COMPOSIÇÕES E MÉTODOS DE EDIÇÃO DE GENOMA PARA O TRATAMENTO DA FIBROSE CÍSTICA

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

[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 3, 2023, each of which is incorporated herein by reference in its entirety. BACKGROUND

[0002] Cystic fibrosis (CF) is one of the most common genetic diseases, particularly in the Caucasian population. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR / ABCC7) gene (chr7: 117480025117668665 (GRCh38 / hg38)). The CFTR protein encoded by the CFTR gene is a chloride channel, which is involved in fluid transport and surface hydration across the epithelial cells of the body's tubular organs (e.g., lungs and intestines). Mutations in the CFTR gene result in defective biosynthesis, trafficking, and / or activity of the CFTR protein, potentially causing severe damage to the lungs, pancreas, liver, intestines, sinuses, and limited respiratory capacity over time. Although technological advances have increased the life expectancy of certain patients with CF, numerous mutations remain unresponsive to currently existing therapies, and there is still no effective cure for the disease. SUMMARY OF THE DISCLOSURE

[0003] This disclosure provides first-line editing methods and compositions for correcting mutations in the CFTR gene associated with cystic fibrosis.

[0004] Methods and compositions for prime editing of alterations in a target sequence in a target gene, for example, a CFTR gene, are provided in this document in some embodiments. The target CFTR gene may comprise stranded DNA. Petition 870260064333, dated 06 / 30 / 2026, p. 7 / 427 2 / 418 double. As exemplified in Figure 1, in some modalities, the target gene, for example, a CFTR gene, is edited by prime editing. In some modalities, the prime editing described in this document results in the efficient correction of one or more pathogenic mutations in the CFTR gene, thus treating cystic fibrosis in an individual.

[0005] Without limiting ourselves to any specific theory, the prime editing process can seek specific targets and edit endogenous sequences in a target gene, for example, the CFTR gene. As exemplified in Figure 1, the spacer sequence of a PEgRNA recognizes and anneals to a search target sequence on a target strand of the target gene. A prime editing complex can generate a cut in the target gene on the editing strand, which is the complementary strand of the target strand. The prime editing complex can then use a free 3' end formed at the cutting site of the editing strand to initiate DNA synthesis, where a primer binding site (PBS) of the PEgRNA forms a complex with the free 3' end, and single-stranded DNA is synthesized using a PEgRNA editing template as a model. The editing template may comprise one or more nucleotide edits compared to the endogenous target CFTR gene sequence.Consequently, the newly synthesized single-stranded DNA also comprises the nucleotide edit(s) encoded by the editing template. Through the removal of a target editing sequence in the editing strand of the target gene and DNA repair, the intended nucleotide edit(s) included in the newly synthesized single-stranded DNA is / are incorporated into the target CFTR gene.

[0006] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence in a first strand of a CF transmembrane conductance regulator (CFTR) gene, wherein the spacer comprises Petition 870260064333, dated 06 / 30 / 2026, p. 8 / 427 3 / 418 its 3' end to 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 a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) comprising 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 the second strand are complementary to each other, wherein the editing template encodes or comprises a G nucleotide at position c.1624 of a wild-type CFTR coding sequence.

[0007] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, 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) comprising 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 the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), where T indicates the presence of a uridine nucleotide.

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

[00010] In some embodiments, the 3' motif is directly connected to the PBS at its 3' end. Petition 870260064333, dated 06 / 30 / 2026, p. 9 / 427 4 / 418

[00011] In some embodiments, the 3' motif is linked to the PBS at its 3' end by means of a linker.

[00012] In some embodiments, the ligand is 4 nucleotides long.

[00013] In some forms, the edit model includes at its 3' end the SEQ ID NO: 68, 76, 84, 91 or 97.

[00014] In some embodiments, the editing template has a length of 20 nucleotides or less.

[00015] In some forms, the editing template has a length of 10, 13, 17, or 20 nucleotides.

[00016] In some modalities, the editing model consists of the sequence TTCTCCA.

[00017] In some embodiments, the spacer is 17 to 22 nucleotides long.

[00018] In some embodiments, the spacer comprises at its 3' end the SEQ ID NO: 10.

[00019] In some forms, the spacer has the sequence SEQ ID NO: 10.

[00020] In some embodiments, the PBS comprises at its 5' end the sequence number 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 or 61.

[00021] In some embodiments, the PBS comprises at its 5' end the sequence number 28, 37, 43 or 49.

[00022] In some embodiments, the PBS comprises at its 5' end the sequence number 19, 22, 25, 55, 58 or 61.

[00023] In some embodiments, PBS has a length of 20 nucleotides or less.

[00024] In some forms, PBS is 8 to 15 nucleotides long.

[00025] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, wherein the PEgRNA comprises a sequence Petition 870260064333, dated 06 / 30 / 2026, p. 10 / 427 5 / 418 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.

[00026] In one aspect, a prime editing system is provided in this document comprising: (a) PEgRNA or one or more polynucleotides of any of the aspects and embodiments described in this document, and (b) an ngRNA or one or more polynucleotides encoding 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 to a Cas9 protein.

[00027] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ ID NO: 473.

[00028] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ ID NO: 475.

[00029] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ IDs 474, 476 or 477.

[00030] In some embodiments, the core of the ngRNA comprises the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGT GAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), where T indicates the presence of a uridine nucleotide.

[00031] In some embodiments, the ngRNA comprises SEQ ID NO: 485, 486, 487, 489, 491, 493, 494, 496, 499, 500, 501, 504, Petition 870260064333, dated 06 / 30 / 2026, p. 11 / 427 6 / 418 505, 506, 507 or 508.

[00032] In some embodiments, the ngRNA comprises SEQ ID NO: 486, 487, 489, 491, 493, 494, 496, 500, 501, 504, 505, 506, 507 or 508.

[00033] In some embodiments, the ngRNA comprises SEQ ID NO: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507 or 508.

[00034] In some embodiments, the ngRNA comprises SEQ ID NO: 491, 493, 494 or 496.

[00035] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA is provided in this document, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence in a first strand of a CF transmembrane conductance regulator (CFTR) gene, wherein the spacer comprises at its 3' end the 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 a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) comprising 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 the second strand are complementary to each other, wherein the editing template encodes or comprises a G nucleotide at position c.1624 of a wild-type CFTR encoding sequence.

[00036] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, 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 to 8 of Petition 870260064333, dated 06 / 30 / 2026, page 12 / 427 7 / 418 sequence number 66, and ii) a primer binding site (PBS) comprising at its 5' end a sequence that is a reverse complement of nucleotides 10 to 14 of SEQ ID NO: 2.

[00037] In some forms, the editing model includes the number sequence 66 at its 3' end.

[00038] In some forms, the edition model comprises at its 3' end the number sequence 66, 67, 69, 71, 75, 77, 78, 83, 87, 88, 89, 92, 93, 94, 96, 98 or 100.

[00039] In some modes, the editing model consists of the number sequence 66.

[00040] In some embodiments, the editing template has a length of 25 nucleotides or less.

[00041] In some forms, the editing template is 11 or 12 nucleotides long.

[00042] In some forms, the editing model includes at its 3' end the SEQ ID NO: 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.

[00043] In some modes, the editing model additionally encodes a PAM muting edit.

[00044] In some modes, the editing model encodes a PAM muting edit from TGA to GGT.

[00045] In some embodiments, the editing template comprises, at its 3' end, nucleotides 7 to 12 of SEQ ID NO: 72.

[00046] In some forms, the edit model includes, at its 3' end, the SEQ ID NO: 72, 80 or 85.

[00047] In some modes, the editing model encodes a PAM silencing edit from TGA to GGG.

[00048] In some embodiments, the editing model comprises, at its 3' end, nucleotides 7 to 12 of the SEQ Petition 870260064333, dated 06 / 30 / 2026, page 13 / 427 8 / 418 ID NO: 73.

[00049] In some forms, the edit model includes, at its 3' end, the SEQ ID NO: 73, 81 or 86.

[00050] In some modes, the editing model encodes a TGA to GGC PAM silencing edit.

[00051] In some embodiments, the editing template comprises, at its 3' end, nucleotides 7 to 12 of SEQ ID NO: 74.

[00052] In some forms, the edit model includes, at its 3' end, the SEQ ID NO: 74 or 82.

[00053] In some modes, the editing model encodes a PAM silencing from GGA to GGC.

[00054] In some forms, the editing model includes at its 3' end the SEQ ID NO: 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.

[00055] In some modes, the editing model encodes a PAM silencing from GGA to GGG.

[00056] In some forms, the editing model comprises at its 3' end the SEQ ID NO: 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.

[00057] In some modes, the editing model encodes a PAM silencing from GGA to GGT.

[00058] In some forms, the editing model comprises at its 3' end the SEQ ID NO: 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, Petition 870260064333, dated 06 / 30 / 2026, page 14 / 427 9 / 418 1012, 1013, 1014, 1015 or 1016.

[00059] In some embodiments, the editing template has a length of 16 nucleotides or less.

[00060] In some forms, the editing template is 12 to 16 nucleotides long.

[00061] In some embodiments, the gRNA core comprises the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGT GAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), where T indicates the presence of a uridine nucleotide.

[00062] In some embodiments, the extension arm additionally comprises a 3' motif comprising the nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), where T indicates the presence of a uridine nucleotide.

[00063] In some embodiments, the 3' motif is directly connected to the PBS at its 3' end.

[00064] In some embodiments, the 3' motif is linked to the PBS at its 3' end by means of a linker.

[00065] In some embodiments, the ligand is 4 nucleotides long.

[00066] In some embodiments, the spacer is 17 to 22 nucleotides long.

[00067] In some embodiments, the spacer comprises at its 3' end the SEQ ID NO: 11. Petition 870260064333, dated 06 / 30 / 2026, p. 15 / 427 10 / 418

[00068] In some modes, the SEQ ID NO: 11.

[00069] In some forms, the spacer has the sequence the PBS is 8 to 15 nucleotides long. PEgRNA comprises a

[00070] In some modalities, Selected sequence 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, , , , , , , , , , , , , , , , , , , , , , , , , , , , , Petition 870260064333, dated 06 / 30 / 2026, p. 16 / 427 11 / 418 822, 823, 824, 825, 826, 828 and 1017.

[00071] In some embodiments, PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 126, 137, 156, 168, 244 and 1184.

[00072] In some embodiments, PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 127, 138, 157, 169, 245 and 1178.

[00073] In some embodiments, PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 128, 139, 158, 170 and 1172.

[00074] In some embodiments, PEgRNA comprises a sequence selected from the group consisting of SEQ ID NO: 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.

[00075] In some embodiments, PEgRNA comprises a sequence selected from the group consisting of SEQ ID NO: 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.

[00076] In some embodiments, 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.

[00077] In some embodiments, PEgRNA comprises a sequence selected from the group consisting of SEQ ID NO: 1036, Petition 870260064333, dated 06 / 30 / 2026, p. 17 / 427 12 / 418 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, 1.207 and 1.208.

[00078] In one aspect, an RNA is provided in this document. Prime editing guide (PEgRNA) or one or more polynucleotides encoding 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, 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, Petition 870260064333, dated 06 / 30 / 2026, p. 18 / 427 13 / 418 470, 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.

[00079] In one aspect, a prime editing system is provided in this document comprising: (a) PEgRNA or one or more polynucleotides of any of the aspects or embodiments described (a) in this document, and (b) an ngRNA or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an 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 to a Cas9 protein.

[00080] In some embodiments, the ngRNA spacer comprises SEQ ID NO: 473.

[00081] In some embodiments, the ngRNA comprises SEQ ID NO: 491, 493, 494 or 496.

[00082] In some embodiments, ngRNA comprises SEQ ID NO: 496.

[00083] In one aspect, a prime editing system is provided in this document comprising (a) PEgRNA or one or more polynucleotides of any of the aspects or embodiments described in this document, and (b) an ngRNA or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 475; and (ii) an ngRNA core capable of binding to a Cas9 protein.

[00084] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ ID NO: 475.

[00085] In some embodiments, the ngRNA comprises SEQ ID NO: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507 or 508. Petition 870260064333, dated 06 / 30 / 2026, p. 19 / 427 14 / 418

[00086] In one aspect, a prime editing system is provided in this document comprising (a) PEgRNA or one or more polynucleotides of any of the aspects or embodiments described (a) in this document, and (b) an ngRNA or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 478; and an ngRNA core capable of binding to a Cas9 protein.

[00087] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ ID NO: 478.

[00088] In some embodiments, ngRNA comprises SEQ ID NO: 502.

[00089] In one aspect, a prime editing system is provided in this document comprising (a) PEgRNA or one or more polynucleotides of any of the aspects or embodiments described in this document, and (b) an ngRNA or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 479; and an ngRNA core capable of binding to a Cas9 protein.

[00090] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ ID NO: 479.

[00091] In some embodiments, ngRNA comprises SEQ ID NO: 503.

[00092] In some embodiments, the core of the ngRNA comprises the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT Petition 870260064333, dated 06 / 30 / 2026, p. 20 / 427 15 / 418 GAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGT GAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), where T indicates the presence of a uridine nucleotide.

[00093] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence in a first strand of a CF transmembrane conductance regulator (CFTR) gene, wherein the spacer comprises at its 3' end the 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 a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) comprising 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 the second strand are complementary to each other, wherein the editing template encodes or comprises a G nucleotide at position c.1624 of a wild CFTR encoding sequence.

[00094] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, 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) comprising at its 5' end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3.

[00095] In some embodiments, the gRNA core comprises the nucleotide sequence Petition 870260064333, dated 06 / 30 / 2026, p. 21 / 427 16 / 418 GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), where T indicates the presence of a uridine nucleotide.

[00096] In some embodiments, the extension arm additionally comprises a 3' motif comprising the nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), where T indicates the presence of a uridine nucleotide.

[00097] In some embodiments, the 3' motif is directly connected to the PBS at its 3' end.

[00098] In some embodiments, the 3' motif is linked to the PBS at its 3' end by means of a linker.

