, the PBS is 9 to 14 nucleotides in length. For example, the PBS can be 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the PBS is 11 or 12 nucleotides in length. In some embodiments, the PBS is 11 to 13 nucleotides in length. In some embodiments, the PBS is 11 to 14 nucleotides in length.

[0347] The PBS may be complementary or substantially complementary to a DNA sequence in the edit strand of the target gene. By annealing with the edit strand at a free hydroxy group, e.g., a free 3’ end generated by prime editor nicking, the PBS may initiate synthesis of a new single stranded DNA encoded by the editing template at the nick site. In some embodiments, the PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the edit strand of the target gene (e.g., the CFTR gene). In some embodiments, the PBS is perfectly complementary, or 100% complementary, to a region of the edit strand of the target gene (e.g., the CFTR gene).

[0348] An extension arm of a PEgRNA may comprise an editing template that serves as a DNA synthesis template for the DNA polymerase in a prime editor during prime editing.

[0349] The length of an editing template may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA. In some embodiments, the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is referred to as a reverse transcription editing template (RTT).

[0350] The editing template (e.g., RTT), in some embodiments, is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length. In some embodiments, the RTT is 10 to 110 nucleotides in length. In some embodiments, the RTT is 10 to 109, 10 to 108, 10 to 107, 10 to 106, 10 to 105, 10 to 104, 10 to 103, 10 to 102, or 10 to 101 nucleotides in length. In some embodiments, the RTT is at least 8 and no more than 50 nucleotides in length. In some embodiments, the RTT is at least 8 and no more than 25 nucleotides in length. In some embodiments, the RTT is about 10 to about 20 nucleotides in length. In some embodiments, the RTT is about 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the RTT is 11 to 17 nucleotides in length. In some embodiments, the RTT is 12 to 17 nucleotides in length. In some embodiments, the RTT is 12 to 16 nucleotides in length. In some embodiments, the RTT is 13 to 17 nucleotides in length. In some embodiments, the RTT is 11, 12, 13, 14, 15, 16, or 17 nucleotides in length. In some embodiments the RTT is 12 nucleotides in length. In some embodiments, the RTT is 16 nucleotides in length. In some embodiments the RTT is 17WSGR Docket No.59761-775.601 nucleotides in length. In some embodiments, the RTT is about 20 to about 30 nucleotides in length. In some embodiments, the RTT is about 20 to about 25 nucleotides in length. In some embodiments, the RTT is about 20 to about 25 nucleotides in length. In some embodiments, the RTT is 21 to 24 nucleotides in length.

[0351] In some embodiments, the editing template (e.g., RTT) sequence is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary to the editing target sequence on the edit strand of the target gene. In some embodiments, the editing template sequence (e.g., RTT) is substantially complementary to the editing target sequence. In some embodiments, the editing template sequence (e.g., RTT) is complementary to the editing target sequence except at positions of the intended nucleotide edits to be incorporated int the target gene. In some embodiments, the editing template comprises a nucleotide sequence comprising about 85% to about 95% complementarity to an editing target sequence in the edit strand in the target gene (e.g., the CFTR gene). In some embodiments, the editing template comprises about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementarity to an editing target sequence in the edit strand of the target gene (e.g., the CFTR gene).

[0352] An intended nucleotide edit in an editing template of a PEgRNA may comprise various types of alterations as compared to the target gene sequence. In some embodiments, the nucleotide edit is a single nucleotide substitution as compared to the target gene sequence. In some embodiments, the nucleotide edit is a deletion as compared to the target gene sequence. In some embodiments, the nucleotide edit is an insertion as compared to the target gene sequence. In some embodiments, the editing template comprises one to ten intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises one or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises three or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises four or more, five or more, or six or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises three single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises four, five, or six single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, a nucleotide substitution comprises an adenine (A)-to-thymine (T) substitution. In some embodiments, a nucleotide substitution comprises an A-to-guanine (G) substitution. In some embodiments, a nucleotide substitution comprises an A-to-cytosine (C) substitution. In some embodiments, a nucleotideWSGR Docket No.59761-775.601 substitution comprises a T-A substitution. In some embodiments, a nucleotide substitution comprises a T-G substitution. In some embodiments, a nucleotide substitution comprises a T-C substitution. In some embodiments, a nucleotide substitution comprises a G-to-A substitution. In some embodiments, a nucleotide substitution comprises a G-to-T substitution. In some embodiments, a nucleotide substitution comprises a G-to-C substitution. In some embodiments, a nucleotide substitution comprises a C-to-A substitution. In some embodiments, a nucleotide substitution comprises a C-to-T substitution. In some embodiments, a nucleotide substitution comprises a C-to-G substitution.

[0353] In some embodiments, a nucleotide insertion is at least 1, at least 2, at least 3, at least 4, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length. In some embodiments, a nucleotide insertion is from 1 to 2 nucleotides, from 1 to 3 nucleotides, from 1 to 4 nucleotides, from 1 to 5 nucleotides, form 2 to 5 nucleotides, from 3 to 5 nucleotides, from 3 to 6 nucleotides, from 3 to 8 nucleotides, from 4 to 9 nucleotides, from 5 to 10 nucleotides, from 6 to 11 nucleotides, from 7 to 12 nucleotides, from 8 to 13 nucleotides, from 9 to 14 nucleotides, from 10 to 15 nucleotides, from 11 to 16 nucleotides, from 12 to 17 nucleotides, from 13 to 18 nucleotides, from 14 to 19 nucleotides, from 15 to 20 nucleotides in length. In some embodiments, a nucleotide insertion is a single nucleotide insertion. In some embodiments, a nucleotide insertion comprises insertion of two nucleotides.

[0354] The editing template of a PEgRNA may comprise one or more intended nucleotide edits, compared to the CFTR gene to be edited. Position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to particular nucleotides (e.g., mutations) in the CFTR target gene may vary. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to or homologous to the protospacer sequence. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to a region of the CFTR gene outside of the protospacer sequence.

[0355] In some embodiments, the position of a nucleotide edit incorporation in the target gene may be referred to relative to position of the nick site. In some embodiments, position of an intended nucleotide edit is 0, 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides apart from the nick site. In some embodiments, position of an intended nucleotide edit is 0, 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides downstream of the nick site on the PAM strand (or the non-target strand, or the edit strand) of the double stranded target DNA. In some embodiments, position of the intended nucleotide edit in the editing template may be referred to by aligning theWSGR Docket No.59761-775.601 editing template with the partially complementary editing target sequence on the edit strand, and referring to nucleotide positions on the editing strand where the intended nucleotide edit is incorporated. Accordingly, in some embodiments, a nucleotide edit in an editing template is at a position corresponding to a position about 0, 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides apart from the nick site. In some embodiments, a nucleotide edit in an editing template is at a position corresponding to a position about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, 20 to 30 nucleotides 30 to 40 nucleotides, 40 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides, 100 to 110 nucleotides, 110 to 120 nucleotides, 120 to 130 nucleotides, 130 to 140 nucleotides, or 140 to 150 nucleotides apart from the nick site.

[0356] In some embodiments, when referred to in the context of the PAM strand (or the non-target strand, or the edit strand) a nucleotide edit in an editing template is at a position corresponding to a position about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, 20 to 30 nucleotides, 30 to 40 nucleotides, 40WSGR Docket No.59761-775.601 to 50 nucleotides, 50 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, 80 to 90 nucleotides, 90 to 100 nucleotides, 100 to 110 nucleotides, 110 to 120 nucleotides, 120 to 130 nucleotides, 130 to 140 nucleotides, or 140 to 150 nucleotides downstream from the nick site. The relative positions of the intended nucleotide edit(s) and nick site may be referred to by numbers. For example, in some embodiments, the nucleotide immediately downstream of the nick site on a PAM strand (or the non-target strand, or the edit strand) may be referred to as at position 0. The nucleotide immediately upstream of the nick site on the PAM strand (or the non-target strand, or the edit strand) may be referred to as at position -1. The nucleotides downstream of position 0 on the PAM strand may be referred to as at positions +1, +2, +3, +4, … +n, and the nucleotides upstream of position -1 on the PAM strand may be referred to as at positions -2, -3, -4, …, -n. Accordingly, in some embodiments, the nucleotide in the editing template that corresponds to position 0 when the editing template is aligned with the partially complementary editing target sequence by complementarity may also be referred to as position 0 in the editing template, the nucleotides in the editing template corresponding to the nucleotides at positions +1, +2, +3, +4, …, +n on the PAM strand of the double stranded target DNA may also be referred to as at positions +1, +2, +3, +4, …, +n in the editing template, and the nucleotides in the editing template corresponding to the nucleotides at positions -1, - 2, -3, -4, …, -n on the PAM strand on the double stranded target DNA may also be referred to as at positions -1, -2, -3, -4, …, -n on the editing template, even though when the PEgRNA is viewed as a is at position +n of the editing template relative to position 0. Accordingly, the intended nucleotide edit may be incorporated at position +n of the PAM strand of the double stranded target DNA (and subsequently, the target strand of the double stranded target DNA) by prime editing. The corresponding positions of the intended nucleotide edit incorporated in the CFTR gene may also be referred to based on the nicking position generated by a prime editor based on sequence homology and complementarity. For example, in embodiments, the distance between the nucleotide edit to be incorporated into the CFTR gene and the nick site (also referred to as the “nick to edit distance”) may be determined by the position of the nick site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence. In certain embodiments, the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand). As used herein, the distance between the nick site and the nucleotide edit, for example, where the nucleotide edit comprises an insertion or deletion, refers to the 5’ most position of the nucleotide edit for a nick that creates a 3’ free end on the edit strand (i.e., the “near position” of the nucleotide edit to the nick site). In some embodiments, the nick-to-edit distance is 2 to 106 nucleotides. In some embodiments, theWSGR Docket No.59761-775.601 nick-to-edit distance is 2 to 105, 2 to 104, 2 to 103, 2 to 102, 2 to 101, 2 to 100, 2 to 99, 2 to 98, or 2 to 97 nucleotides. In some embodiments, the nick-to-edit distance is 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 nucleotides. In some embodiments, the nick-to-edit distance is 2 to 25, 2 to 20, 2 to 15, or 2 to 10 nucleotides. In some embodiments, the nick-to-edit distance is 2, 3, 4, 5, 6, or 7 nucleotides in length. In some embodiments, the nick-to-edit distance is 28 nucleotides. In some embodiments, the nick-to-edit distance is 22 nucleotides. In some embodiments, the nick-to-edit distance is 21 nucleotides. In some embodiments, the nick-to-edit distance is 17 nucleotides. In some embodiments, the nick-to-edit distance is 16 nucleotides. In some embodiments, the nick-to-edit distance is 4 nucleotides.

[0357] The RTT length and the nick-to-edit distance relate to the length of the portion of the RTT that is upstream of (i.e.5’ to) the 5’-most edit in the RTT and is complementary to the edit strand. In some embodiments, the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 nucleotides contiguous are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more contiguous nucleotides of complementarity with the edit strand wherein the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 20-25, 25-30, 30-35, 35-40, 45-45, or 45-50 contiguous nucleotides of complementarity with the edit strand wherein the 20-25, 25-30, 30- 35, 35-40, 45-45, or 45-50 or more contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 9-14 contiguous nucleotides of complementarity with the edit strand wherein the 9-14 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 6-10 contiguous nucleotides of complementarity with the edit strand wherein the 6-10 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 10 contiguous nucleotides of complementarity with the edit strand wherein the 10 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 9 contiguous nucleotides of complementarity with the edit strand wherein the 9 contiguous nucleotides are located upstream of the 5’ most edit in the editing template.

[0358] When referred to within the PEgRNA, positions of the one or more intended nucleotide edits may be referred to relevant to components of the PEgRNA. For example, an intended nucleotide edit may be 5’ or 3’ to the PBS. In some embodiments, a PEgRNA comprises the structure, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, the intended nucleotide edit is 0, 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides upstream to the 5’ most nucleotide of theWSGR Docket No.59761-775.601 PBS. In some embodiments, the intended nucleotide edit is 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, or 20 to 30 nucleotides upstream to the 5’ most nucleotide of the PBS.

