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WO2024163679A1 - Compositions d'édition de génome et méthodes de traitement de la fibrose kystique - Google Patents

Compositions d'édition de génome et méthodes de traitement de la fibrose kystique Download PDF

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WO2024163679A1
WO2024163679A1 PCT/US2024/013877 US2024013877W WO2024163679A1 WO 2024163679 A1 WO2024163679 A1 WO 2024163679A1 US 2024013877 W US2024013877 W US 2024013877W WO 2024163679 A1 WO2024163679 A1 WO 2024163679A1
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Prior art keywords
pegrna
seq
nucleotides
sequence
prime
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Linghe XI
John Benjamin STILLER
Dewi HARJANTO
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Prime Medicine Inc
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Prime Medicine Inc
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Priority to IL322419A priority Critical patent/IL322419A/en
Priority to EP24750984.7A priority patent/EP4658322A1/fr
Priority to AU2024215960A priority patent/AU2024215960A1/en
Publication of WO2024163679A1 publication Critical patent/WO2024163679A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3521—Methyl
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • CF cystic fibrosis transmembrane conductance regulator
  • the CFTR protein encoded by the CFTR gene is a chloride channel, which is involved in fluid transport and surface hydration across epithelial cells of the body’s tubular organs (e.g., lungs and intestines). Mutations in the CFTR gene Mutations in the CFTR gene results in defective biosynthesis, trafficking, and/or activity of the CFTR protein, and may cause severe damage to the lungs, pancreas, liver, intestines, sinuses, and limited ability to breathe overtime.
  • CFTR/ABCC7 cystic fibrosis transmembrane conductance regulator
  • This disclosure provides prime editing methods and compositions for correcting mutations in the CFTR gene associated with Cystic fibrosis.
  • a target gene for example, a CFTR gene.
  • the target CTFR gene may comprise double stranded DNA.
  • the target gene e.g., a CFTR gene, is edited by prime editing.
  • the prime editing described herein results in efficient correction of one or more pathogenic mutations in the CFTR gene, thereby treating cystic fibrosis in a subject.
  • the prime editing process may search specific targets and edit endogenous sequences in a target gene, e.g., the CFTR gene.
  • the spacer sequence of a PEgRNA recognizes and anneals with a search target sequence in a target strand of the target gene.
  • a prime editing complex may generate a nick in the target gene on the edit strand which is the complementary strand of the target strand.
  • the prime editing complex may then use a free 3’ end formed at the nick site of the edit strand to initiate DNA synthesis, where a primer binding site (PBS) of the PEgRNA complexes with the free 3’ end, and a single stranded DNA is synthesized using an editing template of the PEgRNA as a template.
  • the editing template may WSGR Docket No.59761-775.601 comprise one or more nucleotide edits compared to the endogenous target CFTR gene sequence. Accordingly, the newly-synthesized single stranded DNA also comprises the nucleotide edit(s) encoded by the editing template.
  • a prime editing guide RNA or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; and c.
  • an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.
  • PBS primer binding site
  • a spacer comprising at its 3’ end SEQ ID NO: 1; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template comprising at its 3’ end sequence number 64, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 1.
  • PBS primer binding site
  • the gRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), wherein T indicates the presence of a uridine nucleotide.
  • the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.
  • the 3’ motif is directly connected to the PBS at its 3’ end.
  • the 3’ motif is linked to the PBS at its 3’ end via a linker.
  • the linker is 4 nucleotides in length.
  • the editing template comprises at its 3’end SEQ ID NOs: 68, 76, 84, 91, or 97.
  • the editing template has a length of 20 nucleotides or less.
  • the editing template has a length of 10, 13, 17, or 20 nucleotides.
  • the editing template consists of SEQ ID NO: 64.
  • the spacer is from 17-22 nucleotides in length. [0018] In some embodiments, the spacer comprises at its 3’ end SEQ ID NO: 10. [0019] In some embodiments, the spacer has the sequence of SEQ ID NO: 10. [0020] In some embodiments, the PBS comprises at its 5’ end sequence number 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, or 61. [0021] In some embodiments, the PBS comprises at its 5’ end sequence number 28, 37, 43, or 49.