[00099] In some embodiments, the ligand is 4 nucleotides long. [000100] In some forms, the edit model includes at its 3' end the SEQ ID NO: 70, 79, 90, 95 or 99. [000101] In some embodiments, the editing template has a length of 24 nucleotides or less. [000102] In some embodiments, the editing template has a length of 10, 14, 18, 21, or 24 nucleotides. [000103] In some modes, the edition model consists of sequence number 65. [000104] In some embodiments, the spacer is 17 to 22 nucleotides long. [000105] In some embodiments, the spacer comprises at its 3' end the SEQ ID NO: 12. [000106] In some forms, the spacer has the sequence SEQ ID NO: 12. [000107] In some embodiments, the PBS comprises at its 5' end the sequence number 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 or 63. [000108] In some forms, PBS includes in its Petition 870260064333, dated 06 / 30 / 2026, page 22 / 427 17 / 418 end 5' the sequence number 27, 54, 57, 60 or 63. [000109] In some embodiments, the PBS comprises at its 5' end the sequence number 27, 33, 39, 45 or 51. [000110] In some forms, PBS consists of the sequence AGAAGGT. [000111] In some embodiments, PBS has a length of 15 nucleotides or less. [000112] In some forms, PBS is 7 to 15 nucleotides long. [000113] In one aspect, a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA is provided in this document, wherein the PEgRNA comprises a sequence selected from the group consisting of the 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. [000114] In one aspect, a prime editing system is provided in this document comprising: (a) the PEgRNA or one or more polynucleotides of any of the aspects or embodiments described in this document, and (b) an ngRNA or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 480, 11, 481, 482, 483 or 484, and (ii) an ngRNA core capable of binding to a Cas9 protein. [000115] In some embodiments, the ngRNA spacer comprises at its 3' end the SEQ ID NO: 480, 11, 481, 482, 483 or 484. [000116] In some embodiments, the gRNA core comprises the nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), where T indicates the presence of a uridine nucleotide. Petition 870260064333, dated 06 / 30 / 2026, p. 23 / 427 18 / 418 [000117] In some embodiments, the ngRNA comprises SEQ ID NO: 488, 490, 492, 495, 497 or 498. [000118] In some embodiments, PEgRNA comprises 5' to 3', the spacer, the gRNA core, the RTT and the PBS. [000119] In some embodiments, the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule. [000120] In some embodiments, PEgRNA additionally comprises mN*mN*mN*N 3' and mN*mN*mN* 5' modifications, where m indicates that the nucleotide contains a 2'-O-Me modification and a * indicates the presence of a phosphorothioate linkage. [000121] In some embodiments, PEgRNA comprises mT*mT*mT*T 3' and mN*mN*mN* 5' modifications, where m indicates that the nucleotide contains a 2'-O-Me modification, an * indicates the presence of a phosphorothioate linkage, and a T indicates the presence of an additional uridine nucleotide. [000122] In some embodiments, PEgRNA and / or ngRNA additionally comprise(s) mN*mN*mN*N 3' and mN*mN*mN* 5' modifications, where m indicates that the nucleotide contains a 2'-O-Me modification and a * indicates the presence of a phosphorothioate linkage. [000123] In some embodiments, PEgRNA and / or ngRNA comprise modifications with mT*mT*mT*T 3' and mN*mN*mN* 5', where m indicates that the nucleotide contains a 2'O-Me modification, a * indicates the presence of a phosphorothioate linkage, and a T indicates the presence of an additional uridine nucleotide. [000124] In one aspect, a prime editing system is provided in this document comprising (a) the PEgRNA of any of the aspects or embodiments described in this document, or one or more polynucleotides encoding 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 Cas9 nickase, and a Petition 870260064333, dated 06 / 30 / 2026, page 24 / 427 19 / 418 reverse transcriptase, or one or more polynucleotides encoding reverse transcriptase. [000125] In some embodiments, the prime editor 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. [000126] In some embodiments, the prime editor is a fusion protein. [000127] In one aspect, a prime editor system is provided in this document comprising (a) the PEgRNA of any of the aspects or embodiments described in this document 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-terminal and C-terminal exteins (Nintein and C-tein) are capable of autoexcision 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. [000128] In some embodiments, the prime editor system further comprises: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or 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 polynucleotide encoding Petition 870260064333, dated 06 / 30 / 2026, page 25 / 427 20 / 418 the C-terminal extein; wherein the N-intein and C-intein of the N-terminal and C-terminal exteins are capable of autoexcision 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. [000129] In one aspect, a population of viral particles is provided in this document comprising collectively one or more polynucleotides encoding the prime editing system of any of the aspects or embodiments described in this document. [000130] In some modalities, the viral particles are AAV particles. [000131] In one aspect, an LNP is provided in this document which comprises the prime editing system of any of the aspects or modalities described in this document. [000132] In some embodiments, PEgRNA, the polynucleotide encoding Cas9 nickase and the polynucleotide encoding reverse transcriptase. [000133] In some embodiments, the polynucleotide encoding Cas9 nickase and the polynucleotide encoding reverse transcriptase are mRNAs. [000134] In some embodiments, the polynucleotide encoding Cas9 nickase and the d polynucleotide encoding reverse transcriptase are the same molecule. [000135] In one aspect, a method for editing a CFTR gene is provided in this document, the method comprising contacting the CFTR gene with: (a) the PEgRNA of any of the aspects or embodiments described in this document, 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 of the aspects Petition 870260064333, dated 06 / 30 / 2026, p. 26 / 427 21 / 418 or embodiments described in this document. [000136] In some embodiments, the CFTR gene is in a cell. [000137] In some embodiments, the cell is a mammalian cell. [000138] In some embodiments, the cell is a human cell. [000139] In some embodiments, the cell is a primary cell. [000140] In some embodiments, the cell is an epithelial cell. [000141] In some embodiments, the cell is in an individual or was obtained from an individual or a cell bank. [000142] In some embodiments, the individual is a human. [000143] In some embodiments, CFTR gene contact comprises contact of the cell with (i) the particle population viral claims 113 or 114 or (ii) the LNP of any of the aspects or modalities described in this document. [000144] In one aspect, a method is provided in this document for treating cystic fibrosis in an individual in need thereof, the method comprising administering to the individual (i) the PEgRNA of any of the aspects or modalities described in this document, 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 of the aspects or modalities described in this document, (iii) the viral particle population of any of the aspects or modalities described in this document, or (iv) the LNP of any of the aspects or modalities described in this document. [000145] In one aspect, a guide RNA editing primer (PEgRNA) or one or more polynucleotides is provided in this document. Petition 870260064333, dated 06 / 30 / 2026, p. 27 / 427 22 / 418 encoding PEgRNA, wherein the PEgRNA comprises: a spacer comprising at its 3' end a PEgRNA spacer sequence selected from any of Tables 15 to 17; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template comprising at its 3' end an RTT sequence selected from the same Table as the PEgRNA spacer sequence, and a primer binding site (PBS) comprising at its 5' end a PBS sequence selected from the same Table as the PEgRNA spacer sequence. [000146] In some embodiments, the PEgRNA spacer is 17 to 22 nucleotides long. [000147] In some embodiments, the PEgRNA spacer is 20 nucleotides long. [000148] In some embodiments, PEgRNA comprises 5' to 3', the spacer, the gRNA core, the editing template and the PBS. [000149] In some embodiments, the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule. [000150] In some embodiments, the gRNA core comprises a gRNA core sequence selected from Table 10. [000151] In one aspect, a prime editing system is provided in this document comprising: (a) the prime editing guide RNA (PEgRNA) of any of the aspects or embodiments described in this document, or one or more polynucleotides encoding PEgRNA; and optionally, (b) a cutting guide RNA (ngRNA), or one or more polynucleotides encoding 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 an ngRNA core capable of binding to a Cas9 protein. Petition 870260064333, dated 06 / 30 / 2026, p. 28 / 427 23 / 418 [000152] In some embodiments, the ngRNA spacer is 17 to 22 nucleotides long. [000153] In some embodiments, the ngRNA spacer comprises, at its 3' end, nucleotides 3 to 20, 2 to 20, or 1 to 20 of the ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence. [000154] In some embodiments, the ngRNA spacer is 20 nucleotides long. [000155] In some embodiments, the ngRNA core comprises a gRNA core sequence selected from Table 10. [000156] In some embodiments, the prime editor 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. [000157] In some embodiments, the prime editor 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 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 [000158] All publications, patents and patent applications mentioned in this descriptive report are incorporated Petition 870260064333, dated 06 / 30 / 2026, page 29 / 427 24 / 418 in this document by reference, to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS [000159] The new features of the disclosure are presented in detail in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by reference to the following detailed description, which presents illustrative embodiments in which the principles of disclosure are used, and whose attached drawings: FIG. 1 illustrates a schematic diagram of a prime editing guide RNA (PEgRNA) binding to a double-stranded target DNA sequence. [000160] FIG. 2 illustrates an overview of the PEgRNA architecture in an exemplary PEgRNA schematic designed for a prime editor. [000161] FIG. 3 is a diagram showing the spacer and mid-gRNA of an exemplary guide RNA, in two separate molecules. The rest of the PEgRNA structure is not shown. [000162] FIG. 4 shows the restoration of CFTR swelling and function in intestinal organoids derived from patients undergoing prime editing after incubation with 10 μM forskolin. DETAILED DESCRIPTION OF THE DISCLOSURE [000163] Compositions and methods for editing the cystic fibrosis transmembrane conductance regulator (CFTR / ABCC7) target gene with prime editing are provided in this document in some embodiments. In certain embodiments, compositions and methods for correcting mutations in the CFTR gene associated with cystic fibrosis are provided in this document. The compositions provided in this document may comprise prime editors (Pes) that may use manipulated guide polynucleotides, for example, prime-editing guide RNAs (PEgRNAs), Petition 870260064333, dated 06 / 30 / 2026, p. 30 / 427 25 / 418 that can direct Pes to specific DNA targets and can encode DNA edits in the target CFTR gene, which serve a variety of functions, including the direct correction of disease-causing mutations associated with cystic fibrosis. [000164] The following description and examples illustrate embodiments of this disclosure in detail. It should be understood that this disclosure is not limited to the particular embodiments described herein and, as such, may vary. Those skilled in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. While several features of this disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any appropriate combination. Conversely, while this disclosure may be described herein in the context of separate embodiments for clarity, this disclosure may also be implemented in a single embodiment. Definitions [000165] Unless otherwise defined, all technical and scientific terms used in this document have the same meaning that is commonly understood by someone with ordinary skill in the technique. [000166] The terminology used in this document is intended only to describe specific modalities and is not intended to be limiting. As used in this document, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, insofar as the terms including, includes, having, has, with or variants thereof, as used in this document, mean comprising. [000167] Unless otherwise specified, words including, include, includes, having, have, Petition 870260064333, dated 06 / 30 / 2026, p. 31 / 427 26 / 418 has, including, includes, include, containing, contains and contain are inclusive or open-ended and do not exclude additional elements or steps of the method not mentioned. [000168] Reference to some modalities, one modality or other modalities means that a specific feature or resource described in connection with the modalities is included in at least one or more modalities, but not necessarily in all modalities, of this disclosure. [000169] The term about or approximately, in relation to a numerical value, means a range of values ​​that is within 10% more or less of the value. For example, about x means x±(10% * x). [000170] The term substantially, as used in this document, may refer to a value that approximates 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. [000171] The terms protein and polypeptide may be used interchangeably to refer to a polymer of two or more amino acids linked 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 linked by covalent bonds (e.g., amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, enzyme precursor proteins, Petition 870260064333, dated 06 / 30 / 2026, p. 32 / 427 27 / 418 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 a specific biological function). In some embodiments, a protein may be a variant or 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. [000172] In some embodiments, a protein comprises one or more protein domains or subdomains. As used in this document, the term “polypeptide domain,” “protein domain,” or “domain,” when used in the context of a protein or a polypeptide, refers to a polypeptide chain that has one or more biological functions, for example, 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 naturally occurring protein domains. In some embodiments, a protein comprises multiple protein domains from different naturally occurring proteins. For example, in some embodiments, a prime editor might be a fusion protein comprising a Cas9 protein domain of S.pyogenes is a reverse transcriptase protein domain of a retrovirus (e.g., Moloney murine leukemia virus) or a retrovirus variant. A protein comprising sequences. Petition 870260064333, dated 06 / 30 / 2026, page 33 / 427 28 / 418 amino acids from different origins or naturally occurring proteins can be termed a fusion or chimeric protein. [000173] In some embodiments, a protein comprises a functional variant or a functional fragment of a full-length wild-type protein. A “functional fragment” or “functional portion,” as used in this document, refers to any portion of a reference protein (e.g., a wild-type protein) that encompasses less than the complete amino acid sequence of the reference protein while retaining one or more functions, for example, catalytic or binding functions. For example, a functional fragment of a reverse transcriptase may encompass less than the complete amino acid sequence of a wild-type reverse transcriptase but retain 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 functions from at least one of the functional domains.For example, a functional fragment of Cas9 may encompass less than the complete amino acid sequence of a wild-type Cas9, but retains its ability to bind to DNA and lacks its nuclease activity partially or completely. [000174] A “functional variant” or a “functional mutant,” as used in this document, refers to any variant or mutant of a reference protein (e.g., a wild-type protein) that involves one or more changes in the amino acid sequence of the reference protein while retaining one or more functions, for example, catalytic or binding functions. In some embodiments, one or more changes in the amino acid sequence comprise amino acid substitutions, insertions, or deletions, or Petition 870260064333, dated 06 / 30 / 2026, p. 34 / 427 29 / 418 any combination thereof. In some embodiments, one or more amino acid sequence changes comprise 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, for example, an H840A amino acid substitution, compared to the amino acid sequence of a wild-type Cas9, but retains the ability to bind to DNA and does not possess nuclease activity partially or completely. [000175] The term function and its grammatical equivalents, as used in this document, may refer to the ability to operate, have, or serve an intended purpose. Functional may encompass any percentage from reference up to 100% of an intended purpose. For example, functional may encompass or comprise approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even approximately 100% of an intended purpose. In some applications, the term "functional" can mean more or less about 100% of the normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, or even about 1000% of an intended purpose. [000176] In some embodiments, a protein or polypeptide includes naturally occurring amino acids (for example, one of the twenty amino acids commonly found in peptides synthesized in nature and known by Petition 870260064333, dated 06 / 30 / 2026, p. 35 / 427 30 / 418 abbreviations of a letter 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 polypeptide includes non-naturally occurring amino acids (e.g., amino acids that are 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. [000177] In some embodiments, a protein comprises an isolated polypeptide. The term isolated means free or removed to varying degrees from the components that 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 in its natural state, is isolated. [000178] In some embodiments, a protein is present in a cell, a tissue, an organ, or a viral particle. In some embodiments, a protein is present in a cell or part of a cell (e.g., a bacterial 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 a lysate from a single cell. [000179] The terms homologous, homology, or percent homology, as used in this document, 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, for example, polypeptide or DNA sequences that are similar. Homology can mean, for example, Petition 870260064333, dated 06 / 30 / 2026, p. 36 / 427 31 / 418 For example, nucleic acid sequences with at least approximately: 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 with the reference nucleic acid sequence. For example, a “genomic homology region” may be a region of DNA that has a sequence similar to a given genomic region in the genome. A homology region can have any length that is sufficient to promote the binding of a spacer, primer binding site, or protospacer sequence to the genomic region.For example, the homology region may 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, so that the homology region has sufficient homology to link to the corresponding genomic region. [000180] When a percentage of homology or sequence 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 along 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 may be homologous at that position. Unless otherwise indicated, sequence homology or identity is evaluated along the specified length of the nucleic acid, polypeptide, or portion thereof. In some Petition 870260064333, dated 06 / 30 / 2026, page 37 / 427 32 / 418 modalities, homology or identity is evaluated in a functional portion or specified portion of the length. [000181] Sequence alignment for sequence homology assessment can be conducted using algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410, 1990. A publicly available internet interface for performing BLAST analyses is available from the National Center for Biotechnology Information. Additional known algorithms include those published in: Smith & Waterman, “Comparison of Biosequences, Adv. 2:482, 1981; Needleman & Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins J. Mol. Biol. 48:443, 1970; Pearson & Lipman “Improved tools for biological sequence comparison, Proc. Natl. Acad. Sci. EUA 85:2444, 1988; or by the automated implementation of these or similar algorithms.Global alignment programs can also be used to align similar sequences of approximately equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk / Tools / psa / emboss needle / ), which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277), and the GGSEARCH program https: / / fasta.bioch.virginia.edu / fasta_www2 / , which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both programs are based on the Needleman-Wunsch algorithm, used to find the optimal 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 modalities, the alignment between a query sequence and a sequence of... Petition 870260064333, dated 06 / 30 / 2026, p. 38 / 427 Reference 33 / 418 is performed with Needleman-Wunsch alignment with Gap Costs defined as Existence: 11 Extension: 1, where the percentage of identity is calculated by dividing the number of identities by the alignment extent, as 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 tuned substitution matrices”, FEBS J. 272: 5101-5109, 2005). [000182] A qualified person understands that amino acid (or nucleotide) positions can 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. [000183] The term “polynucleotide” or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or chimeric RNA-DNA molecules. In some embodiments, a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA. In some embodiments, a polynucleotide is double-stranded, for example, double-stranded DNA in a gene. In some embodiments, a polynucleotide is single-stranded or substantially single-stranded, for example, single-stranded DNA or mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in the 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 that circulates in the blood. [000184] Polynucleotides can have any three-dimensional structure. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a Petition 870260064333, dated 06 / 30 / 2026, p. 39 / 427 34 / 418 probe, a primer, an 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, isolated RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a small tRNA-derived RNA (tsRNA), an antisense RNA, an shRNA, or a small rDNA-derived RNA (srRNA). [000185] 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 may be transmitted before or after the assembly of the polynucleotide. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. [000186] 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 mechanism as guanine (G). [000187] In some embodiments, a polynucleotide may be modified. As used in this document, the terms modified or modification refer to chemical modification with respect to nucleotides A, C, G, T, and U. In some embodiments, modifications may occur at the nucleoside base. Petition 870260064333, dated 06 / 30 / 2026, p. 40 / 427 35 / 418 and / or in the sugar portion of the nucleosides comprising the polynucleotide. In some embodiments, the modification may occur in the internucleosidic bond (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. [000188] The term complement, complementary, or complementarity, as used in this document, refers to the ability of two polynucleotide molecules to form a base pair with each other. Complementary polynucleotides can form base pairs through hydrogen bonds, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds. For example, an adenine in one polynucleotide molecule will form a base pair with a thymine or uracil in a second polynucleotide molecule, and a cytosine in one polynucleotide molecule will form a base pair with a guanine in a second polynucleotide molecule. Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule, comprising a first nucleotide sequence, can form a base pair with a second polynucleotide molecule, comprising a second nucleotide sequence.For example, 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 complementarity percentage indicates the percentage of nucleotides in a polynucleotide molecule that can form a 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 contiguous nucleotides of one polynucleotide molecule will form a base pair with the same number of contiguous nucleotides in a second molecule. Petition 870260064333, dated 06 / 30 / 2026, page 41 / 427 36 / 418 polynucleotide. “Substantially complementary,” as used in this document, refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over the whole or a portion of two polynucleotide molecules. In some embodiments, the complementary portion can be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. “Substantially complementary” can also refer to 100% complementarity over a portion or region of two polynucleotide molecules. In some embodiments, the complementary portion or region 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 of a functional or defined portion thereof. [000189] As used in this document, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which polynucleotides, for example, transcribed mRNA, are 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, the expression of a polynucleotide, for example, a gene or DNA encoding a protein, is determined by the amount of protein encoded by the gene after transcription and translation of the gene. In some embodiments, the expression of a polynucleotide, for example, a gene or 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 modes, the expression of a gene is determined by the amount of mRNA, or transcript, that is encoded by the gene after transcription. In some modes, the expression of a polynucleotide, for example, an mRNA, is determined by the amount of protein encoded by the mRNA after translation. Petition 870260064333, dated 06 / 30 / 2026, page 42 / 427 37 / 418 mRNA. In some embodiments, the expression of a polynucleotide, for example, a coding mRNA or RNA, is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide. [000190] The term sequencing, as used in this document, may include capillary sequencing, bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite sequencing (TAB), 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 nanosphere sequencing, Heliscope single-molecule sequencing, real-time single-molecule sequencing (SMRT), nanopore sequencing, shotgun sequencing, RNA sequencing, or any combination thereof. [000191] The terms equivalent or biological equivalent are used interchangeably when referring to a specific molecule, or biological or cellular material, and mean a molecule having minimal homology with another molecule, while still maintaining a desired structure or functionality. [000192] The terms equivalent or biological equivalent are used interchangeably when referring to a specific molecule, or a biological or cellular material, and mean a molecule having minimal homology with another molecule, while still maintaining a desired structure or functionality. [000193] The term encoding, as applied to polynucleotides, refers to a polynucleotide that is said to encode another polynucleotide, a polypeptide, or a Petition 870260064333, dated 06 / 30 / 2026, page 43 / 427 38 / 418 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 a template for polynucleotide synthesis, for example, transcribed into RNA, reverse transcribed into DNA or cDNA and / or translated to produce an amino acid, or a polypeptide or fragment thereof. In some embodiments, a polynucleotide comprising three contiguous nucleotides forms a codon that codes for a specific amino acid. In some embodiments, a polynucleotide comprises one or more codons that code for 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 codes for an amino acid substitution in a polypeptide encoded by the polynucleotide compared to a wild-type reference polypeptide. [000194] The term mutation, as used in this document, refers to an alteration and / or change in an amino acid sequence of a protein or a 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 a 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. [000195] The term individual and its equivalents Petition 870260064333, dated 06 / 30 / 2026, page 44 / 427 39 / 418 grammatical terms, as used in this document, may refer to a human or a non-human. An individual may be a mammal. In some modalities, an individual is human. A human individual may be male or female. A human individual may be of any age. An individual may be a human embryo. A human individual may be a newborn, an infant, a child, an adolescent, or an adult. A human individual may require treatment for a genetic disease or disorder. [000196] The terms treatment or treating and their grammatical equivalents may refer to the medical control of an individual with the intention of curing, alleviating, or relieving a symptom of a disease, condition, or disorder. Treatment may include active treatment, that is, treatment specifically aimed at improving a disease, condition, or disorder. Treatment may include causal treatment, that is, treatment aimed at removing the cause of the associated disease, condition, or disorder. In addition, this treatment may include palliative treatment, that is, treatment designed to relieve symptoms rather than cure the disease, condition, or disorder. Treatment may include supportive treatment, that is, treatment used to complement other specific therapy aimed at improving the disease, condition, or disorder. In some modalities, a condition may be pathological.In some modalities, a treatment may not completely cure or prevent a disease, condition, or disorder. In some modalities, a treatment improves but does not completely cure or prevent a disease, condition, or disorder. In some modalities, an individual 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 for the individual's entire life. Petition 870260064333, dated 06 / 30 / 2026, page 45 / 427 40 / 418 [000197] The term improve and its grammatical equivalents mean to decrease, suppress, attenuate, diminish, interrupt or stabilize the development or progression of a disease. [000198] The terms prevent or preventing mean delaying, anticipating, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevention also means reducing the 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, for example, 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 for the life of an individual. [000199] The term effective amount or therapeutically effective amount refers to an amount of a composition, for example, a prime-editing composition comprising a construct, that may be sufficient to result in a desired activity after introduction into an individual, as disclosed in this document. An effective amount of prime-editing compositions may be delivered to the target gene or cell, whether the cell ex vivo or in vivo. An effective amount may be the amount that induces, for example, a change of at least about 2-fold (increase or decrease) or more in the amount of target nucleic acid modulation (e.g., expression of a target CFTR gene to produce the functional CFTR protein) observed relative to a negative control. An effective amount or dose may induce, for example, an increase of about 2-fold, an increase of about 3-fold, an increase of about 4-fold, an increase of about Petition 870260064333, dated 06 / 30 / 2026, p. 46 / 427 41 / 418 times, an increase of about 6 times, an increase of about 7 times, an increase of about 8 times, an increase of about 9 times, an increase of about 10 times, an increase of about 25 times, an increase of about 50 times, an increase of about 100 times, an increase of about 200 times, an increase of about 500 times, an increase of about 700 times, an increase of about 1,000 times, an increase of about 5,000 times, or an increase of about 10,000 times in the modulation of the target gene (e.g., expression of a target CFTR gene to