[0359] The corresponding positions of the intended nucleotide edit incorporated in the target gene may also be referred to based on the nicking position generated by a prime editor based on sequence homology and complementarity. For example, in embodiments, the distance between the nucleotide edit to be incorporated into the target CFTR gene and the nick site (also referred to as the “nick to edit distance”) may be determined by the position of the nick site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence. In certain embodiments, the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand) generated by the prime editor, such that the distance between the nick site and the intended nucleotide edit is 0, 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, 28, 29, or 30 nucleotides in length. In some embodiments, the position of the nucleotide edit is 0, 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, 28, 29, or 30 nucleotides in length. In some embodiments, the position of the nucleotide edit is 0, 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, 28, 29, or 30 nucleotides upstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0, 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, 28, 29, or 30 nucleotides downstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0 base pair from the nick site on the edit strand, that is, the editing position is at the same position as the nick site. As used herein, the distance between the nick site and the nucleotide edit, for example, where the nucleotide edit comprises an insertion or deletion, refers to the 5’ most position of the nucleotide edit for a nick that creates a 3’ free end on the edit strand (i.e., the “near position” of the nucleotide edit to the nick site). Similarly, as used herein, the distance betweenWSGR Docket No.59761-775.601 the nick site and a PAM position edit, for example, where the nucleotide edit comprises an insertion, deletion, or substitution of two or more contiguous nucleotides, refers to the 5’ most position of the nucleotide edit and the 5’ most position of the PAM sequence.

[0360] In some embodiments, the editing template extends beyond a nucleotide edit to be incorporated to the target CFTR gene sequence. For example, in some embodiments, the editing template comprises at least 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

[0361] In some embodiments, the editing template can comprise a second edit relative to a target sequence. The second edit can be designed to mutate or otherwise silence a PAM sequence such that a corresponding nucleic acid guided nuclease or CRISPR nuclease is no longer able to cleave the target sequence (such edits referred to as “PAM silencing edits).

[0362] Without wishing to be bound by any particular theory, PAM silencing edits may prevent the Cas, e.g., Cas9, nickase, from re-nicking the edit strand before the edit is incorporated in the target strand, therefore improving prime editing efficiency. In some embodiments, a PAM silencing edit is a synonymous edit that does not alter the amino acid sequence encoded by the CFTR gene after incorporation of the edit. In some embodiments, a PAM silencing edit is at a position corresponding to a coding region, e.g., an exon, of a CFTR gene. In some embodiments, a PAM silencing edit is at a position corresponding to a non-coding region, e.g., an intron, of a CFTR gene. In some embodiments, the edits in an intron of a CFTR gene is not at a position that corresponds to intron-exon junction and the edit does not affect transcript splicing.

[0363] In some embodiments, the length of the editing template is at least 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides longer than the nick to edit distance. In some embodiments, for example, the nick to edit distance is 8 nucleotides, and the editing template is 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, or 10 to 80 nucleotides in length. In some embodiments, the nick to edit distance is 22 nucleotides, and the editing template is 24 to 28, 24 to 30, 24 to 32, 24 to 34, 24 to 36, 24 to 37, 24 to 38, 24 to 40, 24 to 45, 24 to 50, 24 to 55, 24 to 60, 24 to 65, 24 to 70, 24 to 75, 24 to 80, 24 to 85, 24 to 90, 24 to 95, 24 to 100, 24 to 105, 24 to 100, 24 to 105, or 24 to 110 nucleotides in length.

[0364] In some embodiments, the editing template comprises at least 4 to 30 base pairs 3’ to the nucleotide edit to be incorporated to the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 25 base pairs 3’ to the nucleotide edit to be incorporated to theWSGR Docket No.59761-775.601 target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 20 base pairs 3’ to the nucleotide edit to be incorporated to the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 30 base pairs 5’ to the nucleotide edit to be incorporated to the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 25 base pairs 5’ to the nucleotide edit to be incorporated to the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 20 base pairs 5’ to the nucleotide edit to be incorporated to the target CFTR gene sequence.

[0365] In some embodiments, the editing template comprises an adenine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”). In some embodiments, the editing template comprises a guanine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”). In some embodiments, the editing template comprises an uracil at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”). In some embodiments, the editing template comprises a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”). In some embodiments, the editing template does not comprise a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).

[0366] The editing template of a PEgRNA may encode a new single stranded DNA (e.g., by reverse transcription) to replace an editing target sequence in the target gene. In some embodiments, the editing target sequence in the edit strand of the target gene is replaced by the newly synthesized strand, and the nucleotide edit(s) are incorporated in the region of the target gene. In some embodiments, the target gene is an CFTR gene. In some embodiments, the editing template of the PEgRNA encodes a newly synthesized single stranded DNA that comprises a wild type CFTR gene sequence. In some embodiments, the newly synthesized DNA strand replaces the editing target sequence in the target CFTR gene, wherein the editing target sequence (or the endogenous sequence complementary to the editing target sequence on the target strand of the CFTR gene) comprises a mutation or a nucleotide alteration compared to a wild type CFTR gene. In some embodiments, the mutation is associated with cystic fibrosis.

[0367] In some embodiments, the newly synthesized single stranded DNA encoded by the editing template replaces the editing target sequence and corrects the mutation in the editing target sequence of the target CFTR gene.

[0368] In some embodiments, the editing target sequence comprises position 117587778 in human chromosome 7. In some embodiments, the editing target sequence comprises a mutation as compared to a wild type CFTR gene, wherein the mutation is a nucleotide insertion, a nucleotide deletion, aWSGR Docket No.59761-775.601 nucleotide substitution, two or more nucleotide substitutions, or any combination thereof. In some embodiments, the mutation results in a premature stop codon in a mRNA encoded by the CFTR gene. In some embodiments, the mutation results in an amino acid alteration in the CFTR protein encoded by the CFTR gene. In some embodiments, the mutation results in an amino acid substitution in the CFTR protein encoded by the CFTR gene. In some embodiments, the mutation results in a truncated CFTR polypeptide encoded by the CFTR gene as compared to a wild type CFTR polypeptide. In some embodiments, the mutation results in an aberrant CFTR polypeptide encoded by the CFTR gene. In some embodiments, the mutation results in a CFTR polypeptide encoded by the CFTR gene that has reduced biological activity as compared to a wild type CFTR polypeptide. In some embodiments, the mutation results in a CFTR polypeptide encoded by the CFTR gene that has abolished biological activity as compared to a wild type CFTR polypeptide. In some embodiments, the editing target sequence comprises a mutation corresponding to position 1624 of the coding sequence of the CFTR protein. In some embodiments, the editing target sequence comprises a c.1624G->T mutation (editing target sequence on the sense strand) or a corresponding C->A mutation (editing target sequence on the antisense strand) at position 1624 of the coding sequence of the CFTR protein.

[0369] In some embodiments, the editing template comprises one or more intended nucleotide edits compared to the sequence on the target strand of the CFTR gene that is complementary to the editing target sequence. In some embodiments, the editing template encodes a single stranded DNA that comprises one or more intended nucleotide edits compared to the editing target sequence. In some embodiments, the single stranded DNA replaces the editing target sequence by prime editing, thereby incorporating the one or more intended nucleotide edits. In some embodiments, the one or more intended nucleotide edits encodes a T->G substitution at a position corresponding to position 1624 of the coding sequence of the CFTR protein compared to the editing target sequence (editing target sequence on the sense strand). In some embodiments, the one or more intended nucleotide edits encodes a A->C substitution at a position corresponding to position 1624 of the coding sequence of the CFTR protein compared to the editing target sequence (editing target sequence on the antisense strand). In some embodiments, incorporation of the one or more intended nucleotide edits corrects the mutation in the editing target sequence to wild type nucleotides at corresponding positions in the target CFTR gene. As used herein, “correcting” a mutation means restoring a wild type sequence at the place of the mutation in the double stranded target DNA e.g. target gene, by prime editing. In some embodiments, incorporation of the one or more nucleotide edits can correct any mutations in the CFTR gene that are in the portion of the gene that shares homology with the editing template.

[0370] In some embodiments, incorporation of the one or more intended nucleotide edits results in expression of a functional CFTR protein. For example, in some embodiments, incorporation of the one or more intended nucleotide edits results in a nucleotide substitution, insertion, or deletion that results in a codon that encodes a wild type amino acid as compared to a wild type CFTR polypeptide,WSGR Docket No.59761-775.601 while the codon is not the same as the wild type nucleotide at the corresponding position. In some embodiments, the editing template comprises and / or encodes a wild type CFTR gene sequence.

[0371] In some embodiments, incorporation of the one or more intended nucleotide edits does not correct the mutation in the editing target sequence to wild type sequence, but allows for expression of a functional CFTR protein encoded by the CFTR gene. For example, in some embodiments, incorporation of the one or more intended nucleotide edits results in one or more codons that are different from a wild type codon but encode one or more amino acids same as the wild type CFTR protein. In some embodiments, incorporation of the one or more intended nucleotide edits results in one or more codons that encode one or more amino acids different from the wild type CFTR protein, but allows for expression of a functional CFTR protein. Exemplary amino acid sequence of wild type CFTR protein is provided in SEQ ID NO: 751.

[0372] A guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor. The gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a DNA nickase of the prime editor.

[0373] One of skill in the art will recognize that different prime editors having different DNA binding domains from different DNA binding proteins may require different gRNA core sequences specific to the DNA binding protein. In some embodiments, the gRNA core is capable of binding to a Cas9-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cpf1-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cas12b-based prime editor.

[0374] In some embodiments, the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins. For example, in a Cas9 based prime editing system, the gRNA core of a PEgRNA may comprise one or more regions of a base paired “lower stem” adjacent to the spacer sequence and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs. The gRNA core may further comprise a “nexus” distal from the spacer sequence, followed by a hairpin structure, e.g., at the 3’ end, as exemplified in FIG.3. In some embodiments, the gRNA core comprises modified nucleotides as compared to a wild type gRNA core in the lower stem, upper stem, and / or the hairpin. For example, nucleotides in the lower stem, upper stem, an / or the hairpin regions may be modified, deleted, or replaced. In some embodiments, RNA nucleotides in the lower stem, upper stem, an / or the hairpin regions may be replaced with one or more DNA sequences. In some embodiments, the gRNA core comprises unmodified or wild type RNA sequences in the nexus and / or the bulge regions. In some embodiments, the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU-AAAAC pairing element. In some embodiments, a prime editing system comprises a primeWSGR Docket No.59761-775.601 editor and a PEgRNA, wherein the prime editor comprises a SpCas9 nickase or a variant thereof, and the gRNA core of the PEgRNA comprises a sequence capable of binding to a SpCas9. gRNA core sequences known in the art are also contemplated in the prime editing compositions described herein.

[0375] In some embodiments, the PEgRNA and / or ngRNA comprises a universal gRNA core. A universal gRNA core can be used in a PEgRNA or ngRNA that comprises any spacer that has a PAM sequence compatible with the Cas9 protein capable of binding to the gRNA core, and any PBS and RTT sequences designed to incorporate the intended nucleotide edit(s) based on the spacer. In some embodiments, the PEgRNA and / or ngRNA comprises a universal gRNA core that comprises a nucleic acid sequence selected from the Table 10. In some embodiments, the PEgRNA and / or ngRNA comprises a gRNA core that comprises a nucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences on Table 10.

[0376] Table 10: Exemplary nucleic acid sequences of universal gRNA core (also referred to herein as gRNA scaffold) for PEgRNAs compatible with SpCas9 prime editors. The sequences in Table 10 are annotated with SEQ ID NO as required by ST.26 standard. Although all the sequences provided in Table 10 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.WSGR Docket No.59761-775.601

[0377] In some embodiments, a PEgRNA comprises a sequence specific gRNA core. A sequence specific gRNA core may be designed to form optimal secondary or tertiary structure with other components of the PEgRNA, for example, the spacer, RTT, and / or PBS.