  • the PBS comprises at its 5’ end sequence number 19, 22, 25, 55, 58, or 61. [0023] In some embodiments, the PBS has a length of 20 nucleotides or less. [0024] In some embodiments, the PBS is 8-15 nucleotides in length.
  • PEgRNA prime editing guide RNA
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 306, 309, 310, 314, 317, 318, 322, 328, 335, 336, 345, 353, 363, 364, 371, 382, 390, 399, 400, 410, 425, 426, 443, and 457.
  • a prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of the aspects and embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: an ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 473, 474, 475, 476, or 477, and an ngRNA core capable of binding a Cas9 protein. [0027] In some embodiments, the ngRNA spacer comprises at its 3’ end SEQ ID NO: 473.
  • the ngRNA spacer comprises at its 3’ end SEQ ID NO: 475. [0029] In some embodiments, the ngRNA spacer comprises at its 3’ end SEQ ID NOs: 474, 476, or 477.
  • the ngRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGC GTGAAAACGCGGCACCGAGTCGGTGC (SEQ ID NO
  • the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 491, 493, 494, 496, 499, 500, 501, 504, 505, 506, 507, or 508. [0032] In some embodiments, the ngRNA comprise SEQ ID NOs: 486, 487, 489, 491, 493, 494, 496, 500, 501, 504, 505, 506, 507, or 508. [0033] In some embodiments, the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507, or 508.
  • the ngRNA comprises SEQ ID NOs: 491, 493, 494, or 496.
  • a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 2; b. a gRNA core capable of binding to a Cas9 protein; and c.
  • CFTR CF transmembrane conductance regulator
  • an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 2, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.
  • PBS primer binding site
  • a spacer comprising at its 3’ end SEQ ID NO: 2; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template comprising at its 3’ end nucleotides 4-8 of sequence number 66, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 2.
  • PBS primer binding site
  • the editing template comprises at its 3’ end sequence number 66.
  • the editing template comprises at its 3’ end sequence number 66, 67, 69, 71, 75, 77, 78, 83, 87, 88, 89, 92, 93, 94, 96, 98, or 100.
  • the editing template consists of sequence number 66.
  • the editing template has a length of 25 nucleotides or less.
  • the editing template is 11 or 12 nucleotides in length.
  • the editing template comprises at its 3’ end SEQ ID NOs: 829, 830, 831, 832, 833, 834, 853, 854, 855, 856, 857, 858, 877, 878, 879, 880, 881, 882, 901, 902, 903, 904, 905, 906, 925, 926, 927, 928, 929, 930, 949, 950, 951, 952, 953, 954, 973, 974, 975, 976, 977, 993, 994, 995, 996, 997, or 998.
  • the editing template further encodes a PAM silencing edit.
  • the editing template encodes a TGA-to-GGT PAM silencing edit.
  • the editing template comprises at its 3’ end nucleotides 7-12 of SEQ ID NO: 72.
  • the editing template comprises at its 3’ end SEQ ID NOs: 72, 80 or 85.
  • the editing template encodes a TGA-to-GGG PAM silencing edit.
  • the editing template comprises at its 3’ end nucleotides 7-12 of SEQ ID NO: 73.
  • the editing template comprises at its 3’ end SEQ ID NOs: 73, 81 or 86. [0050] In some embodiments, the editing template encodes a TGA-to-GGC PAM silencing edit. [0051] In some embodiments, the editing template comprises at its 3’ end nucleotides 7-12 of SEQ ID NO: 74. [0052] In some embodiments, the editing template comprises at its 3’ end SEQ ID NO: 74 or 82. [0053] In some embodiments, the editing template encodes a GGA-to-GGC PAM silencing.
  • the editing template comprises at its 3’ end SEQ ID NOs: 835, 836, 837, 838, 839, 840, 859, 860, 861, 862, 863, 864, 883, 884, 885, 886, 887, 888, 907, 908, 909, 910, 911, 912, 931, 932, 933, 934, 935, 936, 955, 956, 957, 958, 959, 960, 978, 979, 980, 981, 982, 999, 1000, 1001, 1002, 1003, or 1004.