produce the functional CFTR protein). The amount of modulation of the target gene can be measured by any suitable method known in the art. In some modalities, the "effective amount" or "therapeutically effective amount" is the quantity of a compound needed to alleviate the symptoms of a disease relative to an untreated patient.In some embodiments, an effective amount is the quantity of a compound sufficient to introduce a change in a gene of interest in a cell (e.g., an in vitro or in vivo cell). [000200] In some embodiments, an effective amount may be an amount that, when administered to a population of cells, induces a certain percentage of the cell population to exhibit a correction of a mutation. For example, in some embodiments, an effective amount may be the amount that induces, when administered to or introduced into a population of cells, the installation of one or more of the 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 cell population. Prime Edition Petition 870260064333, dated 06 / 30 / 2026, p. 47 / 427 42 / 418 [000201] The term “prime editing” refers to the programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate a desired nucleotide edit (also referred to in this document as a nucleotide change) into the target DNA via DNA synthesis initiated at the target. A prime editing target gene may comprise a double-stranded DNA molecule having two complementary strands: a first strand which may be termed the “target strand” or “non-editing strand” and a second strand which may be termed the “non-target strand” or “editing strand”. In some embodiments, in a prime editing guide RNA (PEgRNA), a spacer sequence is complementary or substantially complementary to a specific sequence in the target strand, which may be termed the “search target sequence”. In some embodiments, the spacer sequence anneals to the target strand in the search target sequence.The target strand may also be called the “Non-Protospacer Adjacent Motif (non-PAM strand)”. In some embodiments, the non-target strand may also be called the “PAM strand”. In some embodiments, the PAM strand comprises a protospacer sequence and, optionally, a 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, for example, a Cas nickase or a Cas nuclease. In some embodiments, a specific PAM is characteristic of a specific programmable DNA-binding protein, for example, 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 target sequence being searched for. In a PEgRNA, a spacer sequence can have a substantially longer sequence. Petition 870260064333, dated 06 / 30 / 2026, pp. 48 / 427 43 / 418 identical to the protospacer sequence in the editing strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T). [000202] In some embodiments, the double-stranded target DNA comprises a cleavage site on the PAM strand (or non-target strand). As used in this document, a “cleavage site” refers to a specific position between two nucleotides or two base pairs of the double-stranded target DNA. In some embodiments, the position of a cleavage site is determined relative to the position of a specific PAM sequence. In some embodiments, the cleavage site is the specific position where a cut will occur when the double-stranded target DNA is brought into contact with a nickase, for example, a Cas nickase, which recognizes a specific PAM sequence. In some embodiments, the cleavage site is upstream of a specific PAM sequence on the PAM strand of the double-stranded target DNA. In some embodiments, the cleavage site is downstream of a specific PAM sequence on the PAM strand of the double-stranded target DNA.In some embodiments, the cleavage site is upstream of a PAM sequence recognized by a Cas9 nickase, wherein the Cas9 nickase comprises an active RuvC nuclease domain and an inactive HNH nuclease domain. In some embodiments, the cleavage site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase from Streptococcus pyogenes, a Cas9 nickase from P. lavamentivorans, a Cas9 nickase from C. diphtheriae, a Cas9 from N. cinerea, a Cas9 from S. aureus, or a Cas9 nickase from N. lari. In some embodiments, the cleavage site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase comprises an active RuvC nuclease domain and an inactive HNH nuclease domain. In some forms, the cleavage site is 2 nucleotides upstream of the PAM sequence, e. Petition 870260064333, dated 06 / 30 / 2026, p. 49 / 427 44 / 418 The PAM sequence is recognized by a Cas9 nickase from S. thermophilus comprising an active RuvC nuclease domain and an inactive HNH nuclease domain. By “upstream” and “downstream,” we mean defining relevant positions of at least two regions or sequences in a nucleic acid molecule oriented in a 5' to 3' direction. For example, a first sequence is upstream of a second sequence in a DNA molecule where the first sequence is positioned 5' relative to the second sequence. Consequently, the second sequence is downstream of the first sequence. [000203] A “primer binding site” (also known as PBS or primer binding site sequence) is a single-stranded portion of PEgRNA comprising a region of complementarity to the PAM strand (i.e., the non-target strand or the editing 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 cleavage site. In some embodiments, in the prime editing process, the PEgRNA is complexed with a prime editor and directed to bind the search target sequence on the target strand of the double-stranded target DNA and generates a cleavage at the cleavage site on the non-target strand of the double-stranded target DNA. In some embodiments, the PBS is complementary or substantially complementary to, and may bind to, a free 3' end on the non-target strand of the double-stranded target DNA at the cleavage site.In some embodiments, PBS annealed to the free 3' end on the non-target strand can initiate DNA synthesis started at the target. [000204] A “PEgRNA editing template” is a single-stranded portion of the PEgRNA that is 5' from 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 editing strand) and comprises one or more intended nucleotide edits compared to the endogenous sequence. Petition 870260064333, dated 06 / 30 / 2026, p. 50 / 427 45 / 418 of the double-stranded target DNA. In some embodiments, the editing template and the PBS are immediately adjacent to each other. Consequently, in some embodiments, a primary editing PEgRNA comprises a single-stranded portion comprising 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 in the PAM strand (i.e., the non-target strand or the editing strand) of the double-stranded target DNA, except for one or more non-complementary nucleotides at the intended nucleotide editing position(s).As used in this document, regardless of the 5'-3' relative positioning in other contexts, the relative positions between the PBS and the editing template, and the relative positions between the elements of a PEgRNA, are determined by the 5' to 3' order of the PEgRNA as a single molecule, regardless of the position of sequences in the double-stranded target DNA that may have complementarity or identity with elements of the PEgRNA. In some embodiments, the editing template is complementary or substantially complementary to a sequence in the PAM strand that is immediately downstream of the cleavage site, except for one or more non-complementary nucleotides at the intended nucleotide editing positions.An endogenous sequence, for example, a genomic sequence, that is complementary or substantially complementary to the editing template, except for one or more non-complementary nucleotides at the position corresponding to the intended nucleotide edit, may be called the "editing target sequence". In some embodiments, the editing template has identity or substantial identity with a sequence on the target strand that is complementary to, or has 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... Petition 870260064333, dated 06 / 30 / 2026, p. 51 / 427 In 46 / 418 embodiments, the editing template encodes single-stranded DNA wherein the single-stranded DNA has identity or substantial identity with the target sequence for editing, except for one or more insertions, deletions, or substitutions at the positions of 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 editing 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 with respect to the coding region of the wild-type or non-disease-associated protein.Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence after the desired edit has been installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer). [000205] In some embodiments, a PEgRNA forms a complex with a prime editor and directs it to bind to the target sequence of the target gene. In some embodiments, the bound prime editor creates a cut in the editing strand (PAM strand) of the target gene at the cleavage site. In some embodiments, a primer binding site (PBS) of the PEgRNA anneals to a free 3' end formed at the cleavage site, and the prime editor initiates DNA synthesis from the cleavage site, using the free 3' end as a primer. Subsequently, single-stranded DNA encoded by the PEgRNA editing template is synthesized. In some embodiments, the newly synthesized single-stranded DNA comprises one or more intended nucleotide edits compared to the endogenous sequence of the target gene. Consequently, in some embodiments, the editing template of a PEgRNA is complementary to a sequence in the editing strand, except for one or more mismatches at the positions of Petition 870260064333, dated 06 / 30 / 2026, p. 52 / 427 47 / 418 desired nucleotide editing in the editing template. The endogenous sequence, for example, genomic, that is partially complementary to the editing template may be called the “target editing sequence”. Consequently, in some embodiments, newly synthesized single-stranded DNA has identity or substantial identity with a sequence in the target editing sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide editing positions. In some embodiments, the editing template comprises at least 4 contiguous nucleotides of complementarity with the editing strand, wherein the at least 4 contiguous nucleotides are located upstream of the most edited 5' strand in the editing template. [000206] In some embodiments, newly synthesized single-stranded DNA equilibrates with the editing target on the target gene editing strand for pairing with the target gene strand. In some embodiments, the target gene editing sequence is excised by a tab 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, linked to other prime editor components, or provided trans. In some embodiments, newly synthesized single-stranded DNA, comprising the intended nucleotide editing, replaces the endogenous single-stranded editing target sequence on the target gene editing strand. In some embodiments, the newly synthesized single-stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure in the region corresponding to the target gene editing target sequence.In some embodiments, newly synthesized single-stranded DNA comprising nucleotide editing is paired in the heteroduplex with the target DNA strand that does not comprise nucleotide editing, thus creating a mismatch between the two strands, which would otherwise... Petition 870260064333, dated 06 / 30 / 2026, page 53 / 427 48 / 418 would be complementary. In some modalities, the incompatibility is recognized by DNA repair mechanisms, for example, endogenous DNA repair mechanisms. In some modalities, through DNA repair, the intended nucleotide edit is incorporated into the target gene. Prime Editor [000207] 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 additionally 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 in this document, 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, such as a Cas9 nickase, a Cpf1 nickase, or another Cas CRISPR 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... Petition 870260064333, dated 06 / 30 / 2026, page 54 / 427 49 / 418 5' endonuclease activity, for example, an endogenous 5' DNA flap endonuclease (e.g., FEN1), to assist in driving the prime editing process toward the formation of the edited product. In some embodiments, the prime editor additionally comprises an RNA-linked protein recruitment polypeptide, for example, an MS2 coat protein. [000208] A prime editor can be genetically engineered. In some embodiments, the polypeptide components of a prime editor do not occur naturally 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 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 Cas9 polypeptide from S. pyogenes and a reverse transcriptase polypeptide from Moloney's murine leukemia virus (M-MLV). [000209] In some embodiments, the polypeptide domains of a prime editor can 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 with 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 linked together by an RNA-protein recruitment aptamer, for example, an MS2 aptamer, which can be linked to a PEgRNA. The polypeptide components of the prime editor may be Petition 870260064333, dated 06 / 30 / 2026, page 55 / 427 50 / 418 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 part of a prime editor domain, or a portion of a prime editor fusion protein. For example, a prime editor fusion protein might comprise an N-terminal portion fused to an N-intein and a C-terminal portion fused to a C-intein, each of which is individually encoded by an AAV vector. Prime Editor Nucleotide Polymerase Domain [000210] In some embodiments, a prime editor comprises a nucleotide polymerase domain, for example, a DNA polymerase domain. The DNA polymerase domain may be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, or it 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 depend on a template polynucleotide strand, for example, the editing template sequence, for the synthesis of a new DNA strand. In some embodiments, the prime editor comprises a DNA-dependent DNA polymerase. For example, a prime editor having a DNA-dependent DNA polymerase may synthesize new single-stranded DNA using a PEgRNA editing template comprising a DNA sequence as a template.In these cases, the PEgRNA is a chimeric or hybrid PEgRNA and comprises an extension arm that includes a DNA strand. The chimeric or hybrid PEgRNA may comprise an RNA portion (including the gRNA spacer and core) and a DNA portion (the extension arm comprising the editing template that includes a DNA strand). Petition 870260064333, dated 06 / 30 / 2026, p. 56 / 427 51 / 418 [000211] In some embodiments, DNA polymerases may be wild-type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and / or the polymerases may be modified by genetic engineering, mutagenesis, or processes based on directed evolution. The polymerases may be a T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III, and the like. The polymerases may be thermostable and may include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants, and derivatives thereof. [000212] 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, a 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, a 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 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 Pol II DNA polymerase from Pyrococcus furiosus (Pfu). 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 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 DNA polymerase. Petition 870260064333, dated 06 / 30 / 2026, page 57 / 427 52 / 418 polymerase Pol-alpha. 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-kapka (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 some embodiments, the DNA polymerase is a B family DNA polymerase. In some embodiments, the DNA polymerase is a UL30 DNA polymerase from herpes simplex virus (HSV). In some embodiments, the DNA polymerase is a UL54 DNA polymerase from cytomegalovirus (CMV). [000213] 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 Pfu homolog from Pyrococcus furiosus. In some embodiments, the DNA polymerase is Petition 870260064333, dated 06 / 30 / 2026, p. 58 / 427 53 / 418 a pol II type DNA polymerase. For example, in some embodiments, the DNA polymerase is a homolog of the DP1 / DP2 2-subunit polymerase from P. furiosus. In some embodiments, the DNA polymerase lacks 5' to 3' nuclease activity. Suitable DNA polymerases (pol I or pol II) can be derived from archaea with ideal growth temperatures similar to the desired assay temperatures. [000214] In some embodiments, the DNA polymerase comprises a thermostable archaeal DNA polymerase. In some embodiments, the thermostable DNA polymerase is isolated from or derived from species of Pyrococcus (furiosus, species GB-D, woesii, abysii, horikoshii), species of Thermococcus (kodakaraensis KOD1, litoralis, species 9 degrees North-7, species JDF-3, gorgonarius), Pyrodictium occultum and Archaeoglobus fulgidus. [000215] Polymerases can 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 a Pol I family DNA polymerase from E. coli. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase from Pyrococcus furiosus (Pfu). 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 a Pol IV DNA polymerase from E. coli. In some embodiments, the Pol I DNA polymerase is a functional variant of DNA polymerase that lacks or has reduced 5' to 3' exonuclease activity. [000216] Suitable thermostable Pol I DNA polymerases can be isolated from a variety of thermophilic eubacteria, including Thermus and Thermotoga maritima species, such as Thermus aquaticus (Taq), Thermus thermophilus (Tth), and Thermotoga Petition 870260064333, dated 06 / 30 / 2026, p. 59 / 427 54 / 418 maritime (Tma UlTma). [000217] In some embodiments, a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT). An RT or RT domain may be a wild-type RT domain, a full-length RT domain, or it may be a functional mutant, a functional variant, or a functional fragment thereof. An RT or RT domain of a prime editor may comprise a wild-type RT or it may be engineered or evolved to contain specific amino acid substitutions, truncations, or variants. An engineered RT may comprise amino acid sequences or changes different from a naturally occurring RT. In some embodiments, the engineered RT may have enhanced reverse transcription activity relative to a naturally occurring RT or RT domain.In some embodiments, engineered RT may have improved characteristics compared to naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity. In some embodiments, a prime editor that understands engineered RT has improved prime editing efficiency compared to a prime editor that has a reference naturally occurring RT. [000218] In some embodiments, a prime editor comprises a virus RT, for example, a retrovirus RT. Non-limiting examples of virus RTs include murine leukemia virus Moloney RT (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 helper virus RT. Petition 870260064333, dated 06 / 30 / 2026, p. 60 / 427 55 / 418 (AEV) MCAV, RT of avian myelocytoma virus helper MC29 MCAV, RT of avian reticuloendotheliosis virus helper (REV-T), RT of avian sarcoma virus helper UR2 (UR2AV), RT of avian sarcoma virus helper Y73, RT of Rous-associated virus (RAV) and RT of myeloblastosis-associated virus (MAV), all of which can be suitably used in the methods and compositions described in this document. [000219] A prime editor may comprise a wild-type M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof. Table 1 provides illustrative M-MLV RT sequences suitable for use with the compositions and methods of the present disclosure. [000220] In some embodiments, a prime editor comprises a wild-type M-MLV RT, as presented in SEQ ID NO: 518. In some embodiments, a prime editor comprises a variant M-MLV RT, as presented in SEQ ID NO: 519. In some embodiments, a prime editor comprises a variant M-MLV RT, as presented in SEQ ID NO: 520. In some embodiments, a prime editor comprises a variant M-MLV RT, as presented in SEQ ID NO: 1209. Table 1. Illustrative Sequences of M-MLV SEQ ID NO : Sequence Description Amino acid sequence 518 RT of wild-type M-MLV TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIQPHIQRLLDQGILVPQSPWNTPLLPVKKPGGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQQKAYQ EIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAGWPPCLRMVA AIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATL LPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTEVIWAKAL PAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALL KALFLPKRLSIHCPGHQKRMSAGEKNATCARQATCATQTAL Petition 870260064333, of 30 / 06 / 2026, p. 61 / 427 56 / 418 519 RT of M-MLV variant TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKPGTNDYRPVQDLREVNKRVEDIHPVPNPYPPY LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQ EIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKLDPVAAGWPPCLRMVA AIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLDTDRVQFGPVVALNPATL LPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTEVIWAKAL PAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALL KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP 520 RT of M-MLV variant (D200N, T300P, L303K, T303K, TW313F) TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPVPNPNYLL LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQQQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQ EIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKLDPVAAGWPPCLRMVA AIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLDTDRVQFGPVVALNPATL LPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTEVIWAKAL PAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALL KALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP 1209 RT of M-MLV variant (D200N, T306K, W333F, WT330P e truncamento C-terminal entre D497 / I498) TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQE ARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSG LPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDL ADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEG QRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQ EIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVA AIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATL LPLPEEGLQHNCLDNSRLIN, [000221] In some embodiments, the prime editor comprises an M-MLV RT comprising one or more of the following 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 compared to a reference M-MLV RT, where X is any amino acid different from the original amino acid in the reference M-MLV RT. In some embodiments, the prime editor comprises an M-MLV RT comprising one or more of the following amino acid substitutions: H8Y, P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N compared to an M-MLV RT of Petition 870260064333, dated 06 / 30 / 2026, p. 62 / 427 57 / 418 reference. In some embodiments, the reference M-MLV RT is a variant M-MLV RT, as shown in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is an M-MLV WT RT, as shown in SEQ ID NO: 518. [000222] In some embodiments, a prime editor comprises an M-MLV RT comprising one or more of the amino acid substitutions D200N, T330P, L603W, T306K, or W313F compared to a reference M-MLV RT. In some embodiments, the reference M-MLV RT is a variant M-MLV RT, as shown in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is a WT M-MLV RT, as shown in SEQ ID NO: 518. [000223] In some embodiments, a prime editor comprises an M-MLV RT comprising the amino acid substitutions H8Y, D200N, T330P, L603W, T306K, and W313F, compared to a reference M-MMLV RT. In some embodiments, the reference M-MLV RT is a variant M-MLV RT, as shown in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is a WT M-MLV RT, as shown in SEQ ID NO: 518. [000224] In some embodiments, a prime editor comprises an M-MLV RT comprising 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 shown in Table 1. In some embodiments, the prime editor comprises an M-MLV RT comprising an amino acid sequence 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 comprising an amino acid sequence shown in SEQ ID NO: 520. Petition 870260064333, dated 06 / 30 / 2026, p. 63 / 427 58 / 418 [000225] In some embodiments, a RT variant may be a functional fragment of a reference RT having 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 approximately 70% identical, approximately 80% identical, approximately 90% identical, approximately 95% identical, approximately 96% identical, approximately 97% identical, approximately 98% identical, approximately 99% identical, approximately 99.5% identical, or approximately 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% to the amino acid length of a corresponding reference RT (MMLV reverse transcriptase). A reference RT can be any of the RTs shown in Table 1. [000226] In some embodiments, a functional RT fragment or variant is at least 100 amino acids long. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or even 600 or more amino acids long. [000227] In other embodiments, the functional RT variant is truncated at the N-terminal end or at the C-terminal end, or both, by a specified number of amino acids, resulting in a truncated variant that retains sufficient DNA polymerase function. In some embodiments, the functional RT variant, for example, a functional MMLV RT variant, is truncated at the C-terminus to abolish or reduce RNAase H activity while still retaining DNA polymerase activity. [000228] In some modalities, an editing draft Petition 870260064333, dated 06 / 30 / 2026, p. 64 / 427 59 / 418 prime or a prime editing system disclosed in this document comprises a polynucleotide (e.g., DNA, RNA, e.g., mRNA) that encodes an M-MLV RT. In some embodiments, the polynucleotide encodes an M-MLV RT comprising 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 shown in Table 1. In some embodiments, the polynucleotide encodes an M-MLV RT comprising 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 shown in Table 1. SEQ ID NO: 518, 519 or 520.In some embodiments, the polynucleotide encodes an M-MLV RT comprising an amino acid sequence 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 comprising an amino acid sequence shown in SEQ ID NO: 520. [000229] In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, fruit fly, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a Group II intron RT from Geobacillus stearothermophilus (GsIIIC) or a Group II intron RT from Eubacterium rectale (Eu.re.I2). In some embodiments, the prime editor comprises a retron RT. In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, fruit fly, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a Group II intron RT from Geobacillus stearothermophilus (GsI Petition 870260064333, dated 06 / 30 / 2026, p. 65 / 427 60 / 418 IIC) or an intron RT of Group II Eubacterium rectale (Eu.re.I2). In some embodiments, the prime editor comprises a retron RT. Programmable DNA Binding Domain [000230] In some embodiments, the DNA binding domain of a prime editor is a programmable DNA binding domain. [000231] A programmable DNA-binding domain refers to a protein domain engineered to bind to a specific nucleic acid sequence, for example, a target DNA or 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 DNA sequence, for example, a search target sequence in a target gene. In some embodiments, the DNA-binding domain comprises a clustered regularly spaced short palindromic repeats (CRISPR)-associated protein (Cas). A Cas protein may comprise any Cas protein described in this document 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, for example, a Cas protein. In some embodiments, the DNA-binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator-like effector domain (TALEN) nuclease, where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, for example, a Fok I nuclease domain. Petition 870260064333, dated 06 / 30 / 2026, page 66 / 427 61 / 418 [000232] In some embodiments, the DNA-binding domain comprises nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises an endonuclease domain having single-stranded 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 compared to a wild-type endonuclease domain. For example, the endonuclease domain may comprise one or more amino acid substitutions 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 an HNH domain. In some embodiments, the Cas nickase comprises an amino acid substitution in a RuvC domain. [000233] In some embodiments, the DNA-binding domain comprises a CRISPR-associated protein domain (Cas protein). A Cas protein can be a Class 1 or Class 2 Cas protein. A Cas protein can be a type I, type II, type III, type IV, type V, or type VI Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Petition 870260064333, dated 06 / 30 / 2026, p. 67 / 427 62 / 418 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 Cas effector proteins, CARF, DinG, Cpfl, Cas12b / C2c1, Cas12c / C2c3, Cas12b / C2c1, Cas12c / C2c3, SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, hyperprecise variant of Cas9 (HypaCas9), Cas Φ and Homologous, 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 other polypeptides.A Cas protein can be a chimera of several Cas proteins, for example, comprising Cas protein domains from different organisms. [000234] A Cas protein, for example, Cas9, can be from any suitable organism. In some respects, the organism is Streptococcus pyogenes (S. pyogenes). In some respects, the organism is Staphylococcus aureus (S. aureus). In some respects, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis. [000235] Non-limiting examples of suitable organisms 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, Petition 870260064333, dated 06 / 30 / 2026, page 68 / 427 63 / 418 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, Caldicellulosiruptor 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). [000236] In some embodiments, a Cas protein may 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 Petition 870260064333, dated 30 / 06 / 2026, p. 69 / 427 64 / 418 ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, 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. [000237] In some embodiments, a Cas protein, for example, Cas9, may be a wild type or a modified form of a Cas protein. In some embodiments, a Cas protein, for example, Cas9, may be an active nuclease variant, an inactive nuclease variant, a nickase, or a functional variant or functional fragment of a wild-type Cas protein. In some embodiments, a Cas protein, for example, Cas9, may be a wild type or a modified form of a Cas protein. A Cas protein, for example, Cas9, may be an active nuclease variant, an inactive nuclease variant, a nickase, or a functional variant or functional fragment of Petition 870260064333, dated 06 / 30 / 2026, page 70 / 427 65 / 418 a wild-type Cas protein. In some embodiments, a Cas protein, for example, Cas9, may 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 may 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 similarity to a wild-type Cas protein. [000238] A Cas protein, for example, Cas9, may comprise one or more domains. Non-limiting examples of Cas domains include guide nucleic acid recognition and / or binding domains, nuclease domains (e.g., Dnase or Rnase, RuvC, HNH domains), DNA binding domains, RNA binding domains, 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 that may interact with a guide nucleic acid and one or more nuclease domains comprising catalytic activity for nucleic acid cleavage. [000239] In some embodiments, a Cas protein, for example, Cas9, comprises one or more nuclease domains. A Cas protein may 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 with 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 lack the HNH domain. In some embodiments, a Cas protein comprises two nuclease domains, for example, a Cas9 protein may comprise an HNH nuclease domain and Petition 870260064333, dated 06 / 30 / 2026, page 71 / 427 66 / 418 a RuvC nuclease domain. [000240] In some embodiments, a prime editor comprises a Cas protein, for example, Cas9, in which 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 more nuclease domains (e.g., RuvC, HNH) of a Cas protein may be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity. In some embodiments, a Cas protein, for example, Cas9, comprising mutations in a nuclease domain, has reduced (e.g., nickase) or nullified nuclease activity, while retaining its ability to target a nucleic acid locus in a search target sequence when complexed with a guide nucleic acid, for example, a PEgRNA. [000241] 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 the double-stranded DNA in the target gene, but not a double-strand break. For example, the Cas nickase may cleave either the editing strand