[0378] In some embodiments, a prime editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA). In some embodiments, a ngRNA comprises a spacer (referred to as a ngRNA spacer or ng spacer) and a gRNA core, wherein the spacer of the ngRNA comprises a region of complementarity to the edit strand, and wherein the gRNA core can interact with a Cas, e.g., Cas9, of a prime editor. Without wishing to be bound by any particular theory, an ngRNA may bind to the edit strand and direct Cas nickase to generate a nick on the non- edit strand (or target strand). In some embodiments, the nick on the non-edit strand directs endogenous DNA repair machinery to use the edit strand as a template for repair of the non-edit strand, which may increase efficiency of prime editing. In some embodiments, the non-edit strand is nicked by a prime editor localized to the non-edit strand by the ngRNA. Accordingly, also provided herein are PEgRNA systems comprising at least one PEgRNA and at least one ngRNA.

[0379] A prime editing system comprising a PEgRNA (or one or more polynucleotide encoding the PEgRNA) and a prime editor protein (or one or more polynucleotides encoding the prime editor), may be referred to as a PE2 prime editing system and the corresponding editing approach referred to as PE2 approach or PE2 strategy. A PE2 system does not contain a ngRNA. A prime editing system comprising a PEgRNA (or one or more polynucleotide encoding the PEgRNA), a prime editor protein (or one or more polynucleotides encoding the prime editor), and a ngRNA (or one or more polynucleotides encoding the ngRNA) may be referred to as a “PE3” prime editing system. In some embodiments, an ng spacer sequence is complementary to, and may hybridize with the second search target sequence only after an intended nucleotide edit has been incorporated on the edit strand, by the editing template of a PEgRNA.

[0380] Such ngRNA may be referred to a “PE3b” ngRNA, and the prime editing system a PE3b prime editing system.

[0381] In some embodiments, the ng search target sequence is located on the non-target strand, within 10 base pairs to 100 base pairs of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the ng target search target sequence is within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99WSGR Docket No.59761-775.601 bp, or 100 bp of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the 5’ ends of the ng search target sequence and the PEgRNA search target sequence are within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bp apart from each other. In some embodiments, the 5’ ends of the ng search target sequence and the PEgRNA search target sequence are within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp apart from each other.

[0382] The gRNA core of a PEgRNA or ngRNA can be any gRNA scaffold sequence that is capable of interacting with a Cas protein that recognizes the corresponding PAM of the PEgRNA or ngRNA. In some embodiments, gRNA core of a PEgRNA or a ngRNA comprises a sequence selected from from the sequences in Table 10.

[0383] In some embodiments, the PEgRNA and / or ngRNA comprises a 3’ motif. In some embodiments, the PEgRNA and / or ngRNA comprises a 3’ motif comprising a nucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences provided in Table 11. In some embodiments, the PEgRNA and / or ngRNA comprises a 3’ motif comprising a nucleic acid sequence selected from the group consisting of: the sequences provided in Table 11.

[0384] Table 11: Illustrative nucleic acid sequences for 3’ motif (e.g., universal 3’ motif). The sequences in Table 11 are annotated with SEQ ID NO as required by ST.26 standard. Although all the sequences provided in Table 11 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.

[0385] In some embodiments, a PEgRNA further comprises a nucleotide linker. In some embodiments, the secondary structure is linked to one or more other component of a PEgRNA via a linker. In some embodiments, a secondary structure or a 3’ motif of a PEgRNA is linked to one or more other components of a PEgRNA via a linker. For example, in some embodiments, the secondary structure is at the 3’ end of the PEgRNA (e.g., a RTT, or a PBS) and is linked to the 3’ end of a PBS via a linker. For example, in some embodiments, a 3’ motif is at the 3’ end of the PEgRNA and is linked to the 3’ end of a PEgRNA (e.g., a RTT or a PBS) via a linker. In some embodiments, the secondary structure or a 5’ motif is at the 5’ end of the PEgRNA and is linked to the 5’ end of a spacer via a linker. In some embodiments, the linker is a nucleotide linker that is 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, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, theWSGR Docket No.59761-775.601 linker is 5 to 10 nucleotides in length. In some embodiments, the linker is 10 to 20 nucleotides in length. In some embodiments, the linker is 15 to 25 nucleotides in length. In some embodiments, the linker is 8 nucleotides in length.

[0386] In some embodiments, the linker is designed to minimize base pairing between the linker and another component of the PEgRNA. In some embodiments, the linker is designed to minimize base pairing between the linker and the spacer. In some embodiments, the linker is designed to minimize base pairing between the linker and the PBS. In some embodiments, the linker is designed to minimize base pairing between the linker and the editing template. In some embodiments, the linker is designed to minimize base pairing between the linker and the sequence of the RNA secondary structure. In some embodiments, the linker is optimized to minimize base pairing between the linker and another component of the PEgRNA, in order of the following priority: spacer, PBS, editing template and then scaffold. In some embodiments, base paring probability is calculated using ViennaRNA 2.0 ,as described in Lorenz, R. et al. ViennaRNA package 2.0. Algorithms Mol. Biol.6, incorporated by reference in its entirety herein, under standard parameters (37 °C, 1 M NaCl, 0.05 M MgCl2).

[0387] In certain embodiments, the PEgRNA comprises secondary RNA structure, such as, but not limited to, aptamers, hairpins, stem / loops, toeloops, and / or RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein).In some embodiments, a PEgRNA (or ngRNA) comprises an additional secondary structure at the 5’ end. In some embodiments, a PEgRNA (or ngRNA) comprises an additional secondary structure at the 3’ end. In some embodiments, the secondary structure comprises a pseudoknot. In some embodiments, the secondary structure comprises a pseudoknot derived from a virus. In some embodiments, the secondary structure comprises a pseudoknot of a Moloney murine leukemia virus (M-MLV) genome (a mpknot). In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of sequences provided in Table 12, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence provided in Table 12. In some embodiments, the secondary structure comprises a quadruplex. In some embodiments, the secondary structure comprises a G-quadruplex. In some embodiments, the secondary structure comprises a riboswitch aptamer. In some embodiments, the secondary structure comprises a riboswitch aptamer derived from a prequeosine-1 riboswitch aptamer. In some embodiments, the secondary structure comprises a modified prequeosine-1 riboswitch aptamer. In some embodiments, the PEgRNA comprises a toeloop element having the sequence 5’-GAAANNNNN-3’, wherein N is any nucleobase. In some embodiments, the secondary RNA structure is positioned within the spacer. In some embodiments, the secondary structure is positioned within the extension arm. In some embodiments, the secondary structure is positioned within the gRNA core. In some embodiments, the secondary structure is positioned between the spacer and the gRNA core, between the gRNA core andWSGR Docket No.59761-775.601 the extension arm, or between the spacer and the extension arm. In some embodiments, the secondary structure is positioned between the PBS and the editing template. In some embodiments the secondary structure is positioned at the 3’ end or at the 5’ end of the PEgRNA.

[0388] In some embodiments, the PEgRNA comprises a RNA secondary structure and / or a linker disclosed in Nelson et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. (2021), the entirety of which is incorporated herein by reference.

[0389] Exemplary secondary structure sequences are provided in Table 12.

[0390] Table 12. Illustrative sequences for secondary structures. The sequences in Table 12 are annotated with SEQ ID NO as required by ST.26 standard. Although all the sequences provided in Table 12 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.WSGR Docket No.59761-775.601

[0391] In some embodiments, the PEgRNA comprises a self-cleaving element. In some embodiments, the self-cleaving element improves transcription and / or processing of the PEgRNA when transcribed form the nucleotide encoding the PEgRNA. In some embodiments, the PEgRNA comprises a hairpin or a RNA quadruplex. In some embodiments, the PEgRNA comprises a self- cleaving ribozyme element, for example, a hammerhead, a pistol, a hatchet, a hairpin, a VS, a twister, or a twister sister ribozyme. In some embodiments, the PEgRNA comprises a HDV ribozyme. In some embodiments, the PEgRNA comprises a hairpin recognized by Csy4. In some embodiments, the PEgRNA comprises an ENE motif. In some embodiments, the PEgRNA comprises an element for nuclear expression (ENE) from MALAT1 lnc RNA. In some embodiments, the PEgRNA comprises an ENE element from Kaposi’s sarcoma-associated herpesvirus (KSHV). In some embodiments, the PEgRNA comprises a 3’ box of a U1 snRNA. In some embodiments, the PEgRNA forms a circular RNA.

[0392] In some embodiments, the PEgRNA comprises a RNA secondary structure or a motif that improves binding to the DNA-RNA duple or enhances PEgRNA activity. In some embodiments, the PEgRNA comprises a sequence derived from a native nucleotide element involved in reverse transcription, e.g., initiation of retroviral transcription. In some embodiments, the PEgRNA comprises a sequence of, or derived from, a primer binding site of a substrate of a reverse transcriptase, a polypurine tract (PPT), or a kissing loop. In some embodiments, the PEgRNA comprises a dimerization motif, a kissing loop, or a GNRA tetraloop – tetraloop receptor pair that results in circularization of the PEgRNA. In some embodiments, the PEgRNA comprises a RNA secondary structure of a motif that results in physical separation of the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity. In some embodiments, the PEgRNA comprises a secondary structure or motif, e.g., a 5’ or 3’ extension in the spacer region that form a toehold or hairpin, wherein the secondary structure or motif competes favorably against annealing between the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity.

[0393] In some embodiments, a PEgRNA additionally comprises a sequence provided in Table 13.

[0394] In some embodiments, a PEgRNA comprises the sequence of SEQ ID NO: 735 at the 3’ end. In some embodiments, a PEgRNA comprises the structure [spacer]-[gRNA core]-[editing template]- [PBS]-[3’ motif or secondary structure selected from Tables 11-13] or [spacer]-[gRNA core]-[editing template]-[PBS]- [3’ motif or secondary structure selected from Tables 11-13].

[0395] In some embodiments, the PEgRNA comprises the sequence of SEQ ID NO: 737 at the 5’ end and / or the sequence UGGGAGACGUCCCACC (SEQ ID NO: 738) at the 3’ end. In someWSGR Docket No.59761-775.601 embodiments, the PEgRNA comprises the following structure (M-MLV kissing loop): GGUGGGAGACGUCCCACC (SEQ ID NO: 737)-[spacer]-[gRNA core]-[editing template]-[PBS]- UGGGAGACGUCCCACC (SEQ ID NO: 738), or GGUGGGAGACGUCCCACC (SEQ ID NO: 737)- [spacer]-[gRNA core]-[editing template]-[PBS]-UGGGAGACGUCCCACC-(U)n (SEQ ID NO: 739), wherein n is an integer between 3 and 7. The kissing loop structure is italicized.

[0396] In some embodiments, the PEgRNA comprises the sequence of SEQ ID NO: 740 at the 5’ end and / or the sequence SEQ ID NO: 747 at the 3’ end. In some embodiments, the PEgRNA comprises the following structure (VS ribozyme kissing loop): GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 740)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG (SEQ ID NO: 747), or GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 740)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG-(U)n (SEQ ID NO: 742), wherein n is an integer between 3 and 7. (VS ribozyme kissing loop)

[0397] In some embodiments, the PEgRNA comprises the sequence of SEQ ID NO: 743 at the 5’ end and / or the sequence of SEQ ID NO: 744 at the 3’ end. In some embodiments, the PEgRNA comprises the following structure (tetraloop and receptor): GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 743)-[spacer]-[gRNA core]-[editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG (SEQ ID NO: 744), or GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 743)-[spacer]-[gRNA core]-[editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG-(U)n (SEQ ID NO: 745), wherein n is an integer between 3 and 7. The tetraloop / tetraloop receptor structure is italicized.

[0398] In some embodiments, the PEgRNA comprises the sequence GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGG CGAAUGGGAC (SEQ ID NO: 735) or UCUGCCAUCAAAGCUGCGACCGUGCUCAGUCUGGUGGGAGACGUCCCACCGGCCGGCA UGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGG AC (SEQ ID NO: 746).

[0399] Example sequences of components within PEgRNA are provided in Table 13.

[0400] Table 13. Exemplary sequences of components within PEgRNA.WSGR Docket No.59761-775.601

[0401] In addition to secondary RNA structures, a PEgRNA may comprise one or more linkers. In some embodiments, a PEgRNA comprises a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase. In some embodiments, the chemical linker may function to prevent reverse transcription of the gRNA core.