  • the editing template encodes a GGA-to-GGG PAM silencing.
  • the editing template comprises at its 3’ end SEQ ID NOs: 841, 842, 843, 844, 845, 846, 865, 866, 867, 868, 869, 870, 889, 890, 891, 892, 893, 894, 913, 914, 915, 916, 917, 918, 937, 938, 939, 940, 941, 942, 961, 962, 963, 964, 965, 966, 983, 984, 985, 986, 987, 1005, 1006, 1007, 1008, 1009, or 1010. [0057] In some embodiments, the editing template encodes a GGA-to-GGT PAM silencing.
  • the editing template comprises at its 3’ end SEQ ID NOs: 847, 848, 849, 850, 851, 852, 871, 872, 873, 874, 875, 876, 895, 896, 897, 898, 899, 900, 919, 920, 921, 922, 923, 924, 943, 944, 945, 946, 947, 948, 967, 968, 969, 970, 971, 972, 988, 989, 990, 991, 992, 1011, 1012, 1013, 1014, 1015, or 1016. [0059] In some embodiments, the editing template has a length of 16 nucleotides or less.
  • the editing template is 12-16 nucleotides in length.
  • the gRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), WSGR Docket No.59761-775.601 GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTT
  • the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.
  • the 3’ motif is directly connected to the PBS at its 3’ end.
  • the 3’ motif is linked to the PBS at its 3’ end via a linker.
  • the linker is 4 nucleotides in length.
  • the spacer is from 17-22 nucleotides in length.
  • the spacer comprises at its 3’ end SEQ ID NO: 11. [0068] In some embodiments, the spacer has the sequence of SEQ ID NO: 11. [0069] In some embodiments, the PBS is 8-15 nucleotides in length.
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 129, 130, 131, 132, 133, 134, 135, 136, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 159, 160, 161, 162, 163, 164, 165, 166, 167, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 126, 137, 156, 168, 244, and 1184. [0072] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 127, 138, 157, 169, 245, and 1178. [0073] In some embodiments, the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 128, 139, 158, 170, and 1172.
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1018, 1019, 1020, 1021, 1022, 1023, 1042, 1043, 1044, 1045, 1046, 1047, 1066, 1067, 1068, 1069, 1070, 1071, 1090, 1091, 1092, 1093, 1094, 1095, 1114, 1115, 1116, 1117, 1118, 1119, 1138, 1139, 1140, 1141, 1142, 1143, 1162, 1163, 1164, 1165, 1166, 1185, 1186, 1187, 1188, 1189, or 1190.
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1024, 1025, 1026, 1027, 1028, 1029, 1048, 1049, 1050, 1051, 1052, 1053, 1072, 1073, 1074, 1075, 1076, 1077, 1096, 1097, 1098, 1099, 1100, 1101, 1120, 1121, 1122, 1123, 1124, 1125, 1144, 1145, 1146, 1147, 1148, 1149, 1167, 1168, 1169, 1170, 1171, 1191, 1192, 1193, 1194, 1195, and 1196.
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1030, 1031, 1032, 1033, 1034, 1035, 1054, 1055, 1056, 1057, 1058, 1059, 1078, 1079, 1080, 1081, 1082, 1083, 1102, 1103, 1104, 1105, 1106, 1107, 1126, 1127, 1128, 1129, 1130, 1131, 1150, 1151, 1152, 1153, 1154, 1155, 1173, 1174, 1175, 1176, 1177, 1197, 1198, 1199, 1200, 1201, and 1202.
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 1036, 1037, 1038, 1039, 1040, 1041, 1060, 1061, 1062, 1063, 1064, 1065, 1084, 1085, 1086, 1087, 1088, 1089, 1108, 1109, 1110, 1111, 1112, 1113, 1132, 1133, 1134, 1135, 1136, 1137, 1156, 1157, 1158, 1159, 1160, 1161, 1179, 1180, 1181, 1182, 1183, 1203, 1204, 1205, 1206, 1207, and 1208.