or the non-editing 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 have no catalytic activity or deleted. In some embodiments, the Cas nickase of a prime editor comprises an inactive RuvC nuclease domain and an active HNH nuclease domain. In some embodiments, the Cas nickase of a prime editor comprises an inactive HNH nuclease domain and an active RuvC nuclease domain.In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the RuvC domain, for example, an amino acid substitution that reduces or abolishes the activity of... Petition 870260064333, dated 06 / 30 / 2026, p. 72 / 427 67 / 418 RuvC domain nuclease. In some embodiments, the Cas9 nickase comprises a D10X amino acid substitution compared to a wild-type S. pyogenes Cas9, where 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, for example, an amino acid substitution that reduces or abolishes the nuclease activity of the HNH domain. In some embodiments, the Cas9 nickase comprises an H840X amino acid substitution compared to a wild-type S. pyogenes Cas9, where X is any amino acid other than H. [000242] 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 nullified nuclease activity and may not cleave any of the double-stranded DNA in a target gene. Nullified activity or absent activity may refer to 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% compared to exemplary wild-type activity (e.g., wild-type Cas9 nuclease activity). In some embodiments, a Cas protein of a prime editor does not possess nuclease activity at all. A nuclease, for example, Cas9, that does not possess nuclease activity may be termed an inactive nuclease or “dead nuclease” (abbreviated by “d”).A dead Cas nuclease protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some embodiments, a dead Cas protein is a dead Cas9 protein. In some embodiments, a prime editor comprises a dead Cas protein in which all nuclease domains (e.g., the RuvC and HNH nuclease domains in a Cas9 protein; the RuvC nuclease domain in a Cpf1 protein) are mutated to lose. Petition 870260064333, dated 06 / 30 / 2026, page 73 / 427 68 / 418 catalytic activity or are excluded. [000243] A Cas protein can be modified. A Cas protein, for example, 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 alter 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 protein's function or to optimize (e.g., enhance or reduce) the activity of the Cas protein. [000244] 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 greater or lesser stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein. [000245] 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 homolog, mutant, variant, or a functional fragment of the Cas9 protein. As used in this document, 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 to a guide polynucleotide, for example, a PEgRNA. A Cas9 protein may refer to a wild-type Cas9 protein of Petition 870260064333, dated 06 / 30 / 2026, p. 74 / 427 69 / 418 any organism or to a homolog, ortholog, or paralog of any organism; any mutants or functional variants thereof; or to any fragments or functional domains thereof. In some embodiments, a prime editor comprises a full-length Cas9 protein. In some embodiments, the Cas9 protein may generally comprise at least about 50%, 60%, 70%, 80%, 90%, or 100% sequence identity with a wild-type reference Cas9 protein (e.g., Cas9 from S. pyogenes). In some embodiments, Cas9 comprises an amino acid alteration, such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof, compared to a wild-type reference Cas9 protein. [000246] 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 an SpCas9 polypeptide, for example, comprising an amino acid sequence as shown in NCBI accession number WP_038431314 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is an SaCas9 polypeptide, for example, comprising an amino acid sequence as shown in Uniprot Accession Number J7RUA5 or a fragment or variant thereof.In some embodiments, a Cas9 polypeptide is an ScCas9 polypeptide, for example, comprising an amino acid sequence as presented in Uniprot Accession Number A0A3P5YA78 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is an StCas9 polypeptide, for example, comprising a sequence of... Petition 870260064333, dated 06 / 30 / 2026, page 75 / 427 70 / 418 amino acids as presented in NCBI Accession Number WP_007896501.1 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is an SluCas9 polypeptide, for example, comprising an amino acid sequence as presented in any of NCBI Accession Numbers 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 an NmCas9 polypeptide, for example, comprising an amino acid sequence as presented in any of the NCBI Accession Numbers WP_002238326.1 or WP_061704949.1, or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a CjCas9 polypeptide, for example, comprising an amino acid sequence as presented in any of the NCBI Accession Numbers 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 an FnCas9 polypeptide, for example, comprising the amino acid sequence as presented in Uniprot Accession Number A0Q5Y3 or a fragment or variant thereof. In some embodiments, a Cas9 polypeptide is a TdCas9 polypeptide, for example, comprising the amino acid sequence as presented in NCBI Accession Number 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 in this document or those known in the art. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide from Streptococcus macacae, for example, comprising the amino acid sequence as presented in NCBI Accession Number WP_003079701.1 or a fragment or variant thereof.In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide generated by... Petition 870260064333, dated 06 / 30 / 2026, page 76 / 427 71 / 418 replacement of a PAM interaction domain of a SpCas9 by that of a Cas9 from Streptococcus macacae (Spy-mac Cas9). [000247] Example variants of Cas9 and nickase Cas9 are provided in Table 2. Table 2: Example Cas protein sequences SEQ ID NO : Descrição da Sequência Sequência de aminoacidos 521 Cas9 de Streptococc us Pyogenes de tipo selvagem (SpCas9) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEITITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLKPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 522 SpCas9 nickase H840A MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILSDILRVNTEITKAPLSASMIKRYDEHHQDLTLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH Petition 870260064333, dated 06 / 30 / 2026, page 77 / 427 72 / 418 LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 523 SpCas9 nickase H840A Met () DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILSDILRVNTEITKAPLSASMIKRYDEHHQDLTLTLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRLSKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 524 CAS9 nickase (R221K N394K H840A) Met (-) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEPINASGVDA KAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRLKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMIERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 525 Cas9 de Staphylococ cuslugdunensis (Slu) de tipo selvagem MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLPEANVENNEGRSKRGSRRLKRRRIHRLERVKK LLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIELNDVIELGVKD QLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFHQLDENFINKYELVE MRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAYSADLFNALNDLNNLVIQRDGLSK LEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILE NEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLCCIRLVLEEQWYSSR NQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSPVVKRTFGQAINLINKIIEKYGVPEDIIIELARE NNSKDKQKFINEMQKKNENTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNP NHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKHILNLSKSQDRIS Petition 870260064333, of 30 / 06 / 2026, p. 78 / 427 73 / 418 KKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNNMNVKVKTINGSFTDYLRKVW KFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNYSEMFIIPKQ VQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPEKFLM YQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFK SSTKKLVKLSIKPYRFDVYLDTKGYKFITISYLDVLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFY KNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLG NVFTNTQYTKPQLLFKRGN 526 SluCas9 nickase N582A MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIH RLERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSKEALSKDELVIALLHIAKRRGIHKIDVI DSNDDVGNELSTKEQLNKNSKLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLN VQKNFHQLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHTYFPDELRS VKYYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPE DIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTE LDILLNEEDKENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTA ANKIPKAMIDEFILSPVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVD HIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRI SKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSF TDYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQV DSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKD IYAKDNTTLKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEEETGEYLTKYS KKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYL DVLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYGVKIINSDTRNMIE LDLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN 527 SluCas9 nickase Met (-) NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHR LERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVID SNDDVGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNV QKNFHQLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSV KYAYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPED IKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAA NKIPKAMIDEFILSPVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQ KKNENTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDH IIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRIS KKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFT DYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVD SEDNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKKDNSTYIVQTIKDI YAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSK KNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLDTKGYKFITISYLD VLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIEL DLPDIRYKEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN 528 Cas9 Staphylococcus aureus (SaCas9) MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRH RIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEV EEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKV QKAYHQLDQSFIDTYIDLLETRRTYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVK Petition 870260064333, dated 06 / 30 / 2026, p. 79 / 427 74 / 418 YAYNADLYNALNDLNNLVITRDENEKLEYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDI KGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLN SELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRKLVPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSDAQKMINEMQK RNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISK TKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTS FLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEI ETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLN GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKY SKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKN LDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNDLLNRI EVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG 529 SaCas9 nickase N580A MKRNYILGLDIGITSVGYGIIDRYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRH RIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKV QKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKWYEMLMGHCTYFPEELRSVK YAYNADLYNALNDLNNLVITRDENEKLEYEKYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDI KGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLN SELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRKLVPKKVDLSQQK EIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQK RNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISK TKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLRSYFRVNNLDVKVKSINGGFTS FLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEI ETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLN GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKY SKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKN LDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNDLLNRI EVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG 530 SaCas9 nickase Met (-)KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHR IQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQ KAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKY AYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIK GYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNS ELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKE IPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKR NRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHII PRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKT KKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSF LRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIE TEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNG LYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYS Petition 870260064333, dated 06 / 30 / 2026, page 80 / 427 75 / 418 KKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIE VNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG 531 SpCas9-NG (VRVRFRR) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIELTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQDGSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESIRPKRNSDKLIARKDWDPKKYGGFVSPTVAYSVLVVAKVEKGSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELEGNRKGRMLASARFLQKGNELALPSKYVNFLYLASHY ELKLGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPRAFKYFDTTIDRKKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 532 spCas9-NG Nickase (H840A_VRVR FRR) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILDANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 533 SpCas9-NG Nickase Met (-) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDCADLRLIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH Petition 870260064333, the 30 / 06 / 2026, pág. 81 / 427 76 / 418 LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR KDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 534 spCas9-NGA (VRQR)MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 535 spCas9-NGA Nickase (H840A)_VRQ R) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAETRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG Petition 870260064333, de 30 / 06 / 2026, pág. 82 / 427 77 / 418 FSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY EKLKKGSPEDNEQKQLFVEQHKHYLDEIEQISEFSKRVILDANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 536 spCas9-NGA Nickase Met (-) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFR KDFQFYKVREINNYHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKQIRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 537 Cas9 SpRY MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAERTRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 538 Cas9 Nickase SpRY (HX88) MDKKYSIGLDITNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAERTRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTNFKSNFDLAEDAKLQLSKDTYDDDDLD NLLAQIGDQYADLFLAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI Petition 870260064333, of 30 / 06 / 2026, p. 83 / 427 78 / 418 HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEEETITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 539 Cas9 Nickase SpRY Met (-)DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAERTRLKRTARR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRLKVLSMPQVNIVKKTEVQTGGF SKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 540 sRGN3.1 MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPANVENNEGRRSKRGSRRLKRRRIH RLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVA ADKEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKI IDTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQE LRSVKYAYASADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGV NPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLNLNQIAEILTIYQDKDSIVAE LGQLEYLMSEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYE LKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFI NNLQKKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQ DRISKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTIN GSFTDYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLD IQVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQT IKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLT Petição 870260064333, de 30 / 06 / 2026, pág. 84 / 427 79 / 418 KYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYLTEKGYKFVTI AYLNVFKKDNYYYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRN IIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFK RGL 541 sRGN3.1 Nickase (N585A) MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIH RLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLAKRRGIHNVDVA ADKEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKI IDTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQE LRSVKYAYASADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGV NPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDILLNQIAEILTIYQDKDSIVAE LGQLEYLMSEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYE LKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSSDDRKKFI NNLQKKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHY EVDHIIPRSVSFDNSYNHKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQFKQHINLSKSQ DRISKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTIN GSFTDYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKKDNSTYIVQT IKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLT KYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYLTEKGYKFVTI AYLNVFKKDNYYYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRN IIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFK RGL 542 sRGN3.1 Nickase (N584A) Met (-) NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPANVENNEGRRSKRGSRRLKRRRIHR LERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAA DKEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKII DTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQEL RSVKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVN PEDIKGYRITKSGTPEFTSFKLFHDLKVKDHAILDDILLNQIAEILTIYQDKDSIVAEL GQLEYLMSEADKQSISELTGYTGTHSLSLKCMNMIIDLWHSSMNQMEVFTYLNMRPKKYEL KGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFIN NLQKKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYE VDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQFKQHINLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTING SFTDYLRKVWKFKKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDI QVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTI KDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTK YSKKNNGPIVKSLKKYIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYLTEKGYKFVTIA YLNVFKKDNYYYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNI IELDYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKR GL 543 sRGN3.3 MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIH RLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVA ADKEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVERAKKI IDTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQE Petition 870260064333, de 30 / 06 / 2026, pág. 85 / 427 80 / 418 LRSVKYAYASADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGV NPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDILLNQIAEILTIYQDKDSIVAE LGQLEYLMSEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYE LKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSSDDRKKFI NNLQKKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHY EVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHINLSKSQ DRISKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTIN GSFTNHLRKVWRFDKYRNHGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKK VTVEKEEDYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMCDEHDYIVQT ITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLT KYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTI AYLNVFKKDNYYYIPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRN IIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFK RGL 544 sRGN3.3 Nickase (N585A) MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPANVENNEGRRSKRGSRRLKRRRIH RLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKI IDTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNDGLDLKKWYEMLMGHCTYFPQE LRSVKYAYASADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGV NPEDIKGYRITKSGTPEFTSFKLFHDLKVKDHAILDDILLNQIAEILTIYQDKDSIVAE LGQLEYLMSEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYE LKGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFI NNLQKKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHY EVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQFKQHINLSKSQ DRISKKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTIN GSFTNHLRKVWRFDKYRNHGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKK VTVEKEEDYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQT ITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLT KYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTI AYLNVFKKDNYYYIPKDKYQELKEKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRN IIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFK RGL 545 sRGN3.3 Nickase (N584A) Met (-)NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHR LERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAA DKEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKII DTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQEL RSVKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVN PEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAEL GQLEYLMSEADKQSISELTGYTGTHSLLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYEL KGYQRIPTDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFIN NLQKKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYE VDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQD RISKKKKEYLLEERDINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTING SFTNHLRKVWRFDKYRNHGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKV Petition 870260064333, dated 06 / 30 / 2026, page 86 / 427 81 / 418 TVEKEEDYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTI TDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTK YSKKNNGPIVKKIKLLGNKVNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIA YLNVFKKDNYYYIPKDKYQELKEKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNI IELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKR GL 546 SpG MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEITITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHPVENTQLQNEKLYYLQNGRDMYVDQEL DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 547 SpG Nickase (H840A) MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTAR RRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHL RKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLD NLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQI HLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEITITPWNFEEVVD KGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL DINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKF DNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF RKDFQFYKVREINNYHHAHDAYLNAVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAK YFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH LFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 548 SpG Nickase (H839A) Met (-) DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR KKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEPNASGVDA Petition 870260064333, the 30 / 06 / 2026, pág. 87 / 427 82 / 418 KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDN LLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK YKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSGEQKKAIVDLLF KTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLT LTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANR NFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLSKLVSDFR KDFQFYKVREINNYHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKYDVRKMIAKSEQEIGKATAKY FFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGF SKESILPKRNSDKLIARKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD [000248] In some embodiments, a prime editor comprises a DNA-binding domain comprising 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 of the sequences presented 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, for example, mutations, for example, deletions, substitutions and / or insertions, compared to any of the amino acid sequences shown in Table 2. [000249] In some embodiments, a prime editor comprises a Cas9 protein that is a Cas9 nickase comprising 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 Petition 870260064333, dated 06 / 30 / 2026, page 88 / 427 83 / 418 97%, at least 98%, at least 99%, or 100% identical to any of the nickase sequences shown in Table 2. In some embodiments, a prime editor comprises a Cas9 protein comprising an amino acid sequence selected from the group consisting of the sequences shown in Table 2. In some embodiments, a prime editor comprises a Cas9 protein comprising an amino acid sequence that lacks an N-terminal methionine relative to an amino acid sequence shown in Table 2.In some embodiments, a prime edit composition or a prime edit system disclosed in this document comprises a polynucleotide (e.g., a DNA or an RNA, e.g., an mRNA) encoding a Cas9 protein comprising 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 of the sequences presented in Table 2. [000250] In some embodiments, a Cas9 protein comprises a Cas9 protein from Streptococcus pyogenes (Sp), for example, according to NC_002737.2:854751-858857, or the protein encoded by UniProt Q99ZW2, for example, as per SEQ ID NO: 521. In some embodiments, a prime editor comprises a Cas9 protein (e.g., an SpCas9) according to any of the sequences shown in SEQ ID NOs: 521-524 or a variant thereof. In some embodiments, the Cas9 protein is an SpCas9. In some embodiments, an SpCas9 may be a wild-type SpCas9, a variant of SpCas9, or a nickase SpCas9. In some embodiments, SpCas9 lacks the N-terminal methionine compared to a corresponding SpCas9 (e.g., a wild-type SpCas9, a variant of SpCas9, or a nickase SpCas9). In some embodiments, a prime editor comprises a Petition 870260064333, dated 06 / 30 / 2026, p. 89 / 427 84 / 418 Cas9 protein, having an amino acid sequence according to SEQ ID NO: 521, not including N-terminal methionine. In some embodiments, a wild-type SpCas9 comprises an amino acid sequence shown in SEQ ID NO: 521. 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 SpCas9). In some embodiments, the Cas9 protein comprising one or more mutations relative to a wild-type Cas9 protein (e.g., a wild-type SpCas9 protein) comprises an amino acid sequence shown in SEQ ID NOs: 522, 523, or 524. Example amino acid sequences of Streptococcus pyogenes Cas9 (SpCas9) useful in the prime editors disclosed in this document are provided in Table 2. [000251] In some embodiments, a prime editor comprises a Cas9 protein (e.g., a SluCas9), according to any 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), for example, according to any of the SEQ ID NO: 525 or a variant thereof. In some embodiments, the Cas9 protein is a SluCas9. In some embodiments, a SluCas9 may be a wild-type SluCas9, a variant of SluCas9, or a nickase SluCas9. In some embodiments, SluCas9 lacks the N-terminal methionine relative to a corresponding SluCas9 (e.g., a wild-type SluCas9, a variant of SluCas9, or a nickase SluCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence according to SEQ ID NO: 525, excluding the N-terminal methionine.In some embodiments, a wild-type SluCas9 comprises an amino acid sequence shown in the SEQ ID. Petition 870260064333, dated 06 / 30 / 2026, p. 90 / 427 85 / 418 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 more mutations relative to a wild-type Cas9 protein comprises an amino acid sequence shown in SEQ IDs NOs: 526 or 527. Sample Cas9 amino acid sequences from Staphylococcus lugdunensis (SluCas9) useful in the prime editors disclosed in this document are provided in Table 2. [000252] In some embodiments, a prime editor comprises a Staphylococcus aureus Cas9 protein (SaCas9), for example, according to any of the SEQ ID NOs: 528-530, or a variant thereof. In some embodiments, a prime editor comprises a Staphylococcus aureus Cas9 protein (SaCas9), for example, as shown in Table 2, or a variant thereof. In some embodiments, the Cas9 protein is a SaCas9. In some embodiments, a SaCas9 may be a wild-type SaCas9, a variant of SaCas9, or a SaCas9 nickase. In some embodiments, the SaCas9 lacks the N-terminal methionine relative to a corresponding SaCas9 (e.g., a wild-type SaCas9, a variant of SaCas9, or a SaCas9 nickase). In some embodiments, a prime editor comprises a Cas9 protein, with an amino acid sequence according to SEQ ID NO: 528, excluding the N-terminal methionine.In some embodiments, a wild-type SaCas9 comprises an amino acid sequence shown 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... Petition 870260064333, dated 06 / 30 / 2026, p. 91 / 427 86 / 418 The Cas9 protein comprising one or more mutations relative to a wild-type Cas9 protein comprises an amino acid sequence shown in SEQ ID NOs: 529 or 530. Sample amino acid sequences of Staphylococcus aureus Cas9 (SaCas9) useful in prime editors disclosed in this document are provided in Table 2. [000253] In some embodiments, a prime editor comprises a Cas9 protein according to any of the sequences presented in SEQ ID NOs: 531-539, 546-548 or a variant thereof. In some embodiments, the Cas9 protein is a variant of Cas9, for example, a variant of SpCas9 (e.g., SpCas9-NG, SpCas9-NGA, SpRY, or SpG). In some embodiments, a prime editor comprises a Cas9 protein that lacks the N-terminal methionine relative to a corresponding Cas9 protein (e.g., a variant of Cas9 presented in any of the 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 conforming to any of the following SEQ ID numbers: 531-539, 546-548, excluding the N-terminal 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 presented in any of the SEQ ID NOs: 531-539, 546-548). In some embodiments, the Cas9 protein comprising one or more mutations relative to a corresponding Cas9 protein comprises an amino acid sequence presented in any of the SEQ ID NOs: 532, 533, 535, 536, 538, 539, 547, or 548. [000254] In some embodiments, a Cas9 protein is a chimeric Cas9, for example, a modified Cas9, for example, synthetic RNA-guided nucleases (sRGNs), for example, modified by shuffling of DNA families, for example, Petition 870260064333, dated 06 / 30 / 2026, p. 92 / 427 87 / 418 SRGN3.1, sRGN3. In some embodiments, DNA family shuffling involves the fragmentation and reassembly of precursor Cas9 genes, for example, one or more Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa). In some embodiments, a modified sluCas9 exhibits greater editing efficiency and / or specificity compared to an unmodified sluCas9. In some modes, a modified Cas9, for example, an sRGN, shows at least a 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 an unmodified Cas9.In some modalities, a Cas9, for example, an sRGN shows at least a 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 an unmodified Cas9. In some embodiments, a Cas9, for example, an sRGN, exhibits an increase of 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% in cleavage activity compared to an unmodified Cas9.In some modes, a Cas9, for example, an sRGN, has the ability to cleave a target containing PAM 5,-NNGG-3'. In some modes, a... Petition 870260064333, dated 06 / 30 / 2026, p. 93 / 427 88 / 418 editor prime comprises a Cas9 protein (e.g., a chimeric Cas9), for example, according to any of the sequences presented in SEQ ID NOs: 540-545, or a variant thereof. Example amino acid sequences of the Cas9 protein (e.g., sRGN) useful in the editor primes disclosed in this document are provided in Table 2. In some embodiments, an editor prime comprises a Cas9 protein that lacks an N-terminal methionine relative to SEQ ID NO: 540 or SEQ ID NO: 543. In some embodiments, an editor prime 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 shown in either of the SEQ ID NOs: 540 or 543).In some embodiments, the Cas9 protein comprising one or more mutations relating to a corresponding Cas9 protein comprises an amino acid sequence presented in any of the SEQ ID NOs: 541, 542, 544, or 545. [000255] In some embodiments, a Cas9 protein comprises a variant Cas9 protein containing one or more amino acid substitutions. In some embodiments, a wild-type Cas9 protein comprises a RuvC domain and an HNH domain. In some embodiments, a prime editor comprises an active nuclease Cas9 protein that can cleave both strands of a double-stranded target DNA sequence. In some embodiments, the active nuclease 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 only cleave one strand of a double-stranded target DNA. In some embodiments, the Cas9 nickase comprises only a functional RuvC domain or a functional HNH domain. In some embodiments, a prime editor comprises a Cas9 that has a Petition 870260064333, dated 06 / 30 / 2026, p. 94 / 427 89 / 418 non-functional HNH domain and a functional RuvC domain. In some embodiments, the prime editor can cleave the editing strand (i.e., the PAM strand) but not the non-editing 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 editing strand of a double-stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 that does not have a functional RuvC domain or a functional HNH domain, which may not cleave either strand of a double-stranded target DNA sequence. [000256] 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, Cas9 comprises a mutation in amino acid D10 compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, Cas9 comprises a D10A mutation compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation in amino acids D10, G12, and / or G17 compared to a wild-type SpCas9, as shown 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 compared to a wild-type SpCas9, as presented in SEQ ID NO: 521, or a corresponding mutation thereof. [000257] 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 in the H840 amino acid compared to a wild-type SpCas9, Petition 870260064333, dated 06 / 30 / 2026, page 95 / 427 90 / 418 as shown in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises an H840A mutation compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation in amino acids E762, D839, H840, N854, N856, N863, H982, H983, A984, D986 and / or A987, compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises an E762A, D839A, H840A, N854A, N856A, N863A, H982A, H983A, A984A, and / or D986A mutation, compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or a corresponding mutation thereof.In some embodiments, the Cas9 polypeptide comprises a mutation in the amino acid residues R221, N394, and / or H840 compared to a wild-type SpCas9 (e.g., SEQ ID NO: 521). In some embodiments, the Cas9 polypeptide comprises an R221K, N394L, and / or H840A mutation compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises a mutation in the amino acid residues R220, N393, and / or H839 compared to a wild-type SpCas9 (e.g., SEQ ID NO: 521) lacking N-terminal methionine, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprises an R220K, N393K, and / or H839A mutation compared to a wild-type SpCas9 (as shown in SEQ ID NO: 521) lacking N-terminal methionine or a corresponding mutation thereof. [000258] In some embodiments, a prime editor comprises a Cas9 having one or more amino acid substitutions in both the HNH and RuvC domains that reduce or abolish the nuclease activity of both the HNH and RuvC domains. In some embodiments, the prime editor Petition 870260064333, dated 06 / 30 / 2026, p. 96 / 427 91 / 418 comprises a Cas9 with an inactive nuclease or a Cas9 with a dead nuclease (dCas9). In some embodiments, dCas9 comprises an H840X substitution and a D10X mutation compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or corresponding mutations, where 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 an H840A mutation and a D10A mutation compared to a wild-type SpCas9, as shown in SEQ ID NO: 521, or corresponding mutations thereof. [000259] In some embodiments, the N-terminal methionine is removed from the amino acid sequence of a Cas9 nickase, or any variant, ortholog, or equivalent of Cas9 disclosed or contemplated in this document. For example, methionine-minus (Met(-)) Cas9 nickases include any of the sequences presented in the 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 with them. [000260] In addition to dead Cas9 variants and nickase Cas9, the Cas9 proteins used in this document 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 with any reference Cas9 protein, including any wild-type Cas9 or mutant Cas9 (e.g., a dead Cas9 or nickase Cas9), or Cas9 fragment, or circular permutant Cas9, or other Cas9 variant disclosed in this document or known in the art. In some forms, a Cas9 variant can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Petition 870260064333, dated 06 / 30 / 2026, p. 97 / 427 92 / 418 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, for example, 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. [000261] 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 long. 