[0402] In some embodiments, a secondary structure or a 3’ motif is directly connected to the 3’ end of a PBS. In some embodiments, a secondary structure or a 3’ motif is directly connected to the 3’ end of a PBS via a nucleotide linker. The nucleotide linker may be about 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, 28, 29, or 30 nucleotides in length. In some embodiments, the nucleotide linker is about 4-12 nucleotides in length. In some embodiments, the nucleotide linker is about 4 nucleotides in length. In some embodiments, the nucleotide linker is a universal linker, e.g., as set forth in AACATTGA (SEQ ID NO: 706). In some embodiments, the nucleotide linker is a sequence specific linker, for example, designed to optimize the secondary or tertiary structure of the PEgRNA. Exemplary sequence specific linkers are provided in Table 14.

[0403] Table 14. Exemplary Sequence Specific LinkersWSGR Docket No.59761-775.601

[0404] A PEgRNA may also comprise optional modifiers, e.g. end modifier region. In some embodiments, a PEgRNA comprises at least one nucleotide that is not part of a spacer, a gRNA core, or an extension arm. The optional sequence modifiers could be

[0405] In some embodiments, a PEgRNA comprises a short stretch of uracil at the 5’ end or the 3’ end. For example, in some embodiments, a PEgRNA comprising a 3’ extension arm comprises a “UUU” sequence at the 3’ end of the extension arm.

[0406] In some embodiments, a PEgRNA is transcribed from a nucleotide encoding the PEgRNA, for example, a DNA plasmid encoding the PEgRNA. In some embodiments, the PEgRNA comprises

[0407] A PEgRNA and / or an ngRNA of this disclosure, in some embodiments, may include modified nucleotides, e.g., chemically modified DNA or RNA nucleobases, and may include one or more nucleobase analogs (e.g., modifications which might add functionality, such as temperature resilience). In some embodiments, PEgRNAs and / or ngRNAs as described herein may be chemically modified. The phrase “chemical modifications,” as used herein, can include modifications whichWSGR Docket No.59761-775.601 introduce chemistries which differ from those seen in naturally occurring DNA or RNAs, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in DNA or RNA molecules).

[0408] In some embodiments, the PEgRNAs provided in the disclosure may further comprise nucleotides added to the 5’ of the PEgRNAs. In some embodiments, the PEgRNA further comprises 1, 2, or 3 additional nucleotides added to the 5’ end. The additional nucleotides can be guanine, cytosine, adenine, or uracil. In some embodiments, the additional nucleotide at the 5’ end of the PEgRNA is a guanine or cytosine. In some embodiments, the additional nucleotides can be chemically or biologically modified.

[0409] In some embodiments, the PEgRNAs provided in the disclosure may further comprise nucleotides to the 3’ of the PEgRNAs. In some embodiments, the PEgRNA further comprises 1, 2, or 3 additional nucleotides to the 3’ end. The additional nucleotides can be guanine, cytosine, adenine, or uracil. In some embodiments, the additional nucleotides at the 3’ end of the PEgRNA is a polynucleotide comprising at least 1 uracil. In some embodiments, the additional nucleotides can be chemically or biologically modified.

[0410] In some embodiments, a PEgRNA or ngRNA is produced by transcription from a template nucleotide, for example, a template plasmid. In some embodiments, a polynucleotide encoding the PEgRNA or ngRNA is appended with one or more additional nucleotides that improves PEgRNA or ngRNA function or expression, e.g., expression from a plasmid that encodes the PEgRNA or ngRNA. In some embodiments, a polynucleotide encoding a PEgRNA or ngRNA is appended with one or more additional nucleotides at the 5’ end or at the 3’ end. In some embodiments, the polynucleotide encoding the PEgRNA or ngRNA is appended with a guanine at the 5’ end, for example, if the first nucleotide at the 5’ end of the spacer is not a guanine. In some embodiments, a polynucleotide encoding the PEgRNA or ngRNA is appended with nucleotide sequence CACC at the 5’ end. In some embodiments, the polynucleotide encoding the PEgRNA or ngRNA is appended with an additional nucleotide adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS is a Thymine. In some embodiments, the polynucleotide encoding the PEgRNA or ngRNA is appended with additional nucleotide sequence TTTTTT, TTTTTTT, TTTTT, or TTTT at the 3’ end. In some embodiments, the PEgRNA or ngRNA comprises the appended nucleotides from the transcription template. In some embodiments, the PEgRNA or ngRNA further comprises one or more nucleotides at the 5’ end or the 3’ end in addition to spacer, PBS, and RTT sequences. In some embodiments, the PEgRNA or ngRNA further comprises a guanine at the 5’ end, for example, when the first nucleotide at the 5’ end of the spacer is not a guanine. In some embodiments, the PEgRNA or ngRNA further comprises nucleotide sequence CACC at the 5’ end. In some embodiments, the PEgRNA or ngRNA further comprises an adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBSWSGR Docket No.59761-775.601 is a thymine. In some embodiments, the PEgRNA or ngRNA further comprises nucleotide sequence UUUUUUU, UUUUUU, UUUUU, or UUUU at the 3’ end.

[0411] In some embodiments, a PEgRNA or a nick guide RNA (ngRNA) can be chemically synthesized, or can be assembled or cloned and transcribed from a DNA sequence, e.g., a plasmid DNA sequence, or by any RNA oligonucleotide synthesis method known in the art. In some embodiments, DNA sequence that encodes a PEgRNA (or ngRNA) may be designed to append one or to enhance PEgRNA transcription. For example, in some embodiments, a DNA sequence that encodes end. Accordingly, in some embodiments, the PEgRNA (or nick guide RNA) may comprise an (or nick guide RNA) may be designed to append a sequence that enhances transcription, e.g., a Kozak some embodiments, the PEgRNA (or nick guide RNA) may comprise an appended sequence CACC guide RNA) may be designed to append the sequence TTT, TTTT, TTTTT, TTTTTT, TTTTTTT at end. In some embodiments, a PEgRNA or a ngRNA comprises the sequence TTTTTTT (sequence sequence (e.g., TTTT; sequence number 707) at the 3’ end. In some embodiments, a PEgRNA or a ngRNA comprises a transcription adaptation sequence (e.g., TTTTTTT sequence number 708) at the 3’ end. The sequences in sequence number 707, and sequence number 708 are annotated with a sequence number as required by ST.26 standard. Although the sequences set forth in sequence number 707, and sequence number 708 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.

[0412] In some embodiments, the PEgRNAs and / or ngRNAs provided in this disclosure may have undergone a chemical or biological modifications. Modifications may be made at any position within a PEgRNA or ngRNA and may include modification to a nucleobase or to a phosphate backbone of the PEgRNA or ngRNA. In some embodiments, chemical modifications can be a structure guided modifications. In some embodiments, a chemical modification is at the 5’ end and / or the 3’ end of a PEgRNA. In some embodiments, a chemical modification is at the 5’ end and / or the 3’ end of a ngRNA. In some embodiments, a chemical modification may be within the spacer sequence, the extension arm, the editing template sequence, or the primer binding site of a PEgRNA. In someWSGR Docket No.59761-775.601 embodiments, a chemical modification may be within the spacer sequence or the gRNA core of a PEgRNA or a ngRNA. In some embodiments, a chemical modification may be within the 3’ most nucleotides of a PEgRNA or ngRNA. In some embodiments, a chemical modification may be within the 3’ most end of a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modification may be within the 5’ most end of a PEgRNA or ngRNA. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more contiguous chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is not modified, and the 1, 2, 3, 4, 5, or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3’ order. In some embodiments, a PEgRNA or ngRNA comprises 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, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide isWSGR Docket No.59761-775.601 not modified, and the 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, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3’ order.

[0413] In some embodiments, a PEgRNA or ngRNA comprises one or more chemical modified nucleotides in the gRNA core. As exemplified in FIG.3, the gRNA core of a PEgRNA may comprise one or more regions of a base paired lower stem, a base paired upper stem, where the lower stem and upper stem may be connected by a bulge comprising unpaired RNAs. The gRNA core may further comprise a nexus distal from the spacer sequence. In some embodiments, the gRNA core comprises one or more chemically modified nucleotides in the lower stem, upper stem, and / or the hairpin regions. In some embodiments, all of the nucleotides in the lower stem, upper stem, and / or the hairpin regions are chemically modified.

[0414] phosphorothioate bond modification, any other chemical modifications known in the art, or any combination thereof. A chemical modification may also include, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the PEgRNA and / or ngRNA (e.g., modifications to one or both of the 3’ and 5’ ends of a guide RNA molecule). Such modifications can include the addition of bases to an RNA sequence, complexing the RNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures). Prime Editing Compositions

[0415] Disclosed herein, in some embodiments, are compositions, systems, and methods using a prime editing composition. The term “prime editing composition” or “prime editing system” refers to compositions involved in the method of prime editing as described herein. A prime editing composition may include a prime editor, e.g., a prime editor fusion protein, and a PEgRNA. A prime editing composition may further comprise additional elements, such as second strand nicking ngRNAs. Components of a prime editing composition may be combined to form a complex for prime editing, or may be kept separately, e.g., for administration purposes. In some embodiments, a prime editing composition comprises a prime editor fusion protein complexed with a PEgRNA and optionally complexed with a ngRNA. In some embodiments, the prime editing composition comprises a prime editor comprising a DNA binding domain and a DNA polymerase domain associated with each other through a PEgRNA. For example, the prime editing composition may comprise a prime editor comprising a DNA binding domain and a DNA polymerase domain linked to each other by anWSGR Docket No.59761-775.601 RNA-protein recruitment aptamer RNA sequence, which is linked to a PEgRNA. In some embodiments, a prime editing composition comprises a PEgRNA and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein. In some embodiments, a prime editing composition comprises a PEgRNA, a ngRNA, and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein. In some embodiments, a prime editing composition comprises multiple polynucleotides, polynucleotide constructs, or vectors, each of which encodes one or more prime editing composition components. In some embodiments, the PEgRNA of a prime editing composition is associated with the DNA binding domain, e.g., a Cas9 nickase, of the prime editor. In some embodiments, the PEgRNA of a prime editing composition complexes with the DNA binding domain of a prime editor and directs the prime editor to the target DNA.

[0416] In some embodiments, a prime editing composition comprises one or more polynucleotides that encode prime editor components and / or PEgRNA or ngRNAs. In some embodiments, a prime editing composition comprises a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, and (ii) a PEgRNA or a polynucleotide encoding the PEgRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iii) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, and (iii) a PEgRNA or a polynucleotide encoding the PEgRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iv) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, the polynucleotide encoding the DNA biding domain or the polynucleotide encoding the DNA polymerase domain further encodes an additional polypeptide domain, e.g., an RNA-protein recruitment domain, such as a MS2 coat protein domain. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N and (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and / or (iv) an ngRNA or aWSGR Docket No.59761-775.601 polynucleotide encoding the ngRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain. In some embodiments, the DNA binding domain is a Cas protein domain, e.g., a Cas9 nickase. In some embodiments, the prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and / or (iv) a ngRNA or a polynucleotide encoding the ngRNA.

[0417] In some embodiments, a prime editing system comprises one or more polynucleotides encoding one or more prime editor polypeptides, wherein activity of the prime editing system can be temporally regulated by controlling the timing in which the vectors are delivered. For example, in some embodiments, a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA can be delivered simultaneously. For example, in some embodiments, a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA can be delivered sequentially.

[0418] In some embodiments, a polynucleotide encoding a component of a prime editing system can further comprise an element that is capable of modifying the intracellular half-life of the polynucleotide and / or modulating translational control. In some embodiments, the polynucleotide is a RNA, for example, an mRNA. In some embodiments, the half-life of the polynucleotide, e.g., the RNA may be increased. In some embodiments, the half-life of the polynucleotide, e.g., the RNA may be decreased. In some embodiments, the element may be capable of increasing the stability of the polynucleotide, e.g., the RNA. In some embodiments, the element may be capable of decreasing the UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, e.g., an upstream mRNA or PEgRNA end. In some embodiments, the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription.