  • PEgRNA prime editing guide RNA
  • 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,
  • SEQ ID NOs 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
  • a prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of the aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 473, 474, 476 or 477; and (ii) an ngRNA core capable of binding a Cas9 protein. [0080] In some embodiments, the ngRNA spacer comprises SEQ ID NO: 473.
  • the ngRNA comprises SEQ ID NOs: 491, 493, 494, or 496. [0082] In some embodiments, the ngRNA comprises SEQ ID NO: 496. [0083] In one aspect, provided herein is a prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of the aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 475; and (ii) an ngRNA core capable of binding a Cas9 protein.
  • the ngRNA spacer comprises at its 3’end SEQ ID NO: 475.
  • the ngRNA comprises SEQ ID NOs: 485, 486, 487, 489, 499, 500, 501, 504, 505, 506, 507, or 508.
  • a prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 478; and an ngRNA core capable of binding a Cas9 protein. [0087] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NO: 478. [0088] In some embodiments, the ngRNA comprises SEQ ID NO: 502.
  • a prime editing system comprising (a) the PEgRNA or the one or more polynucleotides of any one of aspects or embodiments herein, and (b) a ngRNA, or one WSGR Docket No.59761-775.601 or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: a ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NO: 479; and an ngRNA core capable of binding a Cas9 protein. [0090] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NO: 479.
  • the ngRNA comprises SEQ ID NO: 503.
  • the ngRNA core comprises nucleotide sequence GTTTAAGAGCTAGAAATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAACG CGGCACCGAGTCGGTGC (SEQ ID NO: 592), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAA AGTGGCACCGAGTCGGTGC (SEQ ID NO: 603), GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 594), or GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAAC TTGAAAA
  • PEgRNA prime editing guide RNA
  • the PEgRNA comprises: a. a spacer that is complementary to a search target sequence on a first strand of a CF transmembrane conductance regulator (CFTR) gene wherein the spacer comprises at its 3’ end SEQ ID NO: 3; b. a gRNA core capable of binding to a Cas9 protein; and c.
  • CFTR CF transmembrane conductance regulator
  • an extension arm comprising: i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the CFTR gene, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3, wherein the first strand and second strand are complementary to each other, wherein the editing template encodes or comprises a nucleotide G at position c.1624 of a wildtype CFTR coding sequence.
  • PBS primer binding site
  • a spacer comprising at its 3’ end SEQ ID NO: 3; b. a gRNA core capable of binding to a Cas9 protein; and c. an extension arm comprising: i) an editing template comprising at its end sequence number 65, and ii) a primer binding site (PBS) that comprises at its 5’ end a sequence that is a reverse complement of nucleotides 10-14 of SEQ ID NO: 3.
  • PBS primer binding site
  • the gRNA core comprises nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA WSGR Docket No.59761-775.601 CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.
  • the extension arm further comprises a 3’ motif comprising nucleotide sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAA (SEQ ID NO: 607), wherein T indicates the presence of a uridine nucleotide.
  • the 3’ motif is directly connected to the PBS at its 3’ end. [0098] In some embodiments, the 3’ motif is linked to the PBS at its 3’ end via a linker. [0099] In some embodiments, the linker is 4 nucleotides in length. [0100] In some embodiments, the editing template comprises at its 3’end SEQ ID NOs: 70, 79, 90, 95, or 99. [0101] In some embodiments, the editing template has a length of 24 nucleotides or less. [0102] In some embodiments, the editing template is a length of 10, 14, 18, 21, or 24 nucleotides. [0103] In some embodiments, the editing template consists of sequence number 65.
  • the spacer is from 17-22 nucleotides in length. [0105] In some embodiments, the spacer comprises at its 3’ end SEQ ID NO: 12. [0106] In some embodiments, the spacer has the sequence of SEQ ID NO: 12. [0107] In some embodiments, the PBS comprises at its 5’ end sequence number 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, or 63. [0108] In some embodiments, the PBS comprises at its 5’ end sequence number 27, 54, 57, 60, or 63. [0109] In some embodiments, the PBS comprises at its 5’ end sequence number 27, 33, 39, 45, or 51.