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 long. In some embodiments, a prime editor comprises a Cas protein, for example, a Cas9 variant, comprising modifications that allow for altered PAM recognition. Amino acid sequences of the example Cas9 protein (by Petition 870260064333, dated 06 / 30 / 2026, page 98 / 427 93 / 418 example, a variant of Cas9 with altered PAM recognition specificities) useful in the Prime editors of this disclosure are provided in Table 2. In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow for altered PAM recognition. In prime editing using a Cas protein-based prime editor, a “protospacer adjacent motif (PAM)”, PAM sequence, or PAM-like motif, can 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 Cas protein.A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides long. In some embodiments, a PAM is between 2 and 6 nucleotides long. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In 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, an SpCas9 recognizes a 5'-NGG-3' PAM. In some embodiments, the Cas protein of a prime editor has altered or non-canonical PAM specificities. Example PAM sequences and corresponding Cas variants are described in Table 3.It should be noted that, for each of the variants provided, the Cas protein comprises one or more of the indicated amino acid substitutions compared to a wild-type Cas protein sequence, for example, Cas9, as presented in SEQ ID NO: 521. The PAM motifs, as shown in Table 3, are in the order of 5' to 3'. Petition 870260064333, dated 06 / 30 / 2026, p. 99 / 427 94 / 418 [000262] In some embodiments, Cas disclosure proteins can also be used to target transcriptional control of target sequences, for example, by silencing transcription through sequence-specific binding to target sequences. In some embodiments, a Cas protein described in this document may have one or more mutations in a PAM recognition motif. In some embodiments, a Cas protein described in this document may have altered PAM specificity. [000263] As used in the PAM sequences in Table 3, N refers to any of the 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. Table 3: Cas protein variants and corresponding PAM sequences Variante PAM spCas9 (type selvagem) NGG, NGA, NAG, NGNGA spCas9- VRVRFRR R1335V, L1111R, D1135V, G1218R, E1219F, A1322R, T1337R NG spCas9-VQR (D1135V, R1335Q, T1337R ) NGA spCas9-EQR (D1135E, R1335Q, T1337R) NGA spCas9-VRER (D1135V, G1218R, R1335E, T1337R) NGCG spCas9-VRQR (D1135V, G1218R, R1335Q, T1337R) NGA Cas9-NG (L1111R, D1135V, G1218R, E1219F, A1322R, T1337R, R1335V) NGN SpG Cas9 (D1135L, S1136W, G1218K, E1219Q, R1335Q, T1337R) NGN SyRY Cas9 (A61R, L1111R, N1317R, A1322R and R1333P) NRN xCas9 (E480K, E543D, E1219V, K294R, Q1256K, A262T, S409I, M694I) NGN SluCa9 NNGG sRGN1, sRGN2, sRGN4, sRGN3.1, sRGN3.3 NNGG saCas9 NNGRRT, NNGRRN saCas9-KKH (E782K, N968K, R1015H) NNNRRT spCas9-MQKSER (D1135M, S1136Q, G1218K, E1219S, R1335E, T1337R) NGCG / NGCN spCas9-LRKIQK (D1135L, S1136R, G1218K, E1219I, R1335Q, T1337K) NGTN Petition 870260064333, de 30 / 06 / 2026, p. 100 / 427 95 / 418 spCas9-LRVSQK (D1135L, S1136R, G1218V, E1219S, R1335Q, T1337K) NGTN spCas9-LRVSQL(D1135L, S1136R, G1218V, E1219S, R1335Q, T1337L) NGTN Cpf1 TTTV Spy-Mac NAA NmCas9 NNNNGATT StCas9 NNAGAAW TdCas9 NAAAAC [000264] In some embodiments, a prime editor comprises a Cas9 polypeptide comprising one or more 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 compared to an SpCas9 polypeptide of Wild type as presented in SEQ ID NO: 521. [000265] In some embodiments, a prime editor comprises a SaCas9 polypeptide. In some embodiments, the SaCas9 polypeptide comprises one or more of the E782K, N968K, and R1015H mutations, compared to a wild-type SaCas9. In some embodiments, a prime editor comprises an FnCas9 polypeptide, for example, a wild-type FnCas9 polypeptide or an FnCas9 polypeptide comprising one or more of the E1369R, E1449H, or R1556A mutations, compared to wild-type FnCas9. In some embodiments, a prime editor comprises a ScCas9, for example, a wild-type ScCas9 or an ScCas9 polypeptide comprising one or more of the I367K, G368D, I369K, H371L, T375S, T376G, and T1227K mutations compared to wild-type ScCas9. In some modalities, an editor Petition 870260064333, dated 06 / 30 / 2026, p. 101 / 427 96 / 418 prime comprises an St1Cas9 polypeptide, an St3Cas9 polypeptide, or an SluCas9 polypeptide. [000266] In some embodiments, a prime editor comprises a Cas polypeptide comprising a circular permutant Cas variant. For example, a Cas9 polypeptide of a prime editor can be engineered so that the N-terminus and C-terminus of a Cas9 protein (e.g., a wild-type Cas9 protein or a Cas9 nickase) are topically rearranged to maintain the ability to bind to DNA when complexed with a guide RNA (gRNA). An example circular permutant configuration might be N-terminus-[original C-terminus]-[original N-terminus]-C-terminus. Any of the Cas9 proteins described in this document, including any naturally occurring or equivalent variant, ortholog, or Cas9, can be reconfigured as a circular permutant variant. [000267] In several embodiments, the circular permutants of a Cas protein, for example, a Cas9, can have the following structure: N-terminal-[original C-terminal]-[optional ligand][original N-terminal]-C-terminal.In some embodiments, a circular permutant Cas9 comprises any of the following structures (amino acid positions as presented in SEQ ID NO: 521): N-[1268-1368]-[optional ligand]-[1-1267]-C-terminal; N-[1168-1368]-[optional ligand]-[1-1167]-C-terminal; N-[1068-1368]-[optional ligand]-[1-1067]-C-terminal; N-[968-1368]-[optional ligand]-[1-967]-C-terminal; N-[868-1368]-[optional ligand]-[1-867]-C-terminal; N-[768-1368]-[optional ligand]-[1-767]-C-terminal; terminal N-[668-1368]-[optional binder]-[1-667]-terminal C; terminal N-[568-1368]-[optional binder]-[1-567]-terminal C; terminal N-[468-1368]-[optional binder]-[1-467]-terminal C; terminal N-[368-1368]-[optional binder]-[1-367]-terminal C; terminal N-[268-1368]-[optional binder]-[1-267]-terminal C;. Petition 870260064333, dated 06 / 30 / 2026, page 102 / 427 97 / 418 terminal N-[168-1368]-[optional ligand]-[1-167]-terminal C; terminal N-[68-1368]-[optional ligand]-[1-67]-terminal C; terminal N-[10-1368]-[optional ligand]-[1-9]-terminal C, or the corresponding circular permutants of other Cas9 proteins (including other orthologs, Cas9 variants, etc.). [000268] In some embodiments, a Cas9 circular permutant comprises any of the following structures (amino acid positions as presented in the SEQ ID NOs: 521-1368 amino acids of UniProtKB - Q99ZW2): N-terminal [102-1368]-[optional ligand]-[1-101]-C-terminal; N-terminal [1028-1368]-[optional ligand]-[1-1027]-C-terminal; N-terminal [1041-1368]-[optional ligand]-[1-1043]-C-terminal; N-terminal [1249-1368]-[optional ligand]-[1-1248]-C-terminal; or the N-[1300-1368]-[optional linker]-[1-1299]-C-terminal terminal, or the corresponding circular permutants of other Cas9 proteins (including other orthologs, variants, etc. of Cas9). [000269] In some embodiments, a Cas9 circular exchanger comprises any of the following structures (amino acid positions as shown in SEQ ID NOs: 521) N-terminal [103-1368]-[optional linker]-[1-102]-C-terminal; N-terminal [1029-1368]-[optional linker]-[1-1028]-C-terminal; N-terminal [1042-1368]-[optional linker]-[1-1041]-C-terminal; N-terminal [1250-1368]-[optional linker]-[1-1249]-C-terminal; or N-terminal [1301-1368]-[optional linker]-[1-1300]-C-terminal, or the corresponding circular exchangers of other Cas9 proteins (including other orthologs, variants, etc. of Cas9). [000270] 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 using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment may correspond to 95% or Petition 870260064333, dated 06 / 30 / 2026, p. 103 / 427 98 / 418 more of the C-terminal amino acids of a Cas9 (for example, amino acids between 1300 and 1368, as presented in SEQ ID NO: 521 or corresponding amino acid positions), or 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or more of the C-terminal amino acids of a Cas9 (for example, SEQ ID NO: 521 or an ortholog or variant thereof). The N-terminal portion may correspond to 95% or more of the N-terminal amino acids of a Cas9 (for example, amino acids between 1 and 1300, as shown in SEQ ID NO: 521 or corresponding amino acid positions), 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 (for example, as shown in SEQ ID NO: 521 or corresponding amino acid positions). [000271] 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 using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to N-terminal includes or corresponds to the C-terminal of 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368, as shown 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 N-terminus in 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 (for example, as shown 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 410 or fewer C-terminal residues than a Cas9 (e.g., as presented in SEQ ID NO: 521 or corresponding amino acid positions thereof). Petition 870260064333, dated 06 / 30 / 2026, p. 104 / 427 99 / 418 In some embodiments, the C-terminal portion that is rearranged to the N-terminal includes or corresponds to the C-terminals 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 (for example, as shown in SEQ ID NO: 521 or in positions of the corresponding amino acids thereof). In some embodiments, the C-terminal portion that is rearranged to the N-terminus includes or corresponds to the 357, 341, 328, 120, or 69 C-terminal residues of a Cas9 (for example, as shown in SEQ ID NO: 521 or at corresponding amino acid positions thereof). [000272] In other embodiments, circular permutant variants of Cas9 may be a topological rearrangement of a primary Cas9 structure based on the following method, which is based on Cas9 from S. pyogenes of SEQ ID NO: 521: (a) select a circular permutant (CP) site corresponding to an internal amino acid residue of the primary Cas9 structure, which dissects the original protein into two halves: an N-terminal region and a C-terminal region; (b) modify the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the amino acid of the CP site) to precede the original N-terminal region, thus forming a new N-terminal Cas9 protein that now begins with the amino acid residue of the CP site. The CP site may be located in any domain of the Cas9 protein, including, for example, the helical domain II, the RuvCIII domain, or the CTD domain.For example, the CP site may be located (as shown in SEQ ID NO: 521 or corresponding amino acid positions thereof) at the 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, the original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041,... Petition 870260064333, dated 06 / 30 / 2026, p. 105 / 427 100 / 418 1247, 1249, or 1282 would become the new N-terminal amino acid. The nomenclature of these CP-Cas9 proteins can be designated 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 limited to the preparation of CP variants from SEQ ID NO: 521, but can be implemented to prepare CP variants in any Cas9 sequence, whether in CP sites that correspond to these positions, or in other CP sites entirely. This description is not intended to limit specific CP sites in any way. Virtually any CP site can be used to form a variant of CP-Cas9. [000273] In some embodiments, a prime editor comprises a functional Cas9 variant with a lower molecular weight than a wild-type SpCas9 protein. In some embodiments, a smaller functional Cas9 variant may facilitate delivery to cells, for example, by an expression vector, nanoparticle, or other delivery means. In certain embodiments, a smaller functional Cas9 variant is a Class 2 Type II Cas protein. In certain embodiments, a smaller functional Cas9 variant is a Class 2 Type V Cas protein. In certain embodiments, a smaller functional Cas9 variant is a Class 2 Type VI Cas protein. [000274] In some embodiments, a prime editor comprises an SpCas9 with a length of 1,368 amino acids and a predicted molecular weight of 158 kilodaltons. In some embodiments, a prime editor comprises a functional variant Cas9 or a functional fragment that has fewer than 1300 amino acids, fewer than 1290 amino acids, fewer than 1280 amino acids, fewer than 1270 amino acids, fewer than 1260 amino acids, fewer than 1250 amino acids, fewer than 1240 amino acids, fewer than 1230 amino acids, fewer than 1220 Petition 870260064333, dated 06 / 30 / 2026, page 106 / 427 101 / 418 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 more than about 400 amino acids and retaining one or more functions, for example, the DNA-binding function of the Cas9 protein. [000275] 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 or 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 several other embodiments, napDNAbp may 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, a SpCas9-NG, a circularly permuted Cas9, or a domain Petition 870260064333, dated 06 / 30 / 2026, p. 107 / 427 102 / 418 Argonaute (Ago), or a functional variant or fragment thereof. [000276] Example Cas proteins and nomenclature are shown in Table 4: Table 4: Example Cas proteins and nomenclature Pre-existing nomenclature Current nomenclature CRISPR-Cas type II enzymes Cas9; Csn1 Cas9 CRISPR-Cas type V enzymes Cpf1 Cas12a CasX Cas12e C2c1 Cas12b1 Cas12b2 same C2c3 Cas12c CasY Cas12d C2c4 same C2c8 same C2c5 same C2c10 Cas12f C2c9 same CRISPR-Cas type VI enzymes C2c2 Cas13a CasRx Cas13d C2c7 Cas13c C2c6 Cas13b [000277] In some embodiments, the prime editors described in this document may also comprise Cas proteins other than Cas9. For example, in some embodiments, a prime editor, as described in this document, may comprise a Cas12a polypeptide (Cpf1) or functional variants thereof. In some embodiments, the Cas12a polypeptide comprises a mutation that reduces or eliminates 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 comprising by Petition 870260064333, dated 06 / 30 / 2026, page 108 / 427 103 / 418 less approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a naturally occurring Cas12a polypeptide. [000278] In some embodiments, a prime editor comprises a Cas protein that is a Cas12b (C2c1) or Cas12c (C2c3) polypeptide. In some embodiments, the Cas protein comprises an amino acid sequence comprising at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a naturally occurring Cas12b (C2c1) or Cas12c (C2c3) protein. In some embodiments, the Cas protein is a Cas12b nickase or a Cas12c nickase. In some embodiments, the Cas protein is a polypeptide Cas12e, Cas12d, Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or CasΦ.In some embodiments, the Cas protein comprises an amino acid sequence comprising at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a naturally occurring Cas12e, Cas12d, Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or CasΦ protein. In some embodiments, the Cas protein is a Cas12e, Cas12d, Cas13, or CasΦ nickase. Nuclear Localization Sequences [000279] In some embodiments, a prime editor additionally comprises one or more nuclear localization sequences (NLSs). In some embodiments, the NLS helps to promote the translocation of a protein to the cell nucleus. In some embodiments, a prime editor comprises a fusion protein, for example, a fusion protein comprising a DNA-binding domain and a DNA polymerase, comprising one or more NLSs. In some embodiments, one or more polypeptides of the prime editor is / are fused or linked to one or more NLSs. In some embodiments, the prime editor comprises a DNA-binding domain and a DNA domain. Petition 870260064333, dated 06 / 30 / 2026, p. 109 / 427 104 / 418 polymerases that are provided in trans, wherein the DNA-binding domain and / or the DNA polymerase domain is / are fused or linked to one or more NLSs. [000280] 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 may be the same NLS or may be different NLSs. In some embodiments, one or more of the NLSs of a prime editor comprise(s) split NLSs. [000281] An NLS can be expressed as part of a prime editor or prime editor complex. In some embodiments, an NLS can be positioned anywhere in the amino acid sequence of a protein and comprise a short sequence of three, four, or more amino acids. The location of the NLS fusion can be at the N-terminus, at 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 the order of N-terminus to C-terminus or C-terminus to N-terminus). In some embodiments, a prime editor is a fusion protein comprising an N-terminus NLS.In some embodiments, a prime editor is a fusion protein comprising an NLS at the C-terminus. In some embodiments, a prime editor is a fusion protein comprising at least one NLS at both the N-terminus and the C-terminus. In some embodiments, the prime editor is a protein. Petition 870260064333, dated 06 / 30 / 2026, p. 110 / 427 105 / 418 fusion comprising two NLSs at the N-terminal and / or C-terminal. [000282] Any NLSs known in the art are covered in this document. NLSs can be any naturally occurring NLS or any non-naturally occurring NLS (for example, an NLS with one or more mutations relative to a wild-type NLS). [000283] In some embodiments, a nuclear localization signal (NLS) is predominantly basic. In some embodiments, one or more of a prime editor's NLSs is / are rich in lysine and arginine residues. In some embodiments, one or more of a prime editor's NLSs comprises proline residues. [000284] Non-limiting examples of NLS sequences suitable for use with disclosure methods and compositions are provided in Table 5. In some embodiments, an 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, an 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 edit composition comprises a polynucleotide encoding an NLS comprising 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 edit composition comprises a polynucleotide encoding an NLS comprising any of the amino acid sequences provided in Table 5. Table 5: Example nuclear localization sequences Petition 870260064333, dated 06 / 30 / 2026, page 111 / 427 106 / 418 Description Sequence SEQ ID NO: SV40 Large T-AG NLS PKKKRKV 549 NLS MKRTADGSEFESPKKKRKV 550 NLS MDSL LMNRRKFLY Q FKNVRWAKGRRE TYLC 551 Nucleoplasmin NLS AVKRPAATKKAGQAKKKKL D 552 EGL-13 NLS MSRRRKANPTKLSENAKKLAKEVEN 553 NLS of C-Myc PAAKRVKLD 554 NLS of Tus Protein KLKIKRPVK 555 NLS of Polyoma Large T-AG VSRKRPRP 556 NLS of Hepatitis D Virus Antigen EGAPPAKRAR 557 NLS of Rev Protein RQARRNRRRRWRERNR 558 NLS of murine p53 PPQPKKKPLDGE 559 NLS KRTADGSEFEPKKKRKV 560 NLS KRTADGSEFE S PKKKRKV 561 NLS NLSKRPAAIKKAGQAKKKK 562 NLS RQRRNELKRSF 563 NLS NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY 564 Nucleoplasmin NLS sequence KRPAATKKAGQAKKKK 565 Xenopus Nucleoplasmin NLS KRXXXXXXXXXXKKKL, where x is any amino acid 566 [000285] In some embodiments, a nuclear locator signal (NLS) comprises the SEQ ID NO: 551. [000286] In some embodiments, an NLS is a single-partite NLS. For example, in some embodiments, an NLS is a large SV40 T antigen NLS comprising the SEQ sequence. ID NO: 549. In some embodiments, an 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, an 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, an NLS is a non-canonical sequence, such as M9 of the hnRNP Al protein, the influenza virus NLS nucleoprotein, and the yeast Gal4 protein NLS. [000287] The components of a prime editor can be connected to each other in any order. In some modes, Petition 870260064333, dated 06 / 30 / 2026, p. 112 / 427 107 / 418 The DNA-binding domain and the DNA polymerase domain of a prime editor can be fused to form a fusion protein, or they can be ligand-linked to a peptide or protein, in any order, from the N-terminus to the C-terminus. In some embodiments, a prime editor comprises a DNA-binding domain fused to or linked to the C-terminal end of a DNA polymerase domain. In some embodiments, a prime editor comprises a DNA-binding domain fused to 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 occurrence of ]-[ indicates the presence of an optional linker sequence.In some embodiments, a prime editor comprises a fusion protein and a trans-supplied DNA polymerase domain, wherein the fusion protein comprises the structure NH2-[DNA-binding domain]-[RNA-recruitment protein polypeptide]-COOH. [000288] In some embodiments, a prime editor fusion protein, a prime editor polypeptide component, or a polynucleotide encoding the prime editor fusion protein or polypeptide component may be split into an N-terminal and a C-terminal half, or polypeptides encoding the N-terminal and C-terminal halves are delivered to a target DNA in a cell separately. For example, in certain embodiments, a prime editor fusion protein may be split into an N-terminal and a C-terminal half for separate dispensing in AAV vectors, Petition 870260064333, dated 06 / 30 / 2026, page 113 / 427 108 / 418 subsequently, translated and colocalized in a target cell to reform the complete polypeptide or prime editor protein. In these cases, separate halves of a protein or fusion protein may each comprise a split intein to facilitate colocalization and reformation of the full-length protein or fusion protein by the mechanism of trans-splicing facilitated by intein. In some embodiments, a prime editor comprises an N-terminal half fused to an N-intein and a C-terminal half fused to a C-intein, or polynucleotides or vectors (e.g., AAV vectors) encoding each of the same. When dispensed and / or expressed in a target cell, the N-intein and C-intein can be excised via protein trans-splicing, resulting in a complete prime editor fusion protein in the target cell.In some embodiments, an exemplary protein described in this document may not possess a methionine residue at the N-terminus. [000289] In some embodiments, a prime editor fusion protein comprises a Cas9 nickase (H840A) and a wild-type M-MLV RT. In some embodiments, a prime editor fusion protein comprises a Cas9 nickase (H840A) and an M-MLV RT comprising the amino acid substitutions D200N, T330P, T306K, W313F, and L603W, compared to a wild-type M-MLV RT. The amino acid sequence of an example prime editor fusion protein and its individual components is shown in Table 6. In some embodiments, a prime editor fusion protein comprises a Cas9 nickase (R221K N394K H840A) and an M-MLV RT comprising the amino acid substitutions D200N, T330P, T306K, W313F, and L603W, compared to a wild-type M-MLV RT. The amino acid sequence of an example prime editor fusion protein and its individual components are shown in Table 7. In some embodiments, an example prime editor protein may Petition 870260064333, dated 06 / 30 / 2026, p. 114 / 427 109 / 418 understand an amino acid sequence as presented in either of the SEQ ID NO: 567 or 568. [000290] 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 in this document (e.g., PE2 or PE3; Table 6, Table 7) or known in the art. [000291] In some embodiments, a prime editor complex comprises a fusion protein comprising a DNA-binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., an RT variant of MMLV) having the following structure: [NLS]-[Cas9(H840A)]-[ligand][MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F), and a desired PEgRNA. In some embodiments, the prime editor complex comprises a prime editor fusion protein having the amino acid sequence SEQ ID NO: 567 (Table 6). The sequence of an example prime editor fusion protein comprising a DNA-binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., an RT variant of MMLV) having the following structure: [NLS]-[Cas9(H840A)]-[ligand][MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 6. [000292] 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% Petition 870260064333, dated 06 / 30 / 2026, p. 115 / 427 110 / 418 identical, at least approximately 99.5% identical, or at least approximately 99.9% identical to the example prime editor sequence in Table 6. [000293] Table 6 lists a sample Prime editor and its components. Table 6. SEQ ID NO. SEQUENCE DESCRIPTION 567 Example Prime Editor [NLS][Cas9(H840A)][ligand][MMLV_RT(D200N)(T3 30P) (L603W) (T306K) (W313F)] — [NLS] MKRTADGSEFSPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFK VLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQE IFSNEMAKVDDS FFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTI YHLRKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGL FGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYAD LFLAAKNLSDAILSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIE KILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFI ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSGE QKKAIVDLLFKTTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIIEERLKTYAHLFDD KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQL IHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLA NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDS PTVAYSVLVVAKVEKGKSK KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN 11HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETR IDLSQLGGDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSTLNIEDEYR LHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHP TSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQ HPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK Petition 870260064333, dated 06 / 30 / 2026, page 116 / 427 111 / 418 QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIP GFAEMAAPL YPL TKPG TLFNWGPDQQKA YQEIKQALLTAPAL GL PDL TKPF ELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKLPKLDPVAAGCLVAWIAW VLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD R VQFGPVVALNPA TLL PL PEEGLQHNCLDILAEAHG TR PDL TDQPL PDADH TWYTDGSSLLQEGQRKAGAAVTTEVIWAKALPAGTSAQALLIALTQAL KMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALL KALFLPKRLSIIHCCPGHQKGHSAEARGNRMADQAARKAAITETETPDTSTLLI ENSSPSGGSKRTADGSEFEFPKKKKV KEY: SEQUÊNCIA DE LOCALIZAÇAR (NUCALZOLS) CAS9(H840A) LIGANT OF 33 AMINOACID REVERSE TRANSCRIPTASE OF M-MLV 550 - NLS N-terminal MKRTADGSEFESPKKKRKV 523 - CAS9 (H840A) FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE SFLVEEDKKKHERHPIFGNIVDEVAYHEKYPTYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIGDLNPDNSDVDKLFIQLVQTYNQLPINAQLFEENSG VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTNFKSNF DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAKNLSDAILLSDILR VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSD NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD Petition 870260064333, dated 06 / 30 / 2026, page 117 / 427 112 / 418 589 - ligand between CAS9 domain and RT domain (33 amino acids) SGGSSGGSSGSETPGTSESATPESSGGSSGGSS 520 - MMLV_RT D200N T330P L603W T306K W313F LKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVK KPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKD AFFCLRLHPTSQPLFAFEWRDPEWRDPEMSQLTWTRLPQGFKNSPTLFNEALFNEH RDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRAS AKKAQICQKQVKYLLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGK AGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQQALQALQALT GLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKLDPVAAGWPP CLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH YQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLT DQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTEVIWAKALPAGTSAQRA ELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIK NKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQALSIGALITE TPDALITE NSPTL-56-99 SGGSKRTADGSEFEPKKRKV [000294] 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., an MMLV RT variant) with the following structure: [NLS]-[Cas9((R221K N394K H840A)][ligand]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA. Cas9((R221K N394K H840A)) and a reverse transcriptase (e.g., an MMLV RT variant) with the following structure: [NLS]-[Cas9 (R221K N394K H840A)]-[binder]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 7. [000295] 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 Petition 870260064333, dated 06 / 30 / 2026, p. 118 / 427 113 / 418 less approximately 96% identical, at least approximately 97% identical, at least approximately 98% identical, at least approximately 99% identical, at least approximately 99.5% identical, or at least approximately 99.9% identical to the example prime editor sequence in Table 7. [000296] Table 7 lists a sample Prime editor and its components. Table 7. SEQ ID NO. DESCRIÇÃO SEQUÊNCIA 568 Editor Prime de Exemplo [NLS][Cas9((R220K) (R393K) (H839A)][ligante][MMLV_RT(D200N)(T330 P) (L603W) (T306K) (W31 3F) ] - [NLS] MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD RHSIKKNLIGALLFDSGETEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDD S FFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFENPINASGVDAKA ILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLS KDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKR YDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE KMDGTEELLVKLKREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNRE KIEKILTFRIPYYVGPLARGNSRFAWMTRKSETITPWNFEEVVDKGASAQSFIERM TNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKVKYVTEGMRKPAFLSGEQKKAIVDLL FKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDFKEDIQKAQVSGQGDSLHEH IANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD VDAIVPQSFLKDD SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRLKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWD PKGYGGFD S PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMITERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFL YLASHYEKLKSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSA YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSKEVLDATLIHQ SITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTNLNIE DEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIK QYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAEWRDPE MGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSE LDCQQGTRALLQTLGNLGYRASACKAQICQKQVKYLGYLLKEGQRWLTEARKETVMG QPTPKTPRQLREFLGKAGFCRLFIPGFEAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAA GWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQ ALLLDTDRVQFGPVVALNPATLLLPPEEEGLQHNCLDILAEAHGTRPDLTDQPLPDAD HTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEG Petition 870260064333, dated 06 / 30 / 2026, page 119 / 427 114 / 418 KKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSI IHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSE FES PKKKRKVGSGPAAKRVKLD KEY: SV40N-Termin C219K N394K H840A) SGGSx2-met-bpSV40NLS-SGGSx2 BINDER M-MLV D200N T306K W313F T330P L603W REVERSE TRANSCRIPTASE binding C-terminal- NLS1 binding C-terminal-NLS2 -NLS terminal N5040 MCRTADGSEFESPKKKRKV 524 - CAS9 (R221K N394K H840A) DKKYSIGLDNVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFKESKVDFEKVDFEKV HPIFGNIVDEVAYHEKYPTYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRKLENLIAQL PGEKKNGLFGNLIALSLGLTNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL PEKYKEIFFDQSKNGYAGYIDGGASQEEFKLFYKDLKLDLEKLLREKLL KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR GNSRFAWMTRKSEETITWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKK IECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKS DGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVK VVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY SVLVVAKVEKGKSKKLKSVKELLGITIMERS S FEKNPIDFLEAKGYKEVKKDLIIKL PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQK QLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 570 - SGGSx2-NLS de bpSV40-ligand SGGSx2 SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGS 520 - MMLV_RT D200N T330P L603W T306K W313FTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATST PVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKPGTNDYRPVQD LREVNKRVEDIHPTVPNPYNLLSGLPPSHQWLTVLDQLTVPLQFLQPLF WRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLL AATSELDCQQGTRALLQTLGNLGYRASAKKAQICQQVKYLGYLLKEGQRWLTEARK ETVMGQPTPKTPRQLREFLGKAGFCRLFIMPGFAQTQPLQTQPLQTG AYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNAR MTHYQALLLDTDRVQFGPVVALNPATLLQPLLDQGLPLDLAQGTPLDALQG LPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQAL Petition 870260064333, of 30 / 06 / 2026, p. 120 / 427 115 / 418 KMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLP KRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSP 571 ligands C-terminal- NLS SGGSKRTADGSEFESPKKRKV C-terminal-NLS5272 ligand GSGPAAKRVKLD [000297] Polypeptides comprising components of a prime editor may be fused via peptide linkers or may be trans-relevant to each other. For example, a reverse transcriptase may be expressed, dispensed, or otherwise provided as an individual component rather than as part of a fusion protein with the DNA-binding domain. In such cases, prime editor components may be associated via non-peptide linkages or colocalization functions. In some embodiments, a prime editor further comprises additional components capable of interacting with, associating with, or recruiting other components of the prime editor or prime editing system. For example, a prime editor may comprise a protein recruitment RNA-polypeptide that can associate with a protein recruitment RNA-aptamer.In some embodiments, a protein recruitment RNA polypeptide can recruit, or be recruited by, a specific RNA sequence. Non-limiting examples of protein recruitment RNA polypeptide and RNA aptamer pairs include an MS2 coat protein and an MS2 RNA hairpin, a PCP polypeptide and a PP7 RNA hairpin, a Com polypeptide and a Com RNA hairpin, a Ku protein and a Ku telomerase-binding RNA motif, and an Sm7 protein and an Sm7 telomerase-binding RNA motif. In some embodiments, the prime editor comprises a DNA-binding domain fused to or bound to a protein recruitment RNA polypeptide. In some embodiments, the prime editor comprises a DNA polymerase domain fused to or bound to a protein recruitment RNA polypeptide. In some embodiments, the DNA-binding domain and the DNA domain. Petition 870260064333, dated 06 / 30 / 2026, p. 121 / 427 116 / 418 polymerase fused to the protein recruitment RNA-polypeptide, or the DNA-binding domain fused to the protein recruitment RNA-polypeptide and the DNA polymerase domain is colocalized by the corresponding protein recruitment RNA-aptamer of the protein recruitment RNA-polypeptide. In some embodiments, the corresponding protein recruitment RNA-aptamer is fused or ligated to a portion of PEgRNA or ngRNA. For example, an MS2 coater protein fused or ligated to DNA polymerase and an MS2 clamp installed on the PEgRNA for colocalization of the DNA polymerase and RNA-guided DNA-binding domain (e.g., a Cas9 nickase). [000298] In certain embodiments, the components of a prime editor are directly merged together. In certain embodiments, the components of a prime editor are associated with each other by means of a linker. [000299] In some embodiments, a prime editor comprises a polypeptide domain, an MS2 coat protein (MCP), that recognizes an MS2 clamp. In some embodiments, the nucleotide sequence of the MS2 clamp (or equivalently called 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. [000300] Table 8: Example MS2 and MCP clamp sequences Table 8. Description Sequence SEQ ID NO: Hairpin MS2 GCCAACATGAGGATCACCCATGTCTGCAGGGCC 573 MCP GSASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT CSVRQSSAQNRKYTIKVEVPKVATQTVGGEELPVAGWRSYLNMELTI PIFATNSDCELIVKAMQGLLKDGNPIPSAIA ANSGIY 574 [000301] As used in this document, a linker can be any chemical group or molecule that links two molecules or fragments, for example, a DNA-binding domain and a polymerase domain of a primed editor. In some Petition 870260064333, dated 06 / 30 / 2026, p. 122 / 427 In 117 / 418 embodiments, a ligand is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the ligand comprises a non-peptidic moiety. The ligand can be as simple as a covalent bond, or it can be a polymeric ligand with many atoms in length, for example, a polynucleotide sequence. In certain embodiments, the ligand is a covalent bond (for example, a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). [000302] In certain embodiments, two or more components of a prime editor are linked together by a peptide linker. In some embodiments, a peptide linker is 5 to 100 amino acids long, 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 long, 24 amino acids long, 64 amino acids long, or 96 amino acids long. [000303] In some embodiments, a ligand comprises from 1 to 100 amino acids. [000304] Non-limiting examples of linkers are provided in Table 9. In some embodiments, a linker comprises any of the amino acid sequences shown in Table 9, or any combination thereof. Table 9. Illustrative Sequences of Peptide Ligands SEQ ID NO. Sequence 575 (GGGGS)n, where n is independently an integer between 1 and 30, where X is any amino acid 576 (G)n, where n is independently an integer between 1 and 30, where X is any amino acid 577 (EAAAK)n, where n is independently an integer between 1 and 30, where X is any amino acid 578 (GGS)n, where n is independently an integer between 1 and 30, where X is any amino acid 579 (SGGS)n, where n is independently an integer between 1 and 30, where X is any amino acid Petition 870260064333, dated 06 / 30 / 2026, pp. 123 / 427 118 / 418 580 (XP)n, where n is independently an integer between 1 and 30, where X is any amino acid 581 (GGS)n, where n is 1, 3 or 7 582 SGSETPGTSESATPES 583 SGGSSGGSSGSETPGTSESATPESSGGSSGGS 584 SGGSGGSGGS 585 SGGS 586 SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS 587 GGSGGS 588 GGSGGSGGS 589 SGGSSGGSSGSETPGTSESATPESSGGSSGGSS [000305] In certain embodiments, two or more components of a primed editor are linked together by a non-peptidic 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 aliphatic or heteroaliphatic linker, substituted or unsubstituted, branched or unbranched. 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 ligand 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 (PEG) moiety. 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 the binding of a nucleophile (e.g., thiol, amino) of the peptide to the linker. Any electrophile may be used as part of the linker. Example electrophiles include, but are not limited to, activated esters, activated amides, acceptors of... Petition 870260064333, dated 06 / 30 / 2026, pp. 124 / 427 119 / 418 Michael, alkyl halides, aryl halides, acyl halides and isothiocyanates. PEgRNA for CFTR gene editing [000306] The term “guide RNA prime editor,” or “PEgRNA,” refers to a guide polynucleotide comprising 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 one or more intended nucleotide edits into the target gene via prime editing. “Nucleotide editing” or “intended nucleotide editing” refers to a specific deletion of one or more nucleotides at a specific position, insertion of one or more nucleotides at a specific position, substitution of a single nucleotide, or other alterations at a specific position to be incorporated into the target gene sequence.The intended nucleotide edit may refer to editing within the editing template compared to the sequence on the target strand of the target gene, or it may refer to editing encoded by the editing template in newly synthesized single-stranded DNA that replaces the target editing sequence, compared to the target editing 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 DNA-binding domain, for example, 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 termed an extension arm. [000307] In certain embodiments, the extension arm comprises a sequence of initiator binding sites (PBS) Petition 870260064333, dated 06 / 30 / 2026, pp. 125 / 427 120 / 418 which can initiate target-initiated DNA synthesis. In some embodiments, the PBS is complementary or substantially complementary to a free 3' end on the target gene editing strand at a cleavage site generated by the prime editor. In some embodiments, the extension arm additionally comprises an editing template comprising one or more nucleotide edits intended to be incorporated into the target gene by prime editing. In some embodiments, the editing template is a template for an RNA-dependent DNA polymerase or polypeptide domain of the prime editor, for example, a reverse transcriptase domain. The reverse transcriptase editing template may also be referred to in this document as an RT, or RTT, template. In some embodiments, the editing template comprises partial complementarity to a target editing sequence in the target gene, for example, a CFTR gene.In some embodiments, the editing model comprises substantial or partial complementarity to the target sequence, except for the position of the nucleotide edits intended to be incorporated into the target gene. An exemplary architecture of a PEgRNA including its components is shown in Figure 2. [000308] 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 may include DNA in the spacer sequence, in the gRNA core, or in 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 comprising a DNA sequence may serve as a synthesis template for Petition 870260064333, dated 06 / 30 / 2026, pp. 126 / 427 121 / 418 DNA is used to create a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase. Thus, PEgRNA can be a chimeric polynucleotide comprising RNA in the spacer, a gRNA core, and / or PBS and DNA sequences in the editing template. [000309] The components of a PEgRNA can be arranged in a modular fashion. In some embodiments, the spacer and extension arm, which comprise a primer binding site (PBS) sequence and an editing template, for example, a reverse transcriptase (RTT) template, can be located interchangeably in the 5' portion of the PEgRNA, in 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 the order of 5' to 3'. In some embodiments, the gRNA core of a PEgRNA of this disclosure can be located between a spacer and an extension arm of the PEgRNA. In some embodiments, the gRNA core of a PEgRNA can be located at the 3' end of a spacer. In some embodiments, the gRNA core of a PEgRNA can be 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. [000310] In some embodiments, a PEgRNA comprises a single polynucleotide molecule comprising the sequence Petition 870260064333, dated 06 / 30 / 2026, pp. 127 / 427 122 / 418 spacer, gRNA core, and extension arm. In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules. In some embodiments, a PEgRNA comprises a first polynucleotide molecule comprising the spacer and a portion of the gRNA core, and a second polynucleotide molecule comprising the remainder 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 partially 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 called crRNA.In some embodiments, PEgRNA comprises a second polynucleotide comprising a second gRNA core portion and the extension arm, wherein the second gRNA core portion may also be called transactivating 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 tracr RNA form a lower stem, an overhang, and an upper stem, as exemplified in Figure 3. [000311] In some embodiments, a spacer sequence comprises a region that has substantial complementarity with a search target sequence on the target strand of a double-stranded target DNA, for example, a CFTR gene. In some embodiments, the PEgRNA spacer sequence is identical or substantially identical to a protospacer sequence on the editing 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 Petition 870260064333, dated 06 / 30 / 2026, pp. 128 / 427 123 / 418 of 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to a target search sequence in the target gene. In some embodiments, the spacer comprises substantially complementary to the target search sequence. [000312] In some embodiments, the spacer length 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 long. In some embodiments, the spacer is 15 to 30 nucleotides long, 15 to 25 nucleotides long, 18 to 22 nucleotides long, 10 to 20 nucleotides long, or 20 to 30 nucleotides long. In some embodiments, the spacer is 16 to 22 nucleotides long, for example, approximately 16, 17, 18, 19, 20, 21, or 22 nucleotides long. In some embodiments, the spacer is 17 to 22 nucleotides long, for example, approximately 17, 18, 19, 20, 21, or 22 nucleotides long. [000313] As used in this document in a PEgRNA or guide RNA sequence, or fragments thereof, such as a spacer, PBS or RTT sequence, unless otherwise indicated, it should be understood that the letter T or thymine indicates a nucleobase in a DNA sequence encoding the PEgRNA or guide RNA sequence, and refers 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. [000314] The extension arm of a PEgRNA may comprise a primer binding site (PBS) and an edit template (e.g., an RTT). The extension arm may be partially complementary to the spacer. In some embodiments, the template of Petition 870260064333, dated 06 / 30 / 2026, pp. 129 / 427 124 / 418 edition (e.g., RTT) is partially complementary to the spacer. In some embodiments, the edition template (e.g., RTT) and the initiator binding site (PBS) are each partially complementary to the spacer. [000315] An extension arm of a PEgRNA may comprise a primer binding site sequence (PBS, or PBS sequence) that comprises complementarity and may hybridize with a free 3' end of single-stranded DNA in the target gene (e.g., the CFTR gene) generated by slicing with a prime editor at the slicing site on the PAM strand. [000316] The length of the PBS sequence may vary depending on, for example, the components of the prime editor, the target search sequence, and other PEgRNA components. [000317] In some embodiments, PBS is about 3 to 19 nucleotides long. In some embodiments, PBS is about 3 to 17 nucleotides long. In some embodiments, 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 long. In some embodiments, PBS is 8 to 17 nucleotides long. In some embodiments, PBS is 8 to 16 nucleotides long. In some embodiments, PBS is 8 to 15 nucleotides long. In some embodiments, PBS is 8 to 14 nucleotides long. In some embodiments, PBS is 8 to 13 nucleotides long. In some embodiments, PBS is 8 to 12 nucleotides long.In some embodiments, PBS is 8 to 11 nucleotides long. In some embodiments, PBS is 8 to 10 nucleotides long. In some embodiments, PBS is 8 or 9 nucleotides long. In some embodiments, PBS is 16 or 17 nucleotides long. Petition 870260064333, dated 06 / 30 / 2026, page 130 / 427 125 / 418 nucleotides in length. In some embodiments, PBS is 15 to 17 nucleotides long. In some embodiments, PBS is 14 to 17 nucleotides long. In some embodiments, PBS is 13 to 17 nucleotides long. In some embodiments, PBS is 12 to 17 nucleotides long. In some embodiments, PBS is 11 to 17 nucleotides long. In some embodiments, PBS is 10 to 17 nucleotides long. In some embodiments, PBS is 9 to 17 nucleotides long. In some embodiments, PBS is approximately 7 to 15 nucleotides long. In some embodiments, PBS is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, PBS is 8 to 14 nucleotides long. In some embodiments, PBS is 9 to 14 nucleotides long. For example, PBS can be 8, 9, 10, 11, 12, 13, or 14 nucleotides long.In some embodiments, PBS is 11 or 12 nucleotides long. In some embodiments, PBS is 11 to 13 nucleotides long. In some embodiments, PBS is 11 to 14 nucleotides long. [000318] PBS can be complementary or substantially complementary to a DNA sequence in the target gene editing strand. By hybridizing with the editing strand at a free hydroxyl group, for example, a free 3' end generated by the prime editor cut, PBS can initiate the synthesis of new single-stranded DNA encoded by the editing template at the cutting site. In some embodiments, PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the target gene editing strand (e.g., the CFTR gene). In some embodiments, PBS is perfectly complementary, or 100% complementary, to a region of the target gene editing strand (e.g., the CFTR gene). [000319] An extension arm of a PEgRNA can Petition 870260064333, dated 06 / 30 / 2026, page 131 / 427 126 / 418 understand an editing model that serves as a DNA synthesis model for DNA polymerase in a prime editor during prime editing. [000320] The length of an editing template can vary depending on, for example, the components of the prime editor, the target sequence being searched, and other components of PEgRNA. In some embodiments, the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is termed a reverse transcription editing (RTT) template. [000321] 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 long. In some embodiments, the RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In some forms, 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 long. In some disciplines, 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 forms, RTT is 10 to 110 nucleotides long.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 long. In some embodiments, the RTT is at least 8 and no more than 50 nucleotides long. In some embodiments, the RTT is at least 8 and no more than 25 nucleotides long. In some embodiments, the RTT is about 10 to about 20 nucleotides long. In some embodiments, the RTT is about 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the RTT... Petition 870260064333, dated 06 / 30 / 2026, p. 132 / 427 127 / 418 has a length of 11 to 17 nucleotides. In some embodiments, RTT has a length of 12 to 17 nucleotides. In some embodiments, RTT has a length of 12 to 16 nucleotides. In some embodiments, RTT has a length of 13 to 17 nucleotides. In some embodiments, RTT has a length of 11, 12, 13, 14, 15, 16, or 17 nucleotides. In some embodiments, RTT has a length of 12 nucleotides. In some embodiments, RTT has a length of 16 nucleotides. In some embodiments, RTT has a length of 17 nucleotides. In some embodiments, RTT has a length of approximately 20 to approximately 30 nucleotides. In some forms, the RTT is about 20 to about 25 nucleotides long. In some forms, the RTT is about 20 to about 25 nucleotides long. In some forms, the RTT is 21 to 24 nucleotides long. [000322] In some embodiments, the editing template sequence (e.g., RTT) is approximately 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary to the target editing sequence in the editing strand of the target gene. In some embodiments, the editing template sequence (e.g., RTT) is substantially complementary to the target editing sequence. In some embodiments, the editing template sequence (e.g., RTT) is complementary to the target editing sequence except at positions of the nucleotide edits intended to be incorporated into the target gene. In some embodiments, the editing template comprises a nucleotide sequence comprising approximately 85% to approximately 95% complementarity with a target editing sequence in the editing strand of the target gene (e.g., the CFTR gene).In some modes, the editing model comprises approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% complementarity with a target editing sequence on the tape. Petition 870260064333, dated 06 / 30 / 2026, p. 133 / 427 128 / 418 editing of the target gene (e.g., the CFTR gene). [000323] An intended nucleotide edit in a PEgRNA editing template may comprise several types of changes compared to the target gene sequence. In some embodiments, the nucleotide edit is a single nucleotide substitution compared to the target gene sequence. In some embodiments, the nucleotide edit is a deletion compared to the target gene sequence. In some embodiments, the nucleotide edit is an insertion compared to the target gene sequence. In some embodiments, the editing template comprises one to ten intended nucleotide edits compared to the target gene sequence. In some embodiments, the editing template comprises one or more intended nucleotide edits compared to the target gene sequence. In some embodiments, the editing template comprises two or more intended nucleotide edits compared to the target gene sequence.In some embodiments, the editing pattern comprises three or more intended nucleotide edits compared to the target gene sequence. In some embodiments, the editing pattern comprises four or more, five or more, or six or more intended nucleotide edits compared to the target gene sequence. In some embodiments, the editing pattern comprises two substitutions, insertions, deletions, or any combination of single nucleotides compared to the target gene sequence. In some embodiments, the editing pattern comprises three substitutions, insertions, deletions, or any combination of single nucleotides compared to the target gene sequence. In some embodiments, the editing pattern comprises four, five, or six substitutions, insertions, deletions, or any combination of single nucleotides compared to the target gene sequence.In some embodiments, a nucleotide substitution comprises a substitution of adenine (A) for... Petition 870260064333, dated 06 / 30 / 2026, page 134 / 427 129 / 418 thymine (T). In some embodiments, a nucleotide substitution comprises a substitution of A for guanine (G). In some embodiments, a nucleotide substitution comprises a substitution of A for cytosine (C). In some embodiments, a nucleotide substitution comprises a substitution of TA. In some embodiments, a nucleotide substitution comprises a substitution of TG. In some embodiments, a nucleotide substitution comprises a substitution of TC. In some embodiments, a nucleotide substitution comprises a substitution of G for A. In some embodiments, a nucleotide substitution comprises a substitution of G for T. In some embodiments, a nucleotide substitution comprises a substitution of G for C. In some embodiments, a nucleotide substitution comprises a substitution of C for A. In some embodiments, a nucleotide substitution comprises a substitution of C for T.In some forms, a nucleotide substitution involves a change from C to G. [000324] In some embodiments, a nucleotide insertion has 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 modalities, a nucleotide insertion has 1 to 2 nucleotides, 1 to 3 nucleotides, 1 to 4 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 3 to 5 nucleotides, 3 to 6 nucleotides, 3 to 8 nucleotides, 4 to 9 nucleotides, 5 to 10 nucleotides, 6 to 11 nucleotides, Petition 870260064333, dated 06 / 30 / 2026, page 135 / 427 130 / 418 to 12 nucleotides, 8 to 13 nucleotides, 9 to 14 nucleotides, 10 to 15 nucleotides, 11 to 16 nucleotides, 12 to 17 nucleotides, 13 to 18 nucleotides, 14 to 19 nucleotides, 15 to 20 nucleotides in length. In some embodiments, a nucleotide insertion is a single nucleotide insertion. In some embodiments, a nucleotide insertion comprises the insertion of two nucleotides. [000325] The editing model of a PEgRNA may comprise one or more intended nucleotide edits, relative to the CFTR gene to be edited. The position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to specific nucleotides (e.g., mutations) in the target CFTR gene, may vary. In some embodiments, nucleotide editing occurs in a region of the PEgRNA corresponding to or homologous to the protospacer sequence. In some embodiments, nucleotide editing occurs in a region of the PEgRNA corresponding to a region of the CFTR gene outside the protospacer sequence. [000326] In some embodiments, the position of a nucleotide editing incorporation in the target gene may be designated relative to the position of the cleavage site. In some embodiments, the position of a desired 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 beyond the slicing site. In some embodiments, the position of a desired 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 cleavage site on the PAM strand (or non-target strand, or editing strand) of the target DNA. Petition 870260064333, dated 06 / 30 / 2026, p. 136 / 427 131 / 418 double. In some embodiments, the position of the intended nucleotide edit in the editing template can be referenced by aligning the editing template with the partially complementary target sequence in the editing strand, and referring to the nucleotide positions in the editing strand where the intended nucleotide edit is embedded. Thus, in some embodiments, a nucleotide edit in an editing model is at a position corresponding to a position around 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 away from the cleavage site. In some embodiments, a nucleotide edit in an editing template is at a position corresponding to approximately 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 8 nucleotides, or 10 nucleotides away.4 nucleotides, 2 nucleotides, 6 nucleotides, 4 to 8 nucleotides, 4 to 14 nucleotides, 6 to 12 nucleotides, 8 to 10 nucleotides, 8 to 16 nucleotides, 10 to 18 nucleotides, 10 to 16 nucleotides, 12 to 22 nucleotides, 14 to 20 nucleotides, 16 to 18 nucleotides, 16 to 24 nucleotides, 18 to 22 nucleotides, 18 to 28 nucleotides, 20 to 26 nucleotides, 30 to 40 nucleotides, 6 nucleotides, 2 to 12 nucleotides, 4 to nucleotides, 4 12 nucleotides, 6 to 10 nucleotides, 6 to 16 nucleotides, 8 to 14 nucleotides, 10 to 12 nucleotides, 16 nucleotides, 10 to 14 nucleotides, 12 to 20 nucleotides, 14 to 18 nucleotides, 14 to 24 nucleotides, 16 to 22 nucleotides, 18 to 20 nucleotides, 18 to 26 nucleotides, 20 to 24 nucleotides, 20 to 30 nucleotides, 10 nucleotides, 2 to nucleotides, 4 to 10 nucleotides, 6 to 8 nucleotides, 6 to 14 nucleotides, 8 to 12 nucleotides, 8 to 18 nucleotides, 10 to 20 nucleotides, 12 to 18 nucleotides, 16 nucleotides, 22 nucleotides, 20 nucleotides,26 nucleotides, 24 nucleotides, 22 nucleotides, 28 nucleotides, 50 nucleotides, Petition 870260064333, dated 06 / 30 / 2026, page 137 / 427 132 / 418 to 60 nucleotides, 60 to 70 nucleotides, 70 to 80 nucleotides, to 90 nucleotides, to 100 nucleotides, 100 to 110 nucleotides, 110 to 120 nucleotides, 120 to 130 nucleotides, 130 to 140 nucleotides or 140 to 150 nucleotides beyond the cleavage site. [000327] In some embodiments, when referred to in the context of the PAM strand (or non-target strand, or editing strand), a nucleotide edit in an editing template is at a position corresponding to a position of approximately nucleotides, nucleotides, 0 to 6 nucleotides, nucleotides, nucleotides, 2 to 4 nucleotides, nucleotides, nucleotides, to 10 nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, to nucleotides, nucleotides, a nucleotides, 10 nucleotides, nucleotides, 12 to 14 nucleotides, ... 120 nucleotides, 120 to 130 nucleotides, 130 to 140 nucleotides or 140 to 150 nucleotides downstream of the cleavage site. The relative positions of the edit(s) of Petition 870260064333, dated 06 / 30 / 2026, page 138 / 427 The intended nucleotide(s) and the slicing site can be designated by numbers. For example, in some embodiments, the nucleotide immediately downstream of the slicing site on a PAM strand (or the non-target strand, or the editing strand) may be designated as being at position 0. The nucleotide immediately upstream of the slicing site on the PAM strand (or the non-target strand, or the editing strand) may be designated as being at position -1. The nucleotides downstream of position 0 on the PAM strand may be designated as being at positions +1, +2, +3, +4, ... +n, and the nucleotides upstream of position -1 on the PAM strand may be designated as being at positions -2, -3, -4, ..., -n.Thus, in some embodiments, the nucleotide in the editing template that corresponds to position 0 when the editing template is aligned with the partially complementary target sequence by complementarity can 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 in the PAM strand of the double-stranded target DNA can 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 in the PAM strand of the double-stranded target DNA can also be referred to as at positions -1, -2, -3, -4, ..., -n in the editing template, even though when PEgRNA is viewed as an autonomous nucleic acid, the positions +1, +2, +3, +4, ..., +n are 5' Position 0 and positions -1, -2, -3, -4, ...