[0419] In some embodiments, the element may include at least one AU-rich element (ARE). The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment. In some embodiments the destabilizing element may promote RNA decay, affect RNA stability, or activate translation. In some embodiments, the ARE may comprise 50 to 150 nucleotides in length. In some embodiments, the ARE may comprise at least one copy of the sequence AUUUA. In some embodiments, at least one ARE may be Virus Posttranscriptional Regulatory Element (WPRE). In further embodiments, the element is a modified and / or truncated WPRE sequence that is capable of enhancing expression from theWSGR Docket No.59761-775.601 In some embodiments, the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts. In some embodiments, the polynucleotide, e.g., a vector, encoding the PE or the PEgRNA may be self-destroyed via cleavage of a target sequence present on the polynucleotide, e.g., a vector. The cleavage may prevent continued transcription of a PE or a PEgRNA.

[0420] Polynucleotides encoding prime editing composition components can be DNA, RNA, or any combination thereof. In some embodiments, a polynucleotide encoding a prime editing composition component is an expression construct. In some embodiments, a polynucleotide encoding a prime editing composition component is a vector. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus vector, e.g., a retroviral vector, adenoviral vector, lentiviral vector, herpesvirus vector, or an adeno-associated virus vector (AAV).

[0421] In some embodiments, polynucleotides encoding polypeptide components of a prime editing composition are codon optimized by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. In some embodiments, a polynucleotide encoding a polypeptide component of a prime editing composition are operably linked to one or more expression regulatory elements, for example, a promoter, a 3’ UTR, a 5’ UTR, or any combination thereof. In some embodiments, a polynucleotide encoding a prime editing composition component is a messenger RNA (mRNA). In some embodiments, the mRNA comprises a Cap at the 5’ end and / or a poly A tail at the 3’ end.

[0422] Unless otherwise indicated, references to nucleotide positions in human chromosomes are as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCF_000001405.38.

[0423] Exemplary combinations of Prime Editing guide RNA (PEgRNA) components, e.g., spacer, PBS, and editing template / RTT, as well as combinations of each PEgRNA and corresponding ngRNA(s) are provided in Tables 18-20. Each of Tables 18-20 contains three columns. The left column is the sequence number. The middle column provides the sequence of the component, labeled with a SEQ ID NO where allowed by the ST.26 standard. Although all the sequences provided in Tables 18-20 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard. The right column contains a description of the sequence. All of the PEgRNAs disclosed in Tables 18-20 are designed to correct a c.1624G->T mutation in the CFTR gene that results in a G542x nonsense mutation (x indicates a premature stop codon; also referred to as G542ter) associated with cystic fibrosis. However, the PEgRNA disclosed in Tables 18-20 are alsoWSGR Docket No.59761-775.601 capable of correcting any other mutations in the CFTR gene that are in the portion of the gene that shares homology or complementarity with the editing template / RTT.

[0424] The PEgRNAs exemplified in Tables 18-20 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to a listed PEgRNA spacer; (b) a gRNA core capable of complexing with a Cas9 protein; and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end any RTT sequence from the same table as the PEgRNA spacer, and (ii) a primer binding site (PBS) comprising at its 5’ end any PBS sequence from the same table as the PEgRNA spacer.

[0425] The PEgRNA spacer can be, for example, 17-22 nucleotides in length. The PEgRNA spacers in Tables 18-20 are annotated in column 3 according to their associated PAM sequence, enabling selection of a prime editor comprising an appropriate Cas9 protein. For example, a prime editor comprising a SpCas9 (H840A) nickase can be used for prime editing with a spacer sequence adjacent to a NGG PAM sequence, and a prime editor comprising a SpCas9 (H840A, D1135V, G1218R, R1335Q, T1337R) nickase variant (the SpCas9 “VRQR” variant) can be used for prime editing with a spacer sequence adjacent to an NGA PAM sequence, wherein N is any nucleotide selected from A, G, C, and T. Exemplary Cas9 protein variants, corresponding PAMs, and amino acid sequences are provided in Tables 2 and 3.

[0426] The editing template can be referred to as a reverse transcription template (RTT). The editing template can encode a wildtype CFTR gene sequence and are annotated in column 3 of Tables 18-20 as simply “RTT”. Alternatively, the editing template can encode one or more synonymous mutations relative to the wildtype CFTR gene. For example, the editing template can be designed to encode one or more PAM silencing mutations besides the nucleotide edit(s) designed to correct the G542X mutation. Editing templates that encode PAM silencing mutations are identified in column 3 of Tables 18-20 by the annotation “XXX-to-YYY PAM silencing”, where XXX is the natural PAM in the target CFTR gene and YYY is the corresponding trinucleotides after incorporation of the PAM silencing edit(s).

[0427] The PBS can be, for example, 5 to 19 nucleotides in length. In some embodiments, the PBS is 8 to 15 nucleotides in length. In some embodiments, the PBS is 7-15 nucleotides in length. In some embodiments, the PBS is 10-15 nucleotides in length. In some embodiments, the PBS is 8 nucleotides in length. In some embodiments, the PBS is 11 nucleotides in length. In some embodiments, the PBS is 7, 9, 11, 13, or 15 nucleotides in length.

[0428] The gRNA core can be any gRNA core capable of binding to a Cas9 protein. For example, the gRNA core can be a canonical SpCas9 guide RNA or a variant thereof. Exemplary gRNA cores can be found in Table 10.

[0429] The PEgRNA provided in Tables 18-20 can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the editing template can be contiguous with the 5’ end of the PBS. The PEgRNA can comprise multiple RNA molecules or can be a single RNAWSGR Docket No.59761-775.601 molecule. Any PEgRNA exemplified in Tables 18-20 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, the PEgRNA comprises 4 U nucleotides at its 3’ end; without being bound by theory, this is believed to increase PEgRNA stability. In some embodiments, the PEgRNA comprises a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. Exemplary 3’ motifs can be found in Tables 11 and 12. In some embodiments, a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ end of the PBS via a linker sequence. Exemplary linker sequences can be found in Table 14. Alternatively, the 3’ motif can be directly connected to the 3’ end of the PBS without a linker sequence.

[0430] PEgRNA sequences exemplified in Tables 18-20 may include adaptations for transcription from a nucleic acid template (e.g., with a U6 promoter). Such transcription adaptations can include the addition of a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, the addition of 6 or 7 U nucleotides at the 3’ end of the extension arm, or both. The 3’ terminal U series may serve as a transcription stop signal; the actual transcribed PEgRNA may therefore contain from 1 to 7 (e.g., 4) 3’ U nucleotides. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series. Alternatively, or additionally, the PEgRNA may include an additional A nucleotide between the PBS and the 3’ terminal U series. The expression adaptations, e.g., a 5’ terminal G, are annotated in Tables 18-20. In some embodiments, for example, when the PEgRNA is chemically synthesized, such adaptation nucleotide(s) may be removed from the PEgRNA sequence. For example, a chemically synthesized PEgRNA sequence may comprise at its 5’ end nucleotides 2-n of a PEgRNA sequence provided in Tables 18-20.

[0431] The PEgRNA sequences exemplified in Tables 18-20 may be chemically synthesized. Such chemically synthesized PEgRNA may comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. In some embodiments, the chemically synthesized PEgRNA comprises an additional 4 U nucleotides on its 3’ end and the chemical modifications, if included, would comprise 3’ mU*mU*mU*U and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

[0432] Any of the PEgRNAs of Tables 18-20 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer and a gRNA coreWSGR Docket No.59761-775.601 capable of complexing with a Cas9 protein. The ngRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the ngRNA spacer comprises at its 5’ end nucleotides 4- 20 of an ngRNA spacer listed in the same table as the PEgRNA. The ngRNA can comprise multiple RNA molecules (e.g., a crRNA containing the ngRNA spacer and a tracrRNA) or can be a single gRNA molecule. The ngRNA and ngRNA spacers in Tables 18-20 are annotated with their corresponding PAM sequences. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins. The ngRNA is capable of directing a complexed Cas9 protein to bind the edit strand of the CFTR gene; thus, a complexed Cas9 nickase containing a nuclease inactivating mutation in the HNH domain will nick the non-edit strand. A PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit. The PE3b ngRNAs annotated with a * followed by a number code in Tables 18-20 have perfect complementarity to the edit strand post-edit with a PEgRNA containing an RTT from the same Table and annotated with the same number code. Such perfect complementarity may be desired, but is not required in a PEgRNA and ngRNA combination.

[0433] Any ngRNA exemplified in Tables 18-20 may comprise, or further comprise, a 3’ motif at the 3’ end of the scaffold, such as a universal motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides. In some embodiments, the ngRNA comprises 4 U nucleotides at its 3’ end; without being bound by theory, this is believed to increase ngRNA stability. In some embodiments, the ngRNA comprises a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used. Exemplary 3’ motifs can be found in Tables 11 and 12. The 3’ structural motif can be connected to the 3’ end of the ngRNA via a linker sequence. Alternatively, the 3’ motif can be directly connected to the 3’ end of the ngRNA without a linker sequence.

[0434] ngRNA sequences exemplified in Tables 18-20 may include adaptations for transcription from a nucleic acid template (e.g., with a U6 promoter). Such transcription adaptations can include the addition of a 5’ terminal G if the spacer of the ngRNA begins with another nucleotide, the addition of 6 or 7 U nucleotides at the 3’ end of the ngRNA, or both. The 3’ terminal U series may serve as a transcription stop signal; the actual transcribed ngRNA may therefore contain from 1 to 7 (e.g., 4) 3’ U nucleotides. Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the gRNA core and the 3’ terminal U series.

[0435] The ngRNA sequences exemplified in Tables 18-20 may be chemically synthesized. Such chemically synthesized ngRNA may comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof. In some embodiments, the ngRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presenceWSGR Docket No.59761-775.601 of a phosphorothioate bond. In some embodiments, the chemically synthesized ngRNA comprises an additional 4 U nucleotides on its 3’ end and the chemical modifications, if included, would comprise 3’ mU*mU*mU*U and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond. Table 18WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601Table 19WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601Table 20WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601WSGR Docket No.59761-775.601 Pharmaceutical compositions

[0436] Disclosed herein are pharmaceutical compositions comprising any of the prime editing composition components, for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, ngRNAs, and / or prime editing complexes described herein.

[0437] The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.

[0438] In some embodiments, a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.)

[0439] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit. Pharmaceutical formulations can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Methods of Editing

[0440] The methods and compositions disclosed herein can be used to edit a target gene of interest by prime editing.

[0441] In some embodiments, the prime editing method comprises contacting a target gene, e.g., a CFTR gene, with a PEgRNA and a prime editor (PE) polypeptide described herein. In some embodiments, the target gene is double stranded, and comprises two strands of DNA complementary to each other. In some embodiments, the contacting with a PEgRNA and the contacting with a prime editor are performed sequentially. In some embodiments, the contacting with a prime editor is performed after the contacting with a PEgRNA. In some embodiments, the contacting with a PEgRNA is performed after the contacting with a prime editor. In some embodiments, the contacting with a PEgRNA, and the contacting with a prime editor are performed simultaneously. In someWSGR Docket No.59761-775.601 embodiments, the PEgRNA and the prime editor are associated in a complex prior to contacting a target gene.

[0442] In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a target strand of the target gene, e.g., a CFTR gene. In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a search target sequence on the target strand of the target gene upon contacting with the PEgRNA. In some embodiments, contacting the target gene with the prime editing composition results in binding of a spacer sequence of the PEgRNA to a search target sequence with the search target sequence on the target strand of the target gene upon said contacting of the PEgRNA.

[0443] In some embodiments, contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, e.g. the target CFTR gene, upon the contacting of the PE composition with the target gene. In some embodiments, the DNA binding domain of the PE associates with the PEgRNA. In some embodiments, the PE binds the target gene, e.g. a CFTR gene, directed by the PEgRNA. Accordingly, in some embodiments, the contacting of the target gene result in binding of a DNA binding domain of a prime editor of the target CFTR gene directed by the PEgRNA.