  • the PBS consists of SEQ ID NO: 27. [0111] In some embodiments, the PBS has a length of 15 nucleotides or less. [0112] In some embodiments, the PBS is 7-15 nucleotides in length.
  • PEgRNA prime editing guide RNA
  • the PEgRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 307, 308, 312, 313, 316, 320, 324, 325, 330, 338, 339, 355, 356, 357, 374, 375, 384, 392, 393, 403, 412, 413, 419, 430, 431, 437, 445, 452, 459, and 467.
  • a prime editing system comprising: (a) the PEgRNA or the one or more polynucleotides of any one of aspects or embodiments herein, and (b) a ngRNA, or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises: (i) an ngRNA spacer comprising at its 3’ end nucleotides 4-20 of SEQ ID NOs: 480, 11, 481, 482, 483, or 484, and (ii) an ngRNA core capable of binding a Cas9 protein. [0115] In some embodiments, the ngRNA spacer comprises at its 3’end SEQ ID NOs: 480, 11, 481, 482, 483, or 484.
  • the gRNA core comprises nucleotide sequence GTTTAAGAGCGGGGAAATCCGCAAGTTTAAATAAGGCTAGTCCGTTATCAGCGTGAAAA WSGR Docket No.59761-775.601 CGCGGCACCGAGTCGGTGC (SEQ ID NO: 593), wherein T indicates the presence of a uridine nucleotide.
  • the ngRNA comprises SEQ ID NOs: 488, 490, 492, 495, 497, or 498.
  • the PEgRNA comprises from 5’ to 3’, the spacer, the gRNA core, the RTT, and the PBS.
  • the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule.
  • the PEgRNA further comprises 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • the PEgRNA comprises s 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.
  • the PEgRNA and/or the ngRNA further comprises 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • the PEgRNA and/or the ngRNA comprises s 3’ mT*mT*mT*T and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification, a * indicates the presence of a phosphorothioate bond, and a T indicates the presence of an additional uridine nucleotide.
  • a prime editing system comprising (a) the PEgRNA of any one of the aspects or embodiments herein, or one or more polynucleotides encoding the PEgRNA, and (b) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.
  • the prime editing system further comprises a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or one or more polynucleotides encoding the Cas9 nickase, and a reverse transcriptase, or one or more polynucleotides encoding the reverse transcriptase.
  • the prime editor is a fusion protein.
  • a prime editing system comprising (a) the PEgRNA of any one of the aspects or embodiments herein or one or more polynucleotides encoding the PEgRNA, (b) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N- intein or a polynucleotide encoding the N-terminal extein; and (c) a C-terminal extein comprising a C- terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the WSGR Docket No.59761-775.601 C-terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises
  • the prime editing system further comprises: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (d) a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C- terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase (RT) domain.
  • RT reverse transcriptase
  • a population of viral particles collectively comprising the one or more polynucleotides encoding the prime editing system of any one of the aspects or embodiments herein.
  • the viral particles are AAV particles.
  • an LNP comprising the prime editing system of any one of the aspects or embodiments herein.
  • the polynucleotide encoding the Cas9 nickase and the polynucleotide encoding the reverse transcriptase are mRNA.
  • the polynucleotide encoding the Cas9 nickase and the polynucleotide d encoding the reverse transcriptase are the same molecule.
  • a method of editing a CFTR gene comprising contacting the CFTR gene with: (a) the PEgRNA of any one of the aspects or embodiments herein, and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase or (b) the prime editing system of any one of the aspects or embodiments herein.
  • the CFTR gene is in a cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a primary cell.
  • the cell is an epithelial cell.
  • the cell is in a subject or obtained from a subject or a cell bank.
  • the subject is a human.
  • contacting the CFTR gene comprises contacting the cell with (i) the population of viral particles of claim 113 or 114 or (ii) the LNP of any one of the aspects or embodiments herein.
  • a method of treating cystic fibrosis in a subject in need thereof comprising administering to the subject (i) the PEgRNA of any one of the aspects or embodiments herein, , and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (ii) the prime editing system of any one of the aspects or embodiments herein, (iii) the population of viral particles of any one of the aspects or embodiments herein, or (iv) the LNP of any one of the aspects or embodiments herein.