-n are 3' away from position 0 in the editing model.In some embodiments, a targeted nucleotide edit is located at the +n position of the editing template relative to the 0 position. Consequently, the targeted nucleotide edit can be incorporated at the +n position of the PAM strand of the target double-stranded DNA (and subsequently, the target strand of the target double-stranded DNA) by prime editing. The corresponding positions of the targeted nucleotide edit incorporated into the CFTR gene are also. Petition 870260064333, dated 06 / 30 / 2026, page 139 / 427 134 / 418 can be named based on the slitting position generated by a prime editor based on sequence homology and complementarity. For example, in some embodiments, the distance between the nucleotide edit to be incorporated into the CFTR gene and the slitting site (also called the “distance between the cut and the edit”) can be determined by the position of the slitting site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying the sequence complementarity between the spacer and the target search sequence and the sequence complementarity between the editing template and the target editing sequence. In certain embodiments, the nucleotide edit position can be anywhere downstream of the slitting site on the editing strand (or on the PAM strand).As used in this document, the distance between the slicing site and the nucleotide edit, for example, when the nucleotide edit comprises an insertion or deletion, refers to the 5' furthest position of the nucleotide edit for a cut that creates a 3' free end in the edit strand (i.e., the “near position” of the nucleotide edit to the slicing site). In some embodiments, the distance between the cut and the edit is 2 to 10⁶ nucleotides. In some embodiments, the distance between the cut and the edit 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 distance between the cut and the edit 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 distance between the cut and the edit is 2 to 25, 2 to 20, 2 to 15, or 2 to 10 nucleotides. In some embodiments, the distance between the cut and the edit is 2, 3, 4, 5, 6, or 7 nucleotides in length.In some modalities, the distance between cutting and editing is 28 nucleotides. In some modalities, the distance between cutting and editing is 22 nucleotides. In some modalities, the distance between cutting and editing is... Petition 870260064333, dated 06 / 30 / 2026, page 140 / 427 135 / 418 nucleotides. In some embodiments, the distance between cutting and editing is 17 nucleotides. In some embodiments, the distance between cutting and editing is 16 nucleotides. In some embodiments, the distance between cutting and editing is 4 nucleotides. [000328] The RTT length and the distance between the cut and the edit refer to the length of the portion of the RTT that is upstream (i.e., 5' relative to) the edit strand plus the 5' end of the RTT and is complementary to the edit strand. In some embodiments, the edit template comprises at least 4 contiguous nucleotides of complementarity with the edit strand, wherein the at least 4 contiguous nucleotides are located upstream of the most edited 5' point in the edit 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 editing strand, wherein 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 most edited 5' point in the editing template.In some embodiments, the editing template comprises 20 to 25, 25 to 30, 30 to 35, 35 to 40, 45 to 45, or 45 to 50 contiguous nucleotides complementary to the editing strand, wherein the 20 to 25, 25 to 30, 30 to 35, 35 to 40, 45 to 45, or 45 to 50 or more contiguous nucleotides are located upstream of the most edited 5' point in the editing template. In some embodiments, the editing template comprises 9 to 14 contiguous nucleotides complementary to the editing strand, wherein the 9 to 14 contiguous nucleotides are located upstream of the most edited 5' point in the editing template. In some embodiments, the editing template comprises 6 to 10 contiguous nucleotides of complementarity with the editing strand, wherein the 6 to 10 contiguous nucleotides are located upstream of the most edited 5' strand in the editing template. In some embodiments, the editing template comprises 10. Petition 870260064333, dated 06 / 30 / 2026, page 141 / 427 136 / 418 contiguous nucleotides of complementarity with the editing strand, wherein the 10 contiguous nucleotides are located upstream of the edit plus 5' in the editing template. In some embodiments the editing template comprises 9 contiguous nucleotides of complementarity with the editing strand, wherein the contiguous nucleotides are located upstream of the edit plus 5' in the editing template. [000329] When referenced within PEgRNA, the positions of one or more intended nucleotide edits can be referenced as relevant to the components of PEgRNA. For example, a desired nucleotide edit might be 5' or 3' from the PBS. In some modalities, a PEgRNA comprises the structure of 5' to 3': a spacer, a gRNA core, an editing template, and a PBS. In some modalities, the intended nucleotide editing 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 or 40 nucleotides upstream of the plus 5' nucleotide of PBS. In some embodiments, the intended nucleotide editing is from 2 nucleotides, 0 to nucleotides, to 6 nucleotides, nucleotides, nucleotides, nucleotides, 2 nucleotides, nucleotides, nucleotides, to 12 nucleotides, nucleotides, nucleotides, to 10 nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, to nucleotides, nucleotides, a nucleotides, 10 nucleotides, nucleotides, 12 to 14 nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, nucleotides, Petition 870260064333, dated 06 / 30 / 2026, page 142 / 427 137 / 418 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 of the nucleotide plus 5' of the PBS. [000330] The corresponding positions of the intended nucleotide edit incorporated into the target gene can also be referenced based on the cleavage position generated by a prime editor based on sequence homology and complementarity. For example, in some embodiments, the distance between the nucleotide edit to be incorporated into the target CFTR gene and the cleavage site (also referred to as the “distance between the cut and the edit”) can be determined by the position of the cleavage site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying the sequence complementarity between the spacer and the target sequence and the sequence complementarity between the editing template and the target sequence.In certain embodiments, the nucleotide edit position can be anywhere downstream of the slicing site on the edit strand (or PAM strand) generated by the prime editor, such that the distance between the slicing 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 nucleotide editing position 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 nucleotide editing position 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 slicing site on the editing strand. In some... Petition 870260064333, dated 06 / 30 / 2026, page 143 / 427 In 138 / 418 embodiments, the nucleotide editing position 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 cleavage site on the editing strand. In some embodiments, the nucleotide editing position is 0 base pairs from the cleavage site on the editing strand, i.e., the editing position is the same position as the cleavage site. As used in this document, the distance between the slicing site and the nucleotide edit, for example, where the nucleotide edit comprises an insertion or deletion, refers to the 5' furthest position of the nucleotide edit for a cut that creates a 3' free end in the edit strand (i.e., the “near position” of the nucleotide edit relative to the slicing site).Similarly, as used in this document, the distance between the splicing 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. [000331] In some embodiments, the editing model extends beyond nucleotide editing to be incorporated into the target CFTR gene sequence. For example, in some modalities, the editing model 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. [000332] In some embodiments, the editing model may comprise a second edit relative to a target sequence. The second edit may be designed to mutate or silence a PAM sequence so that a corresponding nucleic acid-guided nuclease or CRISPR nuclease is no longer able to Petition 870260064333, dated 06 / 30 / 2026, p. 144 / 427 139 / 418 to cleave the target sequence (such edits are called “PAM silencing edits”). [000333] Without wanting to limit ourselves to any specific theory, PAM silencing edits can prevent the Cas nickase, for example, Cas9, from re-cutting the edit strand before the edit is incorporated into the target strand, thus improving the efficiency of the prime edit. 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 the edit is incorporated. In some embodiments, a PAM silencing edit is at a position corresponding to a coding region, for example, an exon, of a CFTR gene. In some embodiments, a PAM silencing edit is at a position corresponding to a non-coding region, for example, an intron, of a CFTR gene. In some embodiments, edits to an intron of a CFTR gene are not at a position corresponding to the intron-exon junction, and the edit does not affect transcript splicing. [000334] In some modes, 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 more than the distance between the cut and the edit. In some modalities, for example, the distance between the cut and the edit is 8 nucleotides, and the edit 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 long. In some modalities, the distance between the cut and the edit is 22 nucleotides, and the editing pattern has 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 Petition 870260064333, dated 06 / 30 / 2026, p. 145 / 427 140 / 418 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. [000335] In some embodiments, the editing template comprises at least 4 to 30 3' base pairs relative to the nucleotide editing to be incorporated into the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 25 3' base pairs relative to the nucleotide editing to be incorporated into the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4-20 3' base pairs relative to the nucleotide editing to be incorporated into the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 30 5' base pairs relative to the nucleotide editing to be incorporated into the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 25 5' base pairs relative to the nucleotide editing to be incorporated into the target CFTR gene sequence.In some modalities, the editing model comprises at least 4-20 5' base pairs in relation to the nucleotide editing to be incorporated into the target CFTR gene sequence. [000336] In some embodiments, the editing template comprises an adenine at the first nucleobase position (e.g., for a PEgRNA following the 5'-spacer-core orientation of gRNA-RTT-PBS-3', the nucleobase plus 5' is the “first base”). In some embodiments, the editing template comprises a guanine at the first nucleobase position (e.g., for a PEgRNA following the 5'-spacer-core orientation of gRNA-RTTPBS-3', the nucleobase plus 5' is the “first base”). In some embodiments, the editing template comprises a uracil at the first nucleobase position (e.g., for a PEgRNA following the 5'-spacer-core orientation of gRNA-RTT-PBS-3', the nucleobase plus 5' is the “first base”). In some Petition 870260064333, dated 06 / 30 / 2026, page 146 / 427 In 141 / 418 embodiments, the editing template comprises a cytosine in the first position of the nucleobase (e.g., for a PEgRNA following the 5'-spacer-core orientation of gRNA-RTT-PBS-3', the nucleobase plus the 5' is the “first base”). In some embodiments, the editing template does not comprise a cytosine in the first position of the nucleobase (e.g., for a PEgRNA following the 5'-spacer-core orientation of gRNA-RTT-PBS-3', the nucleobase plus the 5' is the “first base”). [000337] A PEgRNA editing template can encode new single-stranded DNA (e.g., by reverse transcription) to replace a target editing sequence in the target gene. In some embodiments, the target editing sequence in the target gene's editing strand is replaced by the newly synthesized strand, and the editing nucleotide(s) is / are incorporated into the target gene region. In some embodiments, the target gene is a CFTR gene. In some embodiments, the PEgRNA editing template encodes newly synthesized single-stranded DNA comprising a sequence from the wild-type CFTR gene. In some embodiments, the newly synthesized DNA strand replaces the target editing sequence in the target CFTR gene, wherein the target editing sequence (or the endogenous sequence complementary to the target editing sequence in the target strand of the CFTR gene) comprises a mutation or a nucleotide alteration compared to a wild-type CFTR gene.In some forms, the mutation is associated with cystic fibrosis. [000338] In some embodiments, newly synthesized single-stranded DNA encoded by the editing template replaces the target editing sequence and corrects the mutation in the target editing sequence of the CFTR gene. [000339] In some embodiments, the target sequence for editing comprises position 117587778 on human chromosome 7. In some embodiments, the target sequence for editing comprises a mutation compared to a wild-type CFTR gene, where the mutation is a nucleotide insertion, a deletion of Petition 870260064333, dated 06 / 30 / 2026, p. 147 / 427 142 / 418 nucleotide, a nucleotide substitution, two or more nucleotide substitutions, or any combination thereof. In some embodiments, the mutation results in a premature stop codon in an 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 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 exhibits reduced biological activity compared to a wild-type CFTR polypeptide.In some embodiments, the mutation results in a CFTR polypeptide encoded by the CFTR gene that exhibits nullified biological activity compared to a wild-type CFTR polypeptide. In some embodiments, the target sequence for editing comprises a mutation corresponding to position 1624 of the CFTR protein coding sequence. In some embodiments, the target sequence for editing comprises a c.1624G->T mutation (sense strand target sequence) or a corresponding C->A mutation (antisense strand target sequence) at position 1624 of the CFTR protein coding sequence. [000340] In some embodiments, the editing template comprises one or more intended nucleotide edits compared to the sequence in the target strand of the CFTR gene that is complementary to the target editing sequence. In some embodiments, the editing template encodes single-stranded DNA comprising one or more intended nucleotide edits compared to the target editing sequence. In some embodiments, the single-stranded DNA replaces the target editing sequence by editing. Petition 870260064333, dated 06 / 30 / 2026, pp. 148 / 427 143 / 418 prime, thus incorporating one or more intended nucleotide edits. In some embodiments, one or more intended nucleotide edits encode a T->G substitution at a position corresponding to position 1624 of the CFTR protein coding sequence compared to the target editing sequence (sense strand target editing sequence). In some embodiments, one or more intended nucleotide edits encode an A->C substitution at a position corresponding to position 1624 of the CFTR protein coding sequence compared to the target editing sequence (antisense strand target editing sequence). In some embodiments, the incorporation of one or more intended nucleotide edits corrects the mutation in the target editing sequence to wild-type nucleotides at corresponding positions in the target CFTR gene.As used in this document, correcting a mutation means restoring a wild-type sequence at the mutation site in the target double-stranded DNA, for example, the target gene, through prime editing. In some embodiments, the incorporation of 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. [000341] In some embodiments, the incorporation of one or more intended nucleotide edits results in the expression of a functional CFTR protein. For example, in some embodiments, the incorporation of one or more intended nucleotide edits results in a nucleotide substitution, insertion, or deletion that results in a codon encoding a wild-type amino acid compared to a wild-type CFTR polypeptide, although 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 sequence of the wild-type CFTR gene. Petition 870260064333, dated 06 / 30 / 2026, p. 149 / 427 144 / 418 [000342] In some embodiments, the incorporation of one or more intended nucleotide edits does not correct the mutation in the target edit sequence to the wild-type sequence, but allows the expression of a functional CFTR protein encoded by the CFTR gene. For example, in some embodiments, the incorporation of 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 identical to those of the wild-type CFTR protein. In some embodiments, the incorporation of 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 the expression of a functional CFTR protein. An exemplary amino acid sequence of the wild-type CFTR protein is provided in SEQ ID NO: 751. [000343] A guide RNA core (also referred to in this document as a gRNA core, gRNA scaffold, or gRNA scaffold 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 in this document, for example, by association with a DNA-binding domain, such as a DNA nickase of the prime editor. [000344] A person skilled 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. [000345] In some embodiments, the gRNA core Petition 870260064333, dated 06 / 30 / 2026, p. 150 / 427 145 / 418 comprises regions and secondary structures involved in binding to specific Cas CRISPR 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 “overhang” comprising unpaired RNAs. The gRNA core may additionally comprise a “nexus” distal to the spacer sequence, followed by a hairpin structure, for example, at the 3’ end, as exemplified in Figure 3. In some embodiments, the gRNA core comprises modified nucleotides compared to a wild-type gRNA core in the lower stem, upper stem, and / or hairpin.For example, nucleotides in the lower stem, upper stem, and / or hairpin regions can be modified, deleted, or replaced. In some embodiments, RNA nucleotides in the lower stem, upper stem, and / or hairpin regions can be replaced by one or more DNA sequences. In some embodiments, the gRNA core comprises unmodified or wild-type RNA sequences in the nexus and / or overhang regions. In some embodiments, the gRNA core does not include long stretches of AT pairs, for example, a GUUUUAAAAC pairing element. In some embodiments, a prime editing system comprises a prime editor and a PEgRNA, wherein the prime editor comprises an SpCas9 nickase or a variant thereof, and the gRNA core of the PEgRNA comprises a sequence capable of binding to SpCas9. Known gRNA core sequences are also contemplated in the prime editing compositions described in this document. [000346] In some embodiments, PEgRNA and / or ngRNA comprise a universal gRNA core. A universal gRNA core may be used in a PEgRNA or ngRNA comprising Petition 870260064333, dated 06 / 30 / 2026, p. 151 / 427 146 / 418 any spacer having 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, PEgRNA and / or ngRNA comprises a universal gRNA core comprising a nucleic acid sequence selected from Table 10. In some embodiments, PEgRNA and / or ngRNA comprises a gRNA core comprising a nucleic acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the sequences in Table 10. [000347] Table 10: Example nucleic acid sequences of the universal gRNA core (also referred to in this document as the gRNA scaffold) for PEgRNAs compatible with SpCas9 prime editors. The sequences in Table 10 are annotated with SEQ ID NO, as required by the ST.26 standard. Although all sequences provided in Table 10 are RNA sequences, T is used instead of U in the sequences for consistency with the ST.26 standard. Table 10. SEQ ID NO: Core or scaffold nucleic acid sequence of gRNA 590 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAGTGGCACCGAGTCG GTGC 591 592 GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGGGTGAAAACGCGGCACCGAGTCG GTGC 593 GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGGGTGAAAACGCGGCACCGAGT CGGTGC 594 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGC 595 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGGGTGAAAACGCGG CACCGAGTCGGTGC 596 GTTTTAGAGCTATACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTTACGAAGTGGCACCGAGTCG GTGC Petition 870260064333, of 30 / 06 / 2026, p. 152 / 427 147 / 418 597 GTTTTAGAGCTATACGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTTACGAAGTGGGACCGAGTCG GTCC 598 GTTTTAGAGCTAGCTCATGAAAATGAGCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGGACCGAGTCGGTCC 599 GTTTGAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCG GTCC 601 GTTTTAGAGCTATGCTGGAAACAGCATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGG CACCGAGTCGGTGC 603 GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGT CGGTGC 827 GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGCAGCAAGCACGAAAGTG CTTGCTGCACGCGGCACCGAGTCGGTGC [000348] In some embodiments, a PEgRNA comprises a sequence-specific gRNA core. A sequence-specific gRNA core may be designed to form an ideal secondary or tertiary structure with other PEgRNA components, for example, the spacer, RTT and / or PBS. [000349] In some embodiments, a prime editing system or composition additionally comprises a cutting guide polynucleotide, such as a cutting guide RNA (ngRNA). In some embodiments, an ngRNA comprises a spacer (referred to as an ngRNA spacer or ng spacer) and a gRNA core, wherein the ngRNA spacer comprises a region of complementarity to the editing strand and wherein the gRNA core may interact with a Cas, for example, Cas9, of a prime editor. Without wishing to be limited to any specific theory, an ngRNA may bind to the editing strand and direct the Cas nickase to generate a cut in the non-editing strand (or target strand). In some embodiments, the cut in the non-editing strand directs the endogenous DNA repair mechanism to use the editing strand as a template for repairing the non-editing strand, which may increase the efficiency of prime editing.In some embodiments, the non-editing tape is cut by a prime editor located on the non-editing tape by the ngRNA. Consequently, PEgRNA systems comprising at least one PEgRNA and at least one ngRNA are also provided in this document. Petition 870260064333, dated 06 / 30 / 2026, p. 153 / 427 148 / 418 [000350] A prime editing system comprising a PEgRNA (or one or more polynucleotides encoding PEgRNA) and a prime editor protein (or one or more polynucleotides encoding the prime editor) may be termed a PE2 prime editing system, and the corresponding editing approach termed the PE2 approach or PE2 strategy. A PE2 system does not contain an ngRNA. A prime editing system comprising a PEgRNA (or one or more polynucleotides encoding PEgRNA), a prime editor protein (or one or more polynucleotides encoding the prime editor), and an ngRNA (or one or more polynucleotides encoding ngRNA) may be termed a PE3 prime editing system. In some embodiments, an ng spacer sequence is complementary and may hybridize with the second target sequence only after a desired nucleotide edit has been incorporated into the editing strand, by the PEgRNA editing model. [000351] Such ngRNA may be called ngRNA PE3b”, and the prime editing system, a PE3b prime editing system. [000352] 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 PEgRNA into the editing strand. In some embodiments, the ng search target sequence is 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 from an intended nucleotide edit incorporated by PEgRNA into the editing strand. In some embodiments, the 5' ends of the ng search target sequence and the PEgRNA search target sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bp apart. In some embodiments, the 5' ends of the ng search target sequence and the PEgRNA search target sequence are 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, Petition 870260064333, dated 06 / 30 / 2026, page 154 / 427 149 / 418 sc, 99 sc or 100 sc apart. [000353] The gRNA core of a PEgRNA or ngRNA can be any gRNA scaffold sequence capable of interacting with a Cas protein that recognizes the corresponding PAM of the PEgRNA or ngRNA. In some embodiments, the gRNA core of a PEgRNA or ngRNA comprises a sequence selected from the sequences in Table 10. [000354] In some embodiments, PEgRNA and / or ngRNA comprises a 3' motif. In some embodiments, PEgRNA and / or ngRNA comprises a 3' motif comprising a nucleic acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the sequences provided in Table 11. In some embodiments, 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. [000355] Table 11: Illustrative nucleic acid sequences for the 3' motif (e.g., universal 3' motif). The sequences in Table 11 are annotated with SEQ ID NO as required by ST. 26. Although all sequences provided in Table 11 are RNA sequences, T is used instead of U in the sequences for consistency with the ST. 26 standard. Table 11. SEQ ID NO: Nucleic acid sequence of motif 3' 604 CGCGTCTCTACGTGGGGGCGCG 605 CGGGTCTCTACGTGGGGGCCCG 606 GCGGCACCGTCCGCCCAAACGG 607 CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA [000356] In some embodiments, a PEgRNA additionally comprises a nucleotide linker. In some embodiments, the secondary structure is linked to one or more components of a PEgRNA via a linker. In some embodiments, a secondary structure or a 3' motif of a PEgRNA is linked to a Petition 870260064333, dated 06 / 30 / 2026, p. 155 / 427 150 / 418 or more 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., an 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., an 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 ligand is a nucleotide ligand having 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, the ligand has 5 to 10 nucleotides in length.In some embodiments, the ligand is 10 to 20 nucleotides long. In some embodiments, the ligand is 15 to 25 nucleotides long. In some embodiments, the ligand is 8 nucleotides long. [000357] In some embodiments, the linker is designed to minimize base pairing between the linker and another PEgRNA component. 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 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 RNA secondary structure sequence. In some embodiments, the linker is optimized to minimize base pairing between the linker and another PEgRNA component, in the following priority order: Petition 870260064333, dated 06 / 30 / 2026, page 156 / 427 151 / 418 spacer, PBS, editing template, and then scaffolding. In some embodiments, the base pairing 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 in this document, under standard parameters (37 °C, 1 M NaCl, 0.05 M MgCl2). [000358] In certain embodiments, PEgRNA comprises an RNA secondary structure, such as, but not limited to, aptamers, hairpins, stems / twists, toeloops, and / or RNA-binding protein recruitment domains (e.g., the MS2 aptamer that 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 from a Moloney murine leukemia virus (M-MLV) genome (an mpknot).In some embodiments, the secondary structure comprises a nucleotide sequence selected from the group consisting of the sequences provided in Table 12, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence provided in Table 12. In some embodiments, the secondary structure comprises a quadruplex. In some embodiments, the secondary structure comprises a Gquadruplex. In some embodiments, the secondary structure comprises a riboswitch aptamer. In some embodiments, the secondary structure comprises a riboswitch aptamer derived from a prekeosin-1 riboswitch aptamer. In some embodiments, the secondary structure comprises a modified prekeosin-1 riboswitch aptamer. In some... Petition 870260064333, dated 06 / 30 / 2026, page 157 / 427 In embodiments 152 / 418, PEgRNA comprises a tooloop element with the sequence 5,-GAAANNNNN-3', where N is any nucleobase. In some embodiments, the RNA secondary 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 and 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 the 5' end of the PEgRNA. [000359] In some embodiments, PEgRNA comprises an RNA secondary structure and / or a ligand disclosed in Nelson et al. Engineered PegRNAs improve prime editing efficiency. Nat Biotechnol. (2021), the full text of which is incorporated herein by reference. [000360] Example secondary structure sequences are provided in Table 12. [000361] Table 12. Illustrative sequences for secondary structures. The sequences in Table 12 are annotated with SEQ ID NO, as required by ST. 26. Although all sequences provided in Table 12 are RNA sequences, T is used instead of U in the sequences for consistency with the ST. 26 standard. Table 12. SEQ ID NO: Sequence 709 GGGTCAGGAGCCCCCCCCCTGAACCCAGGATAACCCTCAAAGTCGGGGGGCAACC 710 GTCAGGGTCAGGAGCCCCCCCCCTGAACCCAGGATAACCCTCAAAGTCGGGGGGCAACC C 711 GGGTCAGGAGCCCCCCCCCTGAACCCAGGAAAACCCTCAAAGTCGGGGGGCAACCC 712 GGGTCAGGAGCCCCCCCCCTGCACCCAGGAAAACCCTCAAAGTCGGGGGGCAACCC 713 GGGTCAGGAGCCCCCCCTGCACCCAG GTCAGGGTCAGGAGCCCCCCCCCTGAACCCAGGAAAACCCTCAAAGTCGGGGGCAACC Petition 870260064333, of 30 / 06 / 2026, p. 158 / 427 153 / 418 C 715 GTCAGGGTCAGGAGCCCCCCCCCTGCACCCAGGAAAACCCTCAAAGTCGGGGGGCAACC C 716 GGGTCAGGAGCCCCCCCCCTGAACCCAGGATAACCCTCAAAGTCGGGGGGC 717 TGGTGGTGGTGGT 718 GGGACAGGGCAGGGACAGGG 719 GGGTCCGGGTCTGGGTCTGGG 720 GGGCAGGGTCTGGGCTGGG 721 GGGCTGGGATGGGAAAGGG 722 GGGCTCTGGGTGGGCCGGG 723 GGGCTGGGCTGGGCAGGG 724 GGGTGGGCTGGGAAGGG 725 GGGAGGGAGGGC TAGGG 726 GGGCAGGGCTGGGAGGG 727 GGGTGGGAGGGCTGGG 728 GCGTAACCTCCATCCGAGTTGCAAGAGAGGGAAACGCAGTCTC 729 TTGACGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAAA 730 TTGACGCGGTTCTATCTACTTACGCGTTAAACCAACTAGAAA 731 CGCGAGTCTAGGGGATAACGCGTTAAACTTCCTAGAAGGCGGTT 732 CGCGGATCTAGATTGTAACGCGTTAAACCATCTAGAAGGCGGTT 733 CGCGTCGCTACCGCCCGGCGCGTTAAACACACTAGAAGGCGGTT [000362] In some embodiments, the PEgRNA comprises a self-cleaving element. In some embodiments, the self-cleaving element enhances the transcription and / or processing of the PEgRNA when transcribed from the nucleotide encoding the PEgRNA. In some embodiments, the PEgRNA comprises an RNA hairpin or quadruplex. In some embodiments, the PEgRNA comprises a self-cleaving ribozyme element, for example, a hammerhead, pistol, axe, hairpin, VS, tornado, or tornado sister ribozyme. In some embodiments, the PEgRNA comprises an HDV ribozyme. In some embodiments, the PEgRNA comprises a Csy4-recognized hairpin. In some embodiments, the PEgRNA comprises an ENE motif. In some embodiments, the PEgRNA comprises an element for nuclear expression (ENE) of MALAT1 lnc RNA. In some embodiments, the PEgRNA comprises an ENE element of Kaposi's sarcoma-associated herpesvirus (KSHV).In some embodiments, the PEgRNA comprises a 3' box of a U1 snRNA. In some embodiments, Petition 870260064333, dated 06 / 30 / 2026, p. 159 / 427 154 / 418 PEgRNA forms a circular RNA. [000363] In some embodiments, the PEgRNA comprises a secondary RNA structure or motif that enhances binding to the DNA-RNA double bond or potentiates the activity of the PEgRNA. In some embodiments, the PEgRNA comprises a sequence derived from a native nucleotide element involved in reverse transcription, for example, retroviral transcription initiation. In some embodiments, the PEgRNA comprises a sequence of, or derived from, a primer binding site of a reverse transcriptase substrate, a polypurine tract (PPT), or a kiss fold. In some embodiments, the PEgRNA comprises a dimerization motif, a kiss fold, or a tetrafold-tetrafold GNRA receptor pair that results in PEgRNA circularization.In some embodiments, the PEgRNA comprises a secondary RNA structure of a motif that results in the physical separation of the spacer and the PBS of the PEgRNA, thus preventing spacer occlusion and improving PEgRNA activity. In some embodiments, the PEgRNA comprises a secondary structure or motif, for example, a 5' or 3' extension in the spacer region that forms a hairpin or toehold, wherein the secondary structure or motif competes favorably against hybridization between the spacer and the PBS of the PEgRNA, thus preventing spacer occlusion and improving PEgRNA activity. [000364] In some embodiments, a PEgRNA additionally comprises a sequence given in Table 13. [000365] 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]-[edit template]-[PBS]-[3' motif or selected secondary structure from Tables 11-13] or [spacer]-[gRNA core]-[edit template]-[PBS]-[3' motif or selected secondary structure from Tables 11-13]. Petition 870260064333, dated 06 / 30 / 2026, page 160 / 427 155 / 418 [000366] In some embodiments, PEgRNA comprises the sequence SEQ ID NO: 737 at the 5' end and / or the sequence UGGGAGACGUCCCACC (SEQ ID NO: 738) at the 3' end. In some embodiments, PEgRNA comprises the following structure (M-MLV kiss-folding): GGGGGAGACGUCCCACC (SEQ ID NO: 737)-[spacer]-[gRNA core]-[editing template]-[PBS]UGGGAGACGUCCCACC (SEQ ID NO: 738), or GGUGGG...