[0444] In some embodiments, contacting the target gene with the prime editing composition results in a nick in an edit strand of the target gene, e.g., a CFTR gene by the prime editor upon contacting with the target gene, thereby generating a nicked on the edit strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in a single- stranded DNA comprising a free 3´ end at the nick site of the edit strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in a nick in the edit strand of the target gene by a DNA binding domain of the prime editor, thereby generating a single-stranded DNA comprising a free 3´ end at the nick site. In some embodiments, the DNA binding domain of the prime editor is a Cas domain. In some embodiments, the DNA binding domain of the prime editor is a Cas9. In some embodiments, the DNA binding domain of the prime editor is a Cas9 nickase.

[0445] In some embodiments, contacting the target gene with the prime editing composition results in hybridization of the PEgRNA with the 3’ end of the nicked single-stranded DNA, thereby priming DNA polymerization by a DNA polymerase domain of the prime editor. In some embodiments, the free 3’ end of the single-stranded DNA generated at the nick site hybridizes to a primer binding site sequence (PBS) of the contacted PEgRNA, thereby priming DNA polymerization. In some embodiments, the DNA polymerization is reverse transcription catalyzed by a reverse transcriptase domain of the prime editor. In some embodiments, the method comprises contacting the target gene with a DNA polymerase, e.g., a reverse transcriptase, as a part of a prime editor fusion protein or prime editing complex (in cis), or as a separate protein (in trans).WSGR Docket No.59761-775.601

[0446] In some embodiments, contacting the target gene with the prime editing composition generates an edited single stranded DNA that is coded by the editing template of the PEgRNA by DNA polymerase mediated polymerization from the 3’ free end of the single-stranded DNA at the nick site. In some embodiments, the editing template of the PEgRNA comprises one or more intended nucleotide edits compared to endogenous sequence of the target gene, e.g., a CFTR gene. In some embodiments, the intended nucleotide edits are incorporated in the target gene, by excision of the 5’ single stranded DNA of the edit strand of the target gene generated at the nick site and DNA repair. In some embodiments, the intended nucleotide edits are incorporated in the target gene by excision of the editing target sequence and DNA repair. In some embodiments, excision of the 5’ single stranded DNA of the edit strand generated at the nick site is by a flap endonuclease. In some embodiments, the flap nuclease is FEN1. In some embodiments, the method further comprises contacting the target gene with a flap endonuclease. In some embodiments, the flap endonuclease is provided as a part of a prime editor fusion protein. In some embodiments, the flap endonuclease is provided in trans.

[0447] In some embodiments, contacting the target gene with the prime editing composition generates a mismatched heteroduplex comprising the edit strand of the target gene that comprises the edited single stranded DNA, and the unedited target strand of the target gene. Without being bound by theory, the endogenous DNA repair and replication may resolve the mismatched edited DNA to incorporate the nucleotide change(s) to form the desired edited target gene.

[0448] In some embodiments, the method further comprises contacting the target gene, e.g., a CFTR gene, with a nick guide (ngRNA) disclosed herein. In some embodiments, the ngRNA comprises a spacer that binds a second search target sequence on the edit strand of the target gene. In some embodiments, the contacted ngRNA directs the PE to introduce a nick in the target strand of the target gene. In some embodiments, the nick on the target strand (non-edit strand) results in endogenous DNA repair machinery to use the edit strand to repair the non-edit strand, thereby incorporating the intended nucleotide edit in both strand of the target gene and modifying the target gene. In some embodiments, the ngRNA comprises a spacer sequence that is complementary to, and may hybridize with, the second search target sequence on the edit strand only after the intended nucleotide edit(s) are incorporated in the edit strand of the target gene.

[0449] In some embodiments, the target gene is contacted by the ngRNA, the PEgRNA, and the PE simultaneously. In some embodiments, the ngRNA, the PEgRNA, and the PE form a complex when they contact the target gene. In some embodiments, the target gene is contacted with the ngRNA, the PEgRNA, and the prime editor sequentially. In some embodiments, the target gene is contacted with the ngRNA and / or the PEgRNA after contacting the target gene with the PE. In some embodiments, the target gene is contacted with the ngRNA and / or the PEgRNA before contacting the target gene with the prime editor.WSGR Docket No.59761-775.601

[0450] In some embodiments, the target gene, e.g., a CFTR gene, is in a cell. Accordingly, also provided herein are methods of modifying a target cell, e.g., a mammalian cell, a human cell, or a human primary cell.

[0451] In some embodiments, the prime editing method comprises introducing a PEgRNA, a prime editor, and / or a ngRNA into the cell that has the target gene. In some embodiments, the prime editing method comprises introducing into the cell that has the target gene with a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and / or a ngRNA. In some embodiments, the PEgRNA, the prime editor polypeptide, and / or the ngRNA form a complex prior to the introduction into the cell. In some embodiments, the PEgRNA, the prime editor polypeptide, and / or the ngRNA form a complex after the introduction into the cell. The prime editors, PEgRNA and / or ngRNAs, and prime editing complexes may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, including ribonucleoprotein (RNPs), lipid nanoparticles (LNPs), viral vectors, non-viral vectors, mRNA delivery, and physical techniques such as cell membrane disruption by a microfluidics device. The prime editors, PEgRNA and / or ngRNAs, and prime editing complexes may be introduced into the cell simultaneously or sequentially.

[0452] In some embodiments, the prime editing method comprises introducing into the cell a PEgRNA or a polynucleotide encoding the PEgRNA, a prime editor polynucleotide encoding a prime editor polypeptide, and optionally an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and / or the ngRNA or the polynucleotide encoding the ngRNA into the cell simultaneously. In some embodiments, the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and / or the ngRNA or the polynucleotide encoding the ngRNA into the cell sequentially. In some embodiments, the method comprises introducing the polynucleotide encoding the prime editor polypeptide into the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and / or the ngRNA or the polynucleotide encoding the ngRNA. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into and expressed in the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and / or the ngRNA or the polynucleotide encoding the ngRNA into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into the cell after the PEgRNA or the polynucleotide encoding the PEgRNA and / or the ngRNA or the polynucleotide encoding the ngRNA are introduced into the cell. The polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and / or the ngRNA or the polynucleotide encoding the ngRNA, may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, for example, by RNPs, LNPs, viral vectors, non-viral vectors, mRNA delivery, and physical delivery. In some embodiments, theWSGR Docket No.59761-775.601 polynucleotide is a DNA polynucleotide. In some embodiments, the polynucleotide is a RNA polynucleotide, e.g., mRNA polynucleotide.

[0453] In some embodiments, the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and / or the polynucleotide encoding the ngRNA integrate into the genome of the prime editing target cell after being introduced into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and / or the polynucleotide encoding the ngRNA are introduced into the cell for transient expression. Accordingly, also provided herein are cells modified by prime editing.

[0454] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a non-human primate cell, a rodent cell, a bovine cell, or a porcine cell. In some embodiments, the cell is a human cell.

[0455] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a progenitor cell. In some embodiments, the cell is a pluripotent stem cell. In some embodiments, the cell is an embryonic stem cell. In some embodiments, the cell is a mesenchymal stem cell. In some embodiments, a cell is a bronchioalveolar stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, a cell is a lung progenitor cell.

[0456] In some embodiments, the cell is derived from a stem cell. In some embodiments, the cell is a primary cell. As used herein, the term primary cell can refer to a cell isolated from a subject, which is then grown in tissue culture (i.e., in vitro) for the first time before subdivision and subsequently transferred to a subculture.

[0457] In some embodiments, the cell is a part of or derived from a tissue, an organ, or a cell type. In some embodiments, a cell is in, a part of, or derived from a respiratory organ system of a subject. The respiratory system can include the nose, mouth, throat, voice box, windpipe, or lungs. In some embodiments, a cell is in, part of, or derived from a lung tissue of a subject. In some embodiments, the cell is a part of an organoid, e.g., an intestinal organoid.

[0458] In some embodiments, the cell is a somatic cell. For example, the cell can be an epithelial cell (e.g., a mammary epithelial cell, intestinal epithelial cell, a hepatocyte), a fibroblast, a keratinocyte, an endothelial cell, a glial cell, a neural cell, a muscle cell (e.g., a cardiac muscle cell, a smooth muscle cell, a myosatellite cell), a formed element of the blood (e.g., a lymphocyte, a bone marrow cell), or a precursor of any of these somatic cell types. In some embodiments, the cell is an epithelial cell. In some embodiments, the cell is an airway epithelial cell, a bronchial epithelial cell, a pancreatic epithelial cell, a pancreatic ductal epithelial cell, a pancreatic acinar cell, a kidney epithelial cell, an intestine epithelial cell, or a reproductive tissue epithelial cell (e.g., a sperm canal epithelial cell). In some embodiments, the cell is a tuft cell. In some embodiments, the cell is a neuroendocrine cell. In some embodiments, the cell is a goblet cell. In some embodiments, a cell is a basal cell. In some embodiments, a cell is a basal cell from the respiratory epithelium, e.g., from the bronchioles orWSGR Docket No.59761-775.601 alveoli of the lung. In some embodiments, a cell is a club cell. In some embodiments, a cell is a ciliated cell. In some embodiments, a cell is an ionocyte.

[0459] In some embodiments, the target gene edited by prime editing is in a chromosome of the cell. In some embodiments, the intended nucleotide edits incorporate in the chromosome of the cell and are inheritable by progeny cells. In some embodiments, the intended nucleotide edits introduced to the cell by the prime editing compositions and methods are such that the cell and progeny of the cell also include the intended nucleotide edits. In some embodiments, the cell is autologous, allogeneic, or xenogeneic to a subject. In some embodiments, the cell is from or derived from a subject. In some embodiments, the cell is from or derived from a human subject. In some embodiments, the cell is introduced back into the subject, e.g., a human subject, after incorporation of the intended nucleotide edits by prime editing.

[0460] In some embodiments, the method provided herein comprises introducing the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and / or the ngRNA or the polynucleotide encoding the ngRNA into a plurality or a population of cells that comprise the target gene. In some embodiments, the population of cells is of the same cell type. In some embodiments, the population of cells is of the same tissue or organ. In some embodiments, the population of cells is heterogeneous. In some embodiments, the population of cells is homogeneous. In some embodiments, the population of cells is from a single tissue or organ, and the cells are heterogeneous. In some embodiments, the introduction into the population of cells is ex vivo. In some embodiments, the introduction into the population of cells is in vivo, e.g., into a human subject.

[0461] In some embodiments, the target gene is in a genome of each cell of the population. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and / or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of one or more intended nucleotide edits in the target gene in at least one of the cells in the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and / or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide Edits in the target gene in a plurality of the population of cells.

[0462] In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and / or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in each cell of the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and / or the ngRNA orWSGR Docket No.59761-775.601 the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in sufficient number of cells such that the disease or disorder is treated, prevented or ameliorated.

[0463] In some embodiments, editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition. In some embodiments, the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition. In some embodiments, editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition. In some embodiments, the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition. In some embodiments, the population of cells introduced with the prime editing composition is ex vivo. In some embodiments, the population of cells introduced with the prime editing composition is in vitro. In some embodiments, the population of cells introduced with the prime editing composition is in vivo. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 25% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 35% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein has an editing efficiency of at least 30% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 45% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 50% relative to a suitable control. In some embodiments, editing efficiency of prime the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells after in vivo engraftment of the edited cells. In some embodiments, the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of engraftment. In some embodiments, the editing efficiency is determined after 8 or 16 weeks of engraftment. In some embodiments, prime editing is able to maintain in edited cells at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more than 95% of editing efficiency after 8 or 16 weeks post engraftment.WSGR Docket No.59761-775.601

[0464] In some embodiments, the methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a primary cell (as measured in a population of primary cells) relative to a suitable control.

[0465] In some embodiments, the methods disclosed herein have an editing efficiency of at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a cell as disclosed herein.