  • a prime editing guide RNA or one or more polynucleotides encoding the PEgRNA
  • the PEgRNA comprises: a spacer that comprises at its 3’ end a PEgRNA spacer sequence selected from any one of Tables 18-20; a gRNA core capable of binding to a Cas9 protein; and an extension arm comprising: an editing template that comprises at its 3’ end an RTT sequence selected from same Table as the PEgRNA spacer sequence, and a primer binding site (PBS) that comprises at its 5’ end a PBS sequence selected from same Table as the PEgRNA spacer sequence.
  • PBS primer binding site
  • the spacer of the PEgRNA is from 17 to 22 nucleotides in length. [0147] In some embodiments, the spacer of the PEgRNA is 20 nucleotides in length. [0148] In some embodiments, the PEgRNA comprised from 5’ to 3’, the spacer, the gRNA core, the editing template, and the PBS. [0149] In some embodiments, the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule. [0150] In some embodiments, the gRNA core comprises a gRNA core sequence selected from Table 10.
  • a prime editing system comprising: (a) the prime editing guide RNA (PEgRNA) of any one of the aspects or embodiments herein, or one or more polynucleotides encoding the PEgRNA; and optionally (b) a nick guide RNA (ngRNA), or one or more polynucleotides encoding the ngRNA, wherein the ngRNA comprises a spacer comprising at its 3’ end nucleotides 4-20 of any ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence, and a ngRNA core capable of binding to a Cas9 protein.
  • PEgRNA prime editing guide RNA
  • ngRNA nick guide RNA
  • the ngRNA spacer is from 17 to 22 nucleotides in length.
  • the spacer of the ngRNA comprises at its 3’ end nucleotides 3-20, 2- 20, or 1-20 of the ngRNA spacer sequence selected from the same Table as the PEgRNA spacer sequence.
  • the ngRNA spacer is 20 nucleotides in length.
  • WSGR Docket No.59761-775.601 In some embodiments, the ngRNA core comprises a gRNA core sequence selected from Table 10.
  • the prime editing system further comprises: (c) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or a nucleic acid encoding the Cas9 nickase, and a reverse transcriptase, or a nucleic acid encoding the reverse transcriptase.
  • the prime editing system further comprises: (c) an N-terminal extein comprising an N-terminal fragment of a prime editor fusion protein and an N-intein or a polynucleotide encoding the N-terminal extein; and (d) a C-terminal extein comprising a C-terminal fragment of the prime editor fusion protein and a C-intein, or a polynucleotide encoding the C- terminal extein; wherein the N-intein and the C-intein of the N-terminal and C-terminal exteins are capable of self-excision to join the N-terminal fragment and the C-terminal fragment to form the prime editor fusion protein, and wherein the prime editor fusion protein comprises a Cas9 nickase and a reverse transcriptase (RT) domain.
  • RT reverse transcriptase
  • FIG.1 depicts a schematic of a prime editing guide RNA (PEgRNA) binding to a double stranded target DNA sequence.
  • PEgRNA prime editing guide RNA
  • FIG.2 depicts a PEgRNA architectural overview in an exemplary schematic of PEgRNA designed for a prime editor.
  • FIG.3 is a schematic showing the spacer and gRNA core part of an exemplary guide RNA, in two separate molecules. The rest of the PEgRNA structure is not shown.
  • FIG.4 shows restoration of swelling and CFTR function in prime edited patient-derived intestinal organoids after incubation with 10 ⁇ M of forskolin. DETAILED DESCRIPTION OF THE DISCLOSURE
  • CFTR/ABCC7 cystic fibrosis transmembrane conductance regulator
  • compositions and methods for correction of mutations in the CFTR gene associated with cystic fibrosis can comprise prime editors (Pes) that may use engineered guide polynucleotides, e.g., prime editing guide RNAs (PEgRNAs), that can direct Pes to specific DNA targets and can encode DNA edits on the target gene CFTR that serve a variety of functions, including direct correction of disease-causing mutations associated with cystic fibrosis.