Claims

1. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA, characterized in that the PEgRNA comprises: a. a spacer that is complementary to a search target sequence in a first strand of a CF transmembrane conductance regulator (CFTR) gene, wherein the spacer comprises at its 3' end the 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 a region of complementarity to an editing target sequence in a second strand of the CFTR gene; and ii. a primer binding site (PBS) comprising 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 the second strand are complementary to each other, wherein the editing template encodes or comprises a G nucleotide at position c.1624 of a wild-type CFTR coding sequence.

2. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA, characterized in that 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) comprising at its 5' end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO:

1.

3. PEgRNA, according to claim 1 or 2, characterized in that the gRNA core comprises the nucleotide sequence: GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), wherein T indicates the presence of a uridine nucleotide.

4. PEgRNA, according to any one of claims 1 to 3, characterized in that the extension arm additionally comprises a 3' motif comprising the nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

5. PEgRNA, according to claim 4, characterized in that motif 3' is directly connected to the PBS at its 3' end.

6. PEgRNA, according to claim 4, characterized in that the 3' motif is linked to the PBS at its 3' end by means of a linker.

7. PEgRNA, according to claim 6, characterized in that the ligand is 4 nucleotides long.

8. PEgRNA, according to any one of claims 1 to 7, characterized in that the edition model comprises at its 3' end the SEQ ID NO: 68, 76, 84, 91 or 97.

9. PEgRNA, according to claim 8, characterized in that the editing template has a length of 20 nucleotides or less.

10. PEgRNA, according to claim 8, characterized in that the editing template has a length of 10, 13, 17 or 20 nucleotides.

11. PEgRNA, according to any of claims 1 to 7, characterized in that the edition model consists of Petition 870250066837, dated 07 / 31 / 2025, page 18 / 44 3 / 26 in the sequence TTCTCCA.

12. PEgRNA, according to any one of claims 1 to 11, characterized in that the spacer is 17 to 22 nucleotides long.

13. PEgRNA, according to claim 12, characterized in that the spacer comprises at its 3' end the SEQ ID NO:

10.

14. PEgRNA, according to claim 12, characterized in that the spacer has the sequence SEQ ID NO:

10.

15. PEgRNA, according to any one of claims 1 to 13, characterized in that the PBS comprises at its 5' end the sequence number 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 or 61.

16. PEgRNA, according to claim 15, characterized in that the PBS comprises at its 5' end the sequence number 28, 37, 43 or 49.

17. PEgRNA, according to claim 15, characterized in that the PBS comprises at its 5' end the sequence number 19, 22, 25, 55, 58 or 61.

18. PEgRNA, according to claim 15, characterized in that the PBS has a length of 20 nucleotides or less.

19. PEgRNA, according to claim 15, characterized in that PBS has 8 to 15 nucleotides in length.

20. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the 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. Prime editing system, characterized in that it comprises: (a) PEgRNA or one or more polynucleotides, as defined in any of claims 1 to 20, Petition 870250066837, dated 07 / 31 / 2025, p. 19 / 44 4 / 26 and (b) an ngRNA, or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 473, 474, 475, 476 or 477, and (ii) an ngRNA core capable of binding to a Cas9 protein.

22. Prime editing system, according to claim 21, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO:

473.

23. Prime editing system, according to claim 21, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO:

475.

24. Prime editing system, according to claim 21, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO: 474, 476 or 477.

25. Prime editing system, according to any one of claims 21 to 24, characterized in that the ngRNA core comprises the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGT GAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), where T indicates the presence of a uridine nucleotide.

26. Prime editing system, according to claim 25, characterized in that the ngRNA comprises SEQ ID NO: 485, 486, 487, 489, 491, 493, 494, 496, 499, 500, 501, 504, Petition 870250066837, dated 07 / 31 / 2025, page 20 / 44 5 / 26 505, 506, 507 or 508.

27. Prime editing system, according to claim 25, characterized in that the ngRNA comprises the SEQ ID NO: 486, 487, 489, 491, 493, 494, 496, 500, 501, 504, 505, 506, 507 or 508.

28. Prime editing system, according to claim 25, characterized in that the ngRNA comprises the SEQ ID NO: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507 or 508.

29. Prime editing system, according to claim 25, characterized in that the ngRNA comprises the SEQ ID NO: 491, 493, 494 or 496.

30. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA, characterized in that the PEgRNA comprises: a. a spacer that is complementary to a search target sequence in 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 comprising a region of complementarity to an editing target sequence in a second strand of the CFTR gene, and ii. a primer binding site (PBS) comprising 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 the second strand are complementary to each other, wherein the editing template encodes or comprises a G nucleotide at position c.1624 of a wild-type CFTR coding sequence.Petition 870250066837, dated 07 / 31 / 2025, page 21 / 44 6 / 26.

31. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA, characterized in that 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) comprising at its 5' end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO:

2.

32. PEgRNA, according to claim 30 or 31, characterized in that the editing model comprises at its 3' end the sequence number 66.

33. PEgRNA, according to claim 32, characterized in that the editing model comprises at its 3' end the sequence number 66, 67, 69, 71, 75, 77, 78, 83, 87, 88, 89, 92, 93, 94, 96, 98 or 100.

34. PEgRNA, according to claim 32, characterized in that the editing pattern consists of sequence number 66.

35. PEgRNA, according to claim 33, characterized in that the editing template has a length of 25 nucleotides or less.

36. PEgRNA, according to claim 35, characterized in that the editing template is 11 or 12 nucleotides long.

37. PEgRNA, according to claim 30, characterized in that the editing model comprises at its 3' end the SEQ ID NO: 829, 830, 831, 832, 833, 834, 853, 854, 855, 856, 857, 858, 877, 878, 879, 880, 881, 882, 901, 902, 903, 904, Petition 870250066837, dated 07 / 31 / 2025, page. 22 / 44 7 / 26 905, 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. PEgRNA, according to claim 30 or 31, characterized in that the editing model additionally encodes a PAM silencing edit.

39. PEgRNA, according to claim 38, characterized in that the editing model encodes a TGA to GGT PAM silencing edit.

40. PEgRNA, according to claim 39, characterized in that the editing template comprises, at its 3' end, nucleotides 7 to 12 of SEQ ID NO:

72.

41. PEgRNA, according to claim 40, characterized in that the edition model comprises, at its 3' end, the SEQ ID NO: 72, 80 or 85.

42. PEgRNA, according to claim 30 or 31, characterized in that the editing model encodes a TGA to GGG PAM silencing edit.

43. PEgRNA, according to claim 42, characterized in that the editing template comprises, at its 3' end, nucleotides 7 to 12 of SEQ ID NO:

73.

44. PEgRNA, according to claim 43, characterized in that the edition model comprises, at its 3' end, the SEQ ID NO: 73, 81 or 86.

45. PEgRNA, according to claim 30 or 31, characterized in that the editing model encodes a TGA to GGC PAM silencing edit.

46. ​​PEgRNA, according to claim 45, characterized in that the editing template comprises, at its 3' end, nucleotides 7 to 12 of SEQ ID NO:

74.

47. PEgRNA, according to claim 46, characterized in that the edition model comprises, at its 3' end, the SEQ ID NO: 74 or 82.

48. PEgRNA, according to claim 30, characterized in Petition 870250066837, dated 07 / 31 / 2025, page 23 / 44 8 / 26, by the fact that the editing model encodes a GGA to GGC PAM silencing edit.

49. PEgRNA, according to claim 48, characterized in that the edition model comprises at its 3' end the SEQ ID NO: 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. PEgRNA, according to claim 30, characterized in that the editing model encodes a GGA to GGG PAM silencing.

51. PEgRNA, according to claim 50, characterized in that the edition model comprises at its 3' end the SEQ ID NO: 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. PEgRNA, according to claim 30, characterized in that the editing model encodes a GGA to GGT PAM silencing.

53. PEgRNA, according to claim 52, characterized in that the edition model comprises at its 3' end the SEQ ID NO: 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. PEgRNA, according to any one of claims 37 to 53, characterized in that the editing template has a length of 16 nucleotides or less.

55. PEgRNA, according to claim 54, characterized by the fact that the editing template is 12 to 16 nucleotides long.

56. PEgRNA, according to any one of claims 30 to 55, characterized in that the gRNA core comprises the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGT GAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), where T indicates the presence of a uridine nucleotide.

57. PEgRNA, according to any one of claims 30 to 56, characterized in that the extension arm additionally comprises a 3' motif comprising the nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

58. PEgRNA, according to claim 57, characterized in that the 3' motif is directly connected to the PBS at its 3' end.

59. PEgRNA, according to claim 57, characterized in that the 3' motif is linked to PBS at its 3' end by means of a linker.

60. PEgRNA, according to claim 59, characterized in that the linker is 4 nucleotides long.

61. PEgRNA, according to any one of claims 30 to 60, characterized in that the spacer is 17 to 22 nucleotides long. Petition 870250066837, dated 07 / 31 / 2025, page 25 / 44 10 / 26 62. PEgRNA, according to claim 61, characterized in that the spacer comprises at its 3' end the SEQ ID NO:

11.

63. PEgRNA, according to claim 62, characterized in that the spacer has the sequence SEQ ID NO:

11.

64. PEgRNA, according to any one of claims 30 to 63, characterized in that the PBS is 8 to 15 nucleotides long.

65. PEgRNA, according to any one of claims 32 to 36, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the 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, Petition 870250066837, dated 07 / 31 / 2025, p. 26 / 44 11 / 26 453, 454, 455, 456, 458, 460, 461, 462, 463, 464, 465, 466, 468, 469, 470, 471, 472, 1210 ', 12 11, 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, 66. PEgRNA, 825, 826, according to 828 and 1017. any of claims 39 to 41 or 54 or 55, characterized in that the PEgRNA comprises a sequence selected from the group consisting of SEQ ID Nos: 126, 137, 156, 168, 244 and 1184.

67. PEgRNA, according to any one of claims 42 to 44 or 54 or 55, characterized in that the PEgRNA comprises a sequence selected from the group consisting of SEQ ID Nos: 127, 138, 157, 169, 245 and 1178.

68. PEgRNA, according to any one of claims 45 to 47, characterized in that the PEgRNA comprises a sequence selected from the group consisting of SEQ ID Nos: 128, 139, 158, 170 and 1172.

69. PEgRNA, according to claim 37, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the 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. PEgRNA, according to claim 48 or 49, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the 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. Petition 870250066837, dated 07 / 31 / 2025, p. 27 / 44 12 / 26 71. PEgRNA, according to claim 50 or 51, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the 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. PEgRNA, according to claim 52 or 53, the PEgRNA characterized in that it comprises a sequence selected from the group consisting of the 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. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the following SEQ ID numbers: 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, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, Petition 870250066837, dated 07 / 31 / 2025, p. 28 / 44 13 / 26 276, 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. Prime editing system, characterized in that it comprises: (a) PEgRNA or one or more polynucleotides, as defined in any one of claims 30 to 73, and (b) an ngRNA, or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 473, 474, 476 or 477; and (ii) an ngRNA core capable of binding to a Cas9 protein.

75. Prime editing system, according to claim 74, characterized in that the ngRNA spacer comprises SEQ ID NO:

473.

76. Prime editing system, according to claim 74, characterized in that the ngRNA comprises the SEQ ID NO: 491, 493, 494 or 496.

77. Prime editing system, according to claim 74, characterized in that the ngRNA comprises the SEQ ID Petition 870250066837, dated 07 / 31 / 2025, page 29 / 44 14 / 26 NO:

496.

78. Prime editing system, characterized in that it comprises (a) PEgRNA or one or more polynucleotides, as defined in any one of claims 32 to 36 or 65, and (b) an ngRNA, or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4 to 20 of SEQ ID NO: 475; and (ii) an ngRNA core capable of binding to a Cas9 protein.

79. Prime editing system, according to claim 78, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO:

475.

80. Prime editing system, according to claim 7 8 or 7 9, characterized in that the ngRNA comprises the SEQ ID NO: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507 or 508.

81. Prime editing system, characterized in that it comprises (a) PEgRNA or one or more polynucleotides, as defined in any one of claims 39 to 41, 54 or 55 or 66, and (b) an ngRNA, or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4 to 20 of SEQ ID NO: 478; and (ii) an ngRNA core capable of binding to a Cas9 protein.

82. Prime editing system, according to claim 81, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO:

478.

83. Prime editing system, according to claim 81 or 82, characterized in that the ngRNA comprises SEQ ID NO:

502.

84. Prime editing system, characterized in that Petition 870250066837, dated 07 / 31 / 2025, page 30 / 44 15 / 26 comprises (a) PEgRNA or one or more polynucleotides, as defined in any of claims 42 to 44 or 54 or 55 or 67, and (b) an ngRNA, or one or more polynucleotides encoding (m) the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 479; and (ii) an ngRNA core capable of binding to a Cas9 protein.

85. Prime editing system, according to claim 84, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO:

479.

86. Prime editing system, according to claim 84 or 85, characterized in that the ngRNA comprises SEQ ID NO:

503.

87. Prime editing system, according to any one of claims 74 or 75, 78 or 79, 81 or 82 or 84 or 85, characterized in that the ngRNA core comprises the nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGCGG CACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT GGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGT GAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO: 595), where T indicates the presence of a uridine nucleotide.

88. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA, characterized in that the PEgRNA comprises: Petition 870250066837, dated 07 / 31 / 2025, page 31 / 44 16 / 26 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 the 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 a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii. a primer binding site (PBS) comprising 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 the second strand are complementary to each other, wherein the editing template encodes or comprises a G nucleotide at the c position.1624 of a wild CFTR encoding sequence.

89. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA, characterized in that the PEgRNA comprises: a. a spacer comprising at its 3' end the 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 the sequence number 65, and ii. a primer binding site (PBS) comprising at its 5' end a sequence that is a reverse complement of nucleotides 10-14 of the SEQ ID NO:

3.

90. PEgRNA, according to claim 88 or 89, characterized in that the gRNA core comprises the nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), where T indicates the presence of a uridine nucleotide.

91. PEgRNA, according to any one of claims 88 to 90, characterized in that the extension arm additionally comprises a 3' motif comprising the nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.

92. PEgRNA, according to claim 91, characterized in that motif 3' is directly connected to the PBS at its 3' end.

93. PEgRNA, according to claim 91, characterized in that the 3' motif is linked to PBS at its 3' end by means of a linker.

94. PEgRNA, according to claim 93, characterized in that the linker is 4 nucleotides long.

95. PEgRNA, according to any one of claims 88 to 94, characterized in that the edition model comprises at its 3' end the SEQ ID NO: 70, 79, 90, 95 or 99.

96. PEgRNA, according to claim 95, characterized in that the editing template has a length of 24 nucleotides or less.

97. PEgRNA, according to claim 95, characterized in that the editing template has a length of 10, 14, 18, 21 or 24 nucleotides.

98. PEgRNA, according to claim 88 or 89, characterized in that the edition pattern consists of sequence number 65.

99. PEgRNA, according to any one of claims 88 to 98, characterized in that the spacer has 17 to 22 nucleotides in length. Petition 870250066837, dated 07 / 31 / 2025, page 33 / 44 18 / 26 100. PEgRNA, according to claim 99, characterized in that the spacer comprises at its 3' end the SEQ ID NO:

12.

101. PEgRNA, according to claim 100, characterized in that the spacer has the sequence SEQ ID NO:

12.

102. PEgRNA, according to any one of claims 88 to 101, characterized in that the PBS comprises at its 5' end the sequence number 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 or 63.

103. PEgRNA, according to claim 102, characterized in that the PBS comprises at its 5' end the sequence number 27, 54, 57, 60 or 63.

104. PEgRNA, according to claim 102, characterized in that the PBS comprises at its 5' end the sequence number 27, 33, 39, 45 or 51.

105. PEgRNA, according to any one of claims 77 to 101, characterized in that the PBS consists of SEQ ID NO:

27.

106. PEgRNA, according to any one of claims 102 to 105, characterized in that the PBS has a length of 15 nucleotides or less.

107. PEgRNA, according to claim 106, characterized in that the PBS is 7 to 15 nucleotides long.

108. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding PEgRNA, characterized in that the PEgRNA comprises a sequence selected from the group consisting of the following SEQ IDs: 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. Prime editing system, characterized in that it comprises: (a) PEgRNA or one or more polynucleotides, as defined in any of the claims 88 to Petition 870250066837, dated 07 / 31 / 2025, p. 34 / 44 19 / 26 108, and (b) an ngRNA or one or more polynucleotides encoding ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3' end nucleotides 4-20 of SEQ ID NO: 480, 11, 481, 482, 483 or 484, and (ii) an ngRNA core capable of binding to a Cas9 protein.

110. Prime editing system, according to claim 10 9, characterized in that the ngRNA spacer comprises at its 3' end the SEQ ID NO: 480, 11, 481, 482, 483 or 484.

111. Prime editing system, according to claim 109 or 110, characterized in that the gRNA core comprises the nucleotide sequence: GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACGC GGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.

112. Prime editing system, according to claim 111, characterized in that the ngRNA comprises SEQ ID NO: 488, 490, 492, 495, 497 or 498.

113. PEgRNA, according to any one of claims 1 to 20, 31 to 73 or 88 to 107, characterized in that it comprises 5' to 3', the spacer, the gRNA core, the RTT and the PBS.

114. PEgRNA, according to claim 113, characterized in that the spacer, the gRNA core, the RTT and the PBS form a contiguous sequence in a single molecule.

115. PEgRNA, according to claim 113 or 114, characterized in that it further comprises mN*mN*mN*N modifications at 3' and mN*mN*mN* at 5', where m indicates that the nucleotide contains a modification with 2'-O-Me and a * indicates the presence of a phosphorothioate linkage.

116. PEgRNA, according to claim 115, characterized in Petition 870250066837, dated 07 / 31 / 2025, page 35 / 44 20 / 26, in that the PEgRNA comprises mT*mT*mT*T modifications at 3' and mN*mN*mN* at 5', wherein m indicates that the nucleotide contains a modification with 2'-O-Me, a * indicates the presence of a phosphorothioate linkage, and a T indicates the presence of an additional uridine nucleotide.

117. Prime editing system, according to any one of claims 21 to 30, 74 to 87 or 109 to 112, characterized in that the PEgRNA and / or ngRNA additionally comprises mN*mN*mN*N modifications at 3' and mN*mN*mN* at 5', wherein m indicates that the nucleotide contains a modification with 2'-O-Me and a * indicates the presence of a phosphorothioate linkage.

118. Prime editing system, according to claim 117, characterized in that the PEgRNA and / or ngRNA comprises mT*mT*mT*T modifications at 3' and mN*mN*mN* at 5', where m indicates that the nucleotide contains a 2'O-Me modification, a * indicates the presence of a phosphorothioate linkage, and a T indicates the presence of an additional uridine nucleotide.

119. Prime editing system, characterized in that it comprises (a) PEgRNA, as defined in any one of claims 1 to 20, 31 to 73, 88 to 108 or 113 to 116, or one or more polynucleotides encoding(s) PEgRNA, and (b) a prime editor comprising a Cas9 nickase with a nuclease-inactivating mutation in the HNH domain, or one or more polynucleotides encoding(s) the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding(s) the reverse transcriptase.

120. Prime editing system, according to any one of claims 21 to 29, 74 to 87, 109 to 112 or 117 or 118, characterized in that it 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.

121. Prime editing system, according to claim 119 or 120, characterized in that the prime editor is a fusion protein.

122. Prime editing system, characterized in that it comprises (a) PEgRNA, as defined in any one of claims 1 to 20, 30 to 73, 88 to 108 or 113 to 116, or one or more polynucleotides encoding(s) 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-terminal and C-terminal exteins (Nintein and C-Intein) are capable of autoexcision 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. Prime editing system, according to any one of claims 21 to 29, 74 to 87, 109 to 112 or 117 or 118, characterized in that it 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-terminal and C-terminal extein nintein and C-tein are capable of autoexcision 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 Petition 870250066837, dated 07 / 31 / 2025, page 37 / 44 22 / 26 a Cas9 nickase having a nuclease-inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.

124. Population of viral particles, characterized in that it collectively comprises one or more polynucleotides encoding the prime editing system, as defined in any one of claims 119 to 123.

125. Population of viral particles, according to claim 124, characterized in that the viral particles are AAV particles. 12 6. LNP, characterized in that it comprises the prime editing system, as defined in any one of claims 119 to 123.

127. LNP, according to claim 126, characterized in that it comprises PEgRNA, the polynucleotide encoding Cas9 nickase and the polynucleotide encoding reverse transcriptase.

128. LNP, according to claim 127, characterized in that the polynucleotide encoding Cas9 nickase and the polynucleotide encoding reverse transcriptase are mRNA.

129. LNP, according to claim 127 or 128, characterized in that the polynucleotide encoding Cas9 nickase and the polynucleotide encoding reverse transcriptase are the same molecule.

130. A method for editing a CFTR gene, characterized in that it comprises contacting the CFTR gene with: (a) PEgRNA, as defined in any one of claims 1 to 20, 30 to 73, 88 to 108 or 113 to 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, as defined in any one of claims 109 to 112.

131. Method according to claim 130, characterized in that the CFTR gene is in a cell. Petition 870250066837, dated 07 / 31 / 2025, pp. 38 / 44 23 / 26 132. Method according to claim 131, characterized in that the cell is a mammalian cell.

133. Method according to claim 131, characterized in that the cell is a human cell.

134. Method, according to any one of claims 131 to 133, characterized in that the cell is a primary cell.

135. Method, according to any one of claims 131 to 133, characterized in that the cell is an epithelial cell.

136. A method according to any one of claims 131 to 135, characterized in that the cell is in an individual or was obtained from an individual or from a cell bank.

137. Method according to claim 136, characterized in that the individual is a human.

138. Method according to any one of claims 131 to 137, characterized in that CFTR gene contact comprises cell contact with (i) the viral particle population as defined in claim 124 or 125, or (ii) the LNP as defined in any one of claims 126 to 129.

139. A method for treating cystic fibrosis in an individual in need thereof, the method characterized in that it comprises administering to the individual (i) PEgRNA, as defined in any one of claims 1 to 20, 31 to 73, 88 to 108, or 113 to 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, as defined in any one of claims 119 to 123, (iii) the viral particle population, as defined in claim 124 or 125, or (iv) the LNP as defined in any one of claims 126 to 129. Petition 870250066837, dated July 31, 2025, pp. 39 / 44 24 / 26 140. Prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding(s) the PEgRNA, characterized in that the PEgRNA comprises: a. a spacer comprising at its 3' end a PEgRNA spacer sequence selected from any of Tables 18 to 20; 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 an RTT sequence selected from the same Table as the PEgRNA spacer sequence, and ii. a primer binding site (PBS) comprising at its 5' end a PBS sequence selected from the same Table as the PEgRNA spacer sequence.

141. PEgRNA, according to claim 140, characterized in that the PEgRNA spacer is 17 to 22 nucleotides long.

142. PEgRNA, according to claim 141, characterized in that the PEgRNA spacer is 20 nucleotides long.

143. PEgRNA, according to any one of claims 140 to 142, characterized in that it comprises, from 5' to 3', the spacer, the gRNA core, the editing template and the PBS.

144. PEgRNA, according to claim 143, characterized in that the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule.

145. PEgRNA, according to any one of claims 140 to 144, characterized in that the gRNA core comprises a gRNA core sequence selected from Table 10.

146. Prime editing system, characterized in that Petition 870250066837, dated 07 / 31 / 2025, pp. 40 / 44 25 / 26 comprises: (a) the prime editing guide RNA (PEgRNA), as defined in any of claims 140 to 145, or one or more polynucleotides encoding(s) the PEgRNA; and, optionally, (b) a cutting guide RNA (ngRNA), or one or more polynucleotides encoding(s) the ngRNA, wherein the ngRNA comprises a spacer comprising, at its 3' end, nucleotides 4 to 20 of any ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence, and an ngRNA core capable of binding to a Cas9 protein.

147. Prime editing system, according to claim 146, characterized in that the ngRNA spacer is 17 to 22 nucleotides long.

148. Prime editing system, according to claim 147, characterized in that the ngRNA spacer comprises, at its 3' end, nucleotides 3 to 20, 2 to 20 or 1 to 20 of the ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence.

149. Prime editing system, according to any one of claims 146 to 148, characterized in that the ngRNA spacer is 20 nucleotides long.

150. Prime editing system, according to any one of claims 146 to 148, characterized in that the ngRNA core comprises a gRNA core sequence selected from Table 10.

151. Prime editing system, according to any one of claims 146 to 150, characterized in that it 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.

152. Prime editing system, according to any one of claims 146 to 150, characterized in that Petition 870250066837, dated 07 / 31 / 2025, page 41 / 44 26 / 26 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 C-intein of the N-terminal and C-terminal exteins are capable of autoexcision 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.

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