[0466] In some embodiments, the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits without generating a significant proportion of indels. The term “indel(s)”, as used herein, refers to the insertion or deletion of a nucleotide base within a polynucleotide, for example, a target gene. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. Indel frequency of editing can be calculated by methods known in the art. In some embodiments, indel frequency can be calculated based on sequence alignment such as the CRISPResso 2 algorithm as described in Clement et al., Nat. Biotechnol. 37(3): 224-226 (2019), which is incorporated herein in its entirety. In some embodiments, the prime editing methods disclosed herein can have an indel frequency of less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, or...

Claims

WSGR Docket No.59761-775.601 CLAIMS WHAT IS CLAIMED IS:

1. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.

2. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer comprising at its 3’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template comprising at its 3’ end sequence number 64, and ii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO:

1.

3. The PEgRNA of claim 1 or 2, wherein the gRNA core comprises nucleotide sequence: GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGC (SEQ ID NO: 592), wherein T indicates the presence of a uridine nucleotide.

4. The PEgRNA of any one of claims 1-3, wherein the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

5. The PEgRNA of claim 4, wherein the 3’ motif is directly connected to the PBS at its 3’ end.

6. The PEgRNA of claim 4, wherein the 3’ motif is linked to the PBS at its 3’ end via a linker.

7. The PEgRNA of claim 6, wherein the linker is 4 nucleotides in length.WSGR Docket No.59761-775.601 8. The PEgRNA of any one of claims 1-7, wherein the editing template comprises at its 3’end SEQ ID NOs: 68, 76, 84, 91, or 97.

9. The PEgRNA of claim 8, wherein the editing template has a length of 20 nucleotides or less.

10. The PEgRNA of claim 8, wherein the editing template has a length of 10, 13, 17, or 20 nucleotides.

11. The PEgRNA of any one of claims 1-7, wherein the editing template consists of SEQ ID NO:

64.

12. The PEgRNA of any one of claims 1-11, wherein the spacer is from 17-22 nucleotides in length.

13. The PEgRNA of claim 12, wherein the spacer comprises at its 3’ end SEQ ID NO:

10.

14. The PEgRNA of claim 12, wherein the spacer has the sequence of SEQ ID NO:

10.

15. The PEgRNA of any one of claims 1-13, wherein the PBS comprises at its 5’ end sequence number 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, or 61.

16. The PEgRNA of claim 15, wherein the PBS comprises at its 5’ end sequence number 28, 37, 43, or 49.

17. The PEgRNA of claim 15, wherein the PBS comprises at its 5’ end sequence number 19, 22, 25, 55, 58, or 61.

18. The PEgRNA of claim 15, wherein the PBS has a length of 20 nucleotides or less.

19. The PEgRNA of claim 15, wherein the PBS is 8-15 nucleotides in length.

20. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 306, 309, 310, 314, 317, 318, 322, 328, 335, 336, 345, 353, 363, 364, 371, 382, 390, 399, 400, 410, 425, 426, 443, and 457.

21. A prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of claims 1-20, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 473, 474, 475, 476, or 477, and (ii) an ngRNA core capable of binding a Cas9 protein.

22. The prime editing system of claim 21, wherein the ngRNA spacer comprises at its 3’ end SEQ ID NO:

473.

23. The prime editing system of claim 21, wherein the ngRNA spacer comprises at its 3’ end SEQ ID NO:

475.

24. The prime editing system of claim 21, wherein the ngRNA spacer comprises at its 3’ end SEQ ID NOs: 474, 476, or 477.WSGR Docket No.59761-775.601 25. The prime editing system of any one of claims 21-24, wherein the ngRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTG AAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAGCGTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), wherein T indicates the presence of a uridine nucleotide.

26. The prime editing system of claim 25, wherein the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 491, 493, 494, 496, 499, 500, 501, 504, 505, 506, 507, or 508.

27. The prime editing system of claim 25, wherein the ngRNA comprise SEQ ID NOs: 486, 487, 489, 491, 493, 494, 496, 500, 501, 504, 505, 506, 507, or 508.

28. The prime editing system of claim 25, wherein the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507, or 508.

29. The prime editing system of claim 25, wherein the ngRNA comprises SEQ ID NOs: 491, 493, 494, or 496.

30. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 2; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 2, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.WSGR Docket No.59761-775.601 31. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer comprising at its 3’ end SEQ ID NO: 2; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template comprising at its 3’ end nucleotides 4-8 of sequence number 66, and ii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO:

2.

32. The PEgRNA of claim 30 or 31, wherein the editing template comprises at its 3’ end sequence number 66.

33. The PEgRNA of claim 32, wherein the editing template comprises at its 3’ end sequence number 66, 67, 69, 71, 75, 77, 78, 83, 87, 88, 89, 92, 93, 94, 96, 98, or 100.

34. The PEgRNA of claim 32, wherein the editing template consists of sequence number 66.

35. The PEgRNA of claim 33, wherein the editing template has a length of 25 nucleotides or less.

36. The PEgRNA of claim 35, wherein the editing template is 11 or 12 nucleotides in length.

37. The PEgRNA of claim 30, wherein the editing template comprises at its 3’ end SEQ ID NOs: 829, 830, 831, 832, 833, 834, 853, 854, 855, 856, 857, 858, 877, 878, 879, 880, 881, 882, 901, 902, 903, 904, 905, 906, 925, 926, 927, 928, 929, 930, 949, 950, 951, 952, 953, 954, 973, 974, 975, 976, 977, 993, 994, 995, 996, 997, or 998.

38. The PEgRNA of claim 30 or 31, wherein the editing template further encodes a PAM silencing edit.

39. The PEgRNA of claim 38, wherein the editing template encodes a TGA-to-GGT PAM silencing edit.

40. The PEgRNA of claim 39, wherein the editing template comprises at its 3’ end nucleotides 7- 12 of SEQ ID NO:

72.

41. The PEgRNA of claim 40, wherein the editing template comprises at its 3’ end SEQ ID NOs: 72, 80 or 85.

42. The PEgRNA of claim 30 or 31, wherein the editing template encodes a TGA-to-GGG PAM silencing edit.

43. The PEgRNA of claim 42, wherein the editing template comprises at its 3’ end nucleotides 7- 12 of SEQ ID NO:

73.

44. The PEgRNA of claim 43, wherein the editing template comprises at its 3’ end SEQ ID NOs: 73, 81 or 86.

45. The PEgRNA of claim 30 or 31, wherein the editing template encodes a TGA-to-GGC PAM silencing edit.WSGR Docket No.59761-775.601 46. The PEgRNA of claim 45, wherein the editing template comprises at its 3’ end nucleotides 7- 12 of SEQ ID NO:

74.

47. The PEgRNA of claim 46, wherein the editing template comprises at its 3’ end SEQ ID NOs: 74 or 82.

48. The PEgRNA of claim 30, wherein the editing template encodes a GGA-to-GGC PAM silencing.

49. The PEgRNA of claim 48, wherein the editing template comprises at its 3’ end SEQ ID NOs: 835, 836, 837, 838, 839, 840, 859, 860, 861, 862, 863, 864, 883, 884, 885, 886, 887, 888, 907, 908, 909, 910, 911, 912, 931, 932, 933, 934, 935, 936, 955, 956, 957, 958, 959, 960, 978, 979, 980, 981, 982, 999, 1000, 1001, 1002, 1003, or 1004.

50. The PEgRNA of claim 30, wherein the editing template encodes a GGA-to-GGG PAM silencing.

51. The PEgRNA of claim 50, wherein the editing template comprises at its 3’ end SEQ ID NOs: 841, 842, 843, 844, 845, 846, 865, 866, 867, 868, 869, 870, 889, 890, 891, 892, 893, 894, 913, 914, 915, 916, 917, 918, 937, 938, 939, 940, 941, 942, 961, 962, 963, 964, 965, 966, 983, 984, 985, 986, 987, 1005, 1006, 1007, 1008, 1009, or 1010.

52. The PEgRNA of claim 30, wherein the editing template encodes a GGA-to-GGT PAM silencing.

53. The PEgRNA of claim 52, wherein the editing template comprises at its 3’ end SEQ ID NOs: 847, 848, 849, 850, 851, 852, 871, 872, 873, 874, 875, 876, 895, 896, 897, 898, 899, 900, 919, 920, 921, 922, 923, 924, 943, 944, 945, 946, 947, 948, 967, 968, 969, 970, 971, 972, 988, 989, 990, 991, 992, 1011, 1012, 1013, 1014, 1015, or 1016.

54. The PEgRNA of any one of claims 37-53, wherein the editing template has a length of 16 nucleotides or less.

55. The PEgRNA of claim 54, wherein the editing template is 12-16 nucleotides in length.

56. The PEgRNA of any one of claims 30-55, wherein the gRNA core comprises nucleotide sequenceGTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTG AAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), orWSGR Docket No.59761-775.601 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAGCGTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), wherein T indicates the presence of a uridine nucleotide.

57. The PEgRNA of any one of claims 30-56, wherein the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

58. The PEgRNA of claim 57, wherein the 3’ motif is directly connected to the PBS at its 3’ end.

59. The PEgRNA of claim 57, wherein the 3’ motif is linked to the PBS at its 3’ end via a linker.

60. The PEgRNA of claim 59, wherein the linker is 4 nucleotides in length.

61. The PEgRNA of any one of claims 30-60, wherein the spacer is from 17-22 nucleotides in length.

62. The PEgRNA of claim 61, wherein the spacer comprises at its 3’ end SEQ ID NO:

11.

63. The PEgRNA of claim 62, wherein the spacer has the sequence of SEQ ID NO:

11.

64. The PEgRNA of any one of claims 30-63, wherein the PBS is 8-15 nucleotides in length.

65. The PEgRNA of any one of claims 32-36, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 129, 130, 131, 132, 133, 134, 135, 136, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 159, 160, 161, 162, 163, 164, 165, 166, 167, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 315, 319, 321, 323, 326, 327, 329, 331, 332, 333, 334, 337, 340, 341, 342, 343, 344, 346, 347, 348, 349, 350, 351, 352, 354, 358, 359, 360, 361, 362, 365, 366, 367, 368, 369, 370, 372, 373, 376, 377, 378, 379, 380, 381, 383, 385, 386, 387, 388, 389, 391, 394, 395, 396, 397, 398, 401, 402, 404, 405, 406, 407, 408, 409, 411, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 427, 428, 429, 432, 433, 434, 435, 436, 438, 439, 440, 441, 442, 444, 446, 447, 448, 449, 450, 451, 453, 454, 455, 456, 458, 460, 461, 462, 463, 464, 465, 466, 468, 469, 470, 471, 472, 1210, 1211, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 828, and 1017.WSGR Docket No.59761-775.601 66. The PEgRNA of any one of claims 39-41 or 54-55, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 126, 137, 156, 168, 244, and 1184.

67. The PEgRNA of any one of claims 42-44 or 54-55, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 127, 138, 157, 169, 245, and 1178.

68. The PEgRNA of any one of claims 45-47, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 128, 139, 158, 170, and 1172.

69. The PEgRNA of claim 37, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1018, 1019, 1020, 1021, 1022, 1023, 1042, 1043, 1044, 1045, 1046, 1047, 1066, 1067, 1068, 1069, 1070, 1071, 1090, 1091, 1092, 1093, 1094, 1095, 1114, 1115, 1116, 1117, 1118, 1119, 1138, 1139, 1140, 1141, 1142, 1143, 1162, 1163, 1164, 1165, 1166, 1185, 1186, 1187, 1188, 1189, and 1190.

70. The PEgRNA of any one of claims 48-49, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1024, 1025, 1026, 1027, 1028, 1029, 1048, 1049, 1050, 1051, 1052, 1053, 1072, 1073, 1074, 1075, 1076, 1077, 1096, 1097, 1098, 1099, 1100, 1101, 1120, 1121, 1122, 1123, 1124, 1125, 1144, 1145, 1146, 1147, 1148, 1149, 1167, 1168, 1169, 1170, 1171, 1191, 1192, 1193, 1194, 1195, and 1196.

71. The PEgRNA of any one of claims 50-51, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1030, 1031, 1032, 1033, 1034, 1035, 1054, 1055, 1056, 1057, 1058, 1059, 1078, 1079, 1080, 1081, 1082, 1083, 1102, 1103, 1104, 1105, 1106, 1107, 1126, 1127, 1128, 1129, 1130, 1131, 1150, 1151, 1152, 1153, 1154, 1155, 1173, 1174, 1175, 1176, 1177, 1197, 1198, 1199, 1200, 1201, and 1202.

72. The PEgRNA of any one of claims 52-53, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1036, 1037, 1038, 1039, 1040, 1041, 1060, 1061, 1062, 1063, 1064, 1065, 1084, 1085, 1086, 1087, 1088, 1089, 1108, 1109, 1110, 1111, 1112, 1113, 1132, 1133, 1134, 1135, 1136, 1137, 1156, 1157, 1158, 1159, 1160, 1161, 1179, 1180, 1181, 1182, 1183, 1203, 1204, 1205, 1206, 1207, and 1208.

73. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,WSGR Docket No.59761-775.601 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 315, 319, 321, 323, 326, 327, 329, 331, 332, 333, 334, 337, 340, 341, 342, 343, 344, 346, 347, 348, 349, 350, 351, 352, 354, 358, 359, 360, 361, 362, 365, 366, 367, 368, 369, 370, 372, 373, 376, 377, 378, 379, 380, 381, 383, 385, 386, 387, 388, 389, 391, 394, 395, 396, 397, 398, 401, 402, 404, 405, 406, 407, 408, 409, 411, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 427, 428, 429, 432, 433, 434, 435, 436, 438, 439, 440, 441, 442, 444, 446, 447, 448, 449, 450, 451, 453, 454, 455, 456, 458, 460, 461, 462, 463, 464, 465, 466, 468, 469, 470, 471, 472, 1210, 1211, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 828, and 1017.

74. A prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of claims 30-73, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 473, 474, 476 or 477; and (ii) an ngRNA core capable of binding a Cas9 protein.

75. The prime editing system of claim 74, wherein the ngRNA spacer comprises SEQ ID NO:

473.

76. The prime editing system of claim 74, wherein the ngRNA comprises SEQ ID NOs: 491, 493, 494, or 496.

77. The prime editing system of claim 74, wherein the ngRNA comprises SEQ ID NO:

496.

78. A prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of claims 32-36 or 65, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 475; and (ii) an ngRNA core capable of binding a Cas9 protein.

79. The prime editing system of claim 78, wherein the ngRNA spacer comprises at its 3’end SEQ ID NO:

475.

80. The prime editing system of claim 78 or 79, wherein the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507, or 508.

81. A prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of claims 39-41, 54-55 or 66, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises:WSGR Docket No.59761-775.601 (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 478; and (ii) an ngRNA core capable of binding a Cas9 protein.

82. The prime editing system of claim 81, wherein the ngRNA spacer comprises at its 3’end SEQ ID NO:

478.

83. The prime editing system of claim 81 or 82, wherein the ngRNA comprises SEQ ID NO:

502.

84. A prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of claims 42-44 or 54-55 or 67, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 479; and (ii) an ngRNA core capable of binding a Cas9 protein.

85. The prime editing system of claim 84, wherein the ngRNA spacer comprises at its 3’end SEQ ID NO:

479.

86. The prime editing system of claim 84 or 85, wherein the ngRNA comprises SEQ ID NO:

503.

87. The prime editing system of any one of claims 74-75, 78-79, 81-82, or 84-85, wherein the ngRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAA AACGCGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTG AAAACGCGGCACCGAGTCGGTGC (SEQ ID NO:593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTG AAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTA TCAGCGTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), wherein T indicates the presence of a uridine nucleotide.

88. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 3; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, andWSGR Docket No.59761-775.601 ii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.

89. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer comprising at its 3’ end SEQ ID NO: 3; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template comprising at its end sequence number 65, and ii. a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO:

3.

90. The PEgRNA of claim 88 or 89, wherein the gRNA core comprises nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTG AAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.

91. The PEgRNA of any one of claims 88-90, wherein the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

92. The PEgRNA of claim 91, wherein the 3’ motif is directly connected to the PBS at its 3’ end.

93. The PEgRNA of claim 91, wherein the 3’ motif is linked to the PBS at its 3’ end via a linker.

94. The PEgRNA of claim 93, wherein the linker is 4 nucleotides in length.

95. The PEgRNA of any one of claims 88-94, wherein the editing template comprises at its 3’end SEQ ID NOs: 70, 79, 90, 95, or 99.

96. The PEgRNA of claim 95, wherein the editing template has a length of 24 nucleotides or less.

97. The PEgRNA of claim 95, wherein the editing template is a length of 10, 14, 18, 21, or 24 nucleotides.

98. The PEgRNA of claim 88 or 89, wherein the editing template consists of sequence number 65.

99. The PEgRNA of any one of claims 88-98, wherein the spacer is from 17-22 nucleotides in length.

100. The PEgRNA of claim 99, wherein the spacer comprises at its 3’ end SEQ ID NO:

12.

101. The PEgRNA of claim 100, wherein the spacer has the sequence of SEQ ID NO:

12.

102. The PEgRNA of any one of claims 88-101, wherein the PBS comprises at its 5’ end sequence number 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, or 63.WSGR Docket No.59761-775.601 103. The PEgRNA of claim 102, wherein the PBS comprises at its 5’ end sequence number 27, 54, 57, 60, or 63.

104. The PEgRNA of claim 102, wherein the PBS comprises at its 5’ end sequence number 27, 33, 39, 45, or 51.

105. The PEgRNA of any one of claims 77-101, wherein the PBS consists of SEQ ID NO:

27.

106. The PEgRNA of any one of claims 102-105, wherein the PBS has a length of 15 nucleotides or less.

107. The PEgRNA of claim 106, wherein the PBS is 7-15 nucleotides in length.

108. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 307, 308, 312, 313, 316, 320, 324, 325, 330, 338, 339, 355, 356, 357, 374, 375, 384, 392, 393, 403, 412, 413, 419, 430, 431, 437, 445, 452, 459, and 467.

109. A prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of claims 88-108, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 480, 11, 481, 482, 483, or 484, and (ii) an ngRNA core capable of binding a Cas9 protein.

110. The prime editing system of claim 109, wherein the ngRNA spacer comprises at its 3’end SEQ ID NOs: 480, 11, 481, 482, 483, or 484.

111. The prime editing system of claim 109 or 110, wherein the gRNA core comprises nucleotide sequence: GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTG AAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.

112. The prime editing system of claim 111, wherein the ngRNA comprises SEQ ID NOs: 488, 490, 492, 495, 497, or 498.

113. The PEgRNA of any one of claims 1-20, 31-73, or 88-107, comprising from 5’ to 3’, the spacer, the gRNA core, the RTT, and the PBS.

114. The PEgRNA of claim 113, wherein the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule.

115. The PEgRNA of claim 113 or 114, further comprising 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

116. The PEgRNA of claim 115, wherein the PEgRNA comprises s 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-MeWSGR Docket No.59761-775.601 modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.

117. The prime editing system of any one of claims 21-30, 74-87, or 109-112, wherein the PEgRNA and / or the ngRNA further comprises 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.

118. The prime editing system of claim 117, wherein the PEgRNA and / or the ngRNA comprises s 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.

119. A prime editing system comprising (a) the PEgRNA of any one of claims 1-20, 31-73, 88- 108, or 113-116, or one or more polynucleotides encoding the PEgRNA, and (b) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.

120. The prime editing system of claim 21-29, 74-87, 109-112, or 117-118, further comprising a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.

121. The prime editing system of claim 119 or 120, wherein the prime editor is a fusion protein.

122. A prime editing system comprising (a) the PEgRNA of any one of claims 1-20, 30-73, 88- 108, or 113-116 or one or more polynucleotides encoding the PEgRNA, (b) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (c) a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C- terminal exteins are capable of self-excision to join the N-terminal fragment and the C- terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.

123. The prime editing system of any one of claims 21-29, 74-87, 109-112, or 117-118, further comprising: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (d) a C- terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C- intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C- intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-WSGR Docket No.59761-775.601 terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.

124. A population of viral particles collectively comprising the one or more polynucleotides encoding the prime editing system of any one of claims 119-123.

125. The population of viral particle of claim 124, wherein the viral particles are AAV particles.

126. An LNP comprising the prime editing system of any one of claims 119-123.

127. The LNP of claim 126, comprising the PEgRNA, the polynucleotide encoding the Cas9 nickase, and the polynucleotide encoding the reverse transcriptase.

128. The LNP of claim 127, wherein the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are mRNA.

129. The LNP of claim 127 or 128, wherein the polynucleotide encoding the Cas9 nickase and the polynucleotide d encoding the reverse transcriptase are the same molecule.

130. A method of editing a CFTR gene, the method comprising contacting the CFTR gene with: (a) the PEgRNA of any one of claims 1-20, 30-73, 88-108, or 113-116, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase or (b) the prime editing system of any one of claims 109-112.

131. The method of claim 130, wherein the CFTR gene is in a cell.

132. The method of claim 131, wherein the cell is a mammalian cell.

133. The method of claim 131, wherein the cell is a human cell.

134. The method of any one of claims 131-133, wherein the cell is a primary cell.

135. The method of any one of claims 131-133, wherein the cell is an epithelial cell.

136. The method of any one of claims 131-135, wherein the cell is in a subject or obtained from a subject or a cell bank.

137. The method of claim 136, wherein the subject is a human.

138. The method of any one of claims 131-137, wherein contacting the CFTR gene comprises contacting the cell with (i) the population of viral particles of claim 124 or 125 or (ii) the LNP of any one of claims 126-129.

139. A method of treating cystic fibrosis in a subject in need thereof, the method comprising administering to the subject (i) the PEgRNA of any one of claims 1-20, 31-73, 88-108, or 113- 116, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (ii) the prime editing system of any one of claims 119-123, (iii) the population of viral particles of claim 124 or 125, or (iv) the LNP of any one of claims 126-129.

140. A prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises:WSGR Docket No.59761-775.601 a. a spacer that comprises at its 3’ end a PEgRNA spacer sequence selected from any one of Tables 18-20; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i. an editing template that comprises at its 3’ end an RTT sequence selected from same Table as the PEgRNA spacer sequence, and ii. a primer binding site (PBS) that comprises at its 5’ end a PBS sequence selected from same Table as the PEgRNA spacer sequence.

141. The PEgRNA of claim 140, wherein the spacer of the PEgRNA is from 17 to 22 nucleotides in length.

142. The PEgRNA of claim 141, wherein the spacer of the PEgRNA is 20 nucleotides in length.

143. The PEgRNA of any one of claims 140-142, comprising from 5’ to 3’, the spacer, the gRNA core, the editing template, and the PBS.

144. The PEgRNA of claim 143, wherein the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule.

145. The PEgRNA of any one of claims 140-144, wherein the gRNA core comprises a gRNA core sequence selected from Table 10.

146. A prime editing system comprising: (a) the prime editing guide RNA (PEgRNA) of any one of claims 140-145, or one or more polynucleotides encoding the PEgRNA; and optionally (b) a nick guide RNA (ngRNA), or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises a spacer comprising at its 3’ end nucleotides 4-20 of any ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence, and a ngRNA core capable of binding to a Cas9 protein.

147. The prime editing system of claim 146, wherein the ngRNA spacer is from 17 to 22 nucleotides in length.

148. The prime editing system of claim 147, wherein the spacer of the ngRNA comprises at its 3’ end nucleotides 3-20, 2-20, or 1-20 of the ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence.

149. The prime editing system of any one of claims 146-148, wherein the ngRNA spacer is 20 nucleotides in length.

150. The prime editing system of any one of claims 146-148, wherein the ngRNA core comprises a gRNA core sequence selected from Table 10.

151. The prime editing system of any one of claims 146-150, further comprising: (c) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or a nucleic acid encoding the Cas9 nickase, and a reverse transcriptase, or a nucleic acid encoding the reverse transcriptase.WSGR Docket No.59761-775.601 152. The prime editing system of any one of claims 146-150, further comprising: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (d) a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C- terminal exteins are capable of self-excision to join the N-terminal fragment and the C- terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase and a reverse transcriptase (RT) domain.

Images