  • Pes prime editors
  • PEgRNAs prime editing guide RNAs
  • references to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure.
  • the term “about” or “approximately” in relation to a numerical means a range of values that fall within 10% greater than or less than the value. For example, about x means x ⁇ (10% * x).
  • the term “substantially” as used herein may refer to a value approaching 100% of a given value.
  • the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.
  • WSGR Docket No.59761-775.601 The terms “protein” and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three- dimensional conformation.
  • a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody. In some embodiments, a protein may be a full-length protein (e.g., a fully processed protein having certain biological function).
  • a protein may be a variant or a fragment of a full-length protein.
  • a variant of a protein or enzyme comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.
  • a protein comprises one or more protein domains or subdomains.
  • polypeptide domain when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function.
  • a protein comprises multiple protein domains.
  • a protein comprises multiple protein domains that are naturally occurring.
  • a protein comprises multiple protein domains from different naturally occurring proteins.
  • a prime editor may be a fusion protein comprising a Cas9 protein domain of S.
  • a protein comprises a functional variant or functional fragment of a full-length wild type protein.
  • a “functional fragment” or “functional portion”, as used herein, refers to any portion of a reference protein (e.g., a wild type protein) that encompasses less than the entire amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions.
  • a functional fragment of a reverse transcriptase may encompass less than the entire amino acid sequence of a wild type reverse transcriptase, but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide.
  • a functional fragment thereof may retain one or more of the functions of at least one of the functional domains.
  • a WSGR Docket No.59761-775.601 functional fragment of a Cas9 may encompass less than the entire amino acid sequence of a wild type Cas9 but retains its DNA binding ability and lacks its nuclease activity partially or completely.
  • a “functional variant” or “functional mutant”, as used herein, refers to any variant or mutant of a reference protein (e.g., a wild type protein) that encompasses one or more alterations to the amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions.
  • the one or more alterations to the amino acid sequence comprises amino acid substitutions, insertions or deletions, or any combination thereof.
  • the one or more alterations to the amino acid sequence comprises amino acid substitutions.
  • 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.
  • a functional variant thereof may retain one or more of the functions of at least one of the functional domains.
  • a functional fragment of a Cas9 may comprise one or more amino acid substitutions in a nuclease domain, e.g., an H840A amino acid substitution, compared to the amino acid sequence of a wild type Cas9, but retains the DNA binding ability and lacks the nuclease activity partially or completely.
  • the term “function” and its grammatical equivalents as used herein may refer to a capability of operating, having, or serving an intended purpose. Functional may comprise any percent from baseline to 100% of an intended purpose.
  • functional may comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose.
  • the term functional may mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.
  • a protein or polypeptides includes naturally occurring amino acids (e.g., one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).
  • a protein or polypeptides includes non-naturally occurring amino acids (e.g., amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics).
  • a protein or polypeptide is modified.
  • a protein comprises an isolated polypeptide.
  • isolated means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.
  • WSGR Docket No.59761-775.601 [0179]
  • a protein is present within a cell, a tissue, an organ, or a virus particle.
  • a protein is present within a cell or a part of a cell (e.g., a bacteria cell, a plant cell, or an animal cell).
  • a protein is present in a mixture of analytes (e.g., a lysate). In some embodiments, the protein is present in a lysate from a plurality of cells or from a lysate of a single cell.
  • the terms “homologous,” “homology,” or “percent homology” as used herein refer to the degree of sequence identity between an amino acid and a corresponding reference amino acid sequence or a polynucleotide sequence and a corresponding reference polynucleotide sequence. “Homology” can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar.
  • Homology can mean, for example, nucleic acid sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.
  • a “homologous sequence” of nucleic acid sequences may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid sequence.
  • a “region of homology to a genomic region” can be a region of DNA that has a similar sequence to a given genomic region in the genome.
  • a region of homology can be of any length that is sufficient to promote binding of a spacer, a primer binding site, or protospacer sequence to the genomic region.
  • the region of homology can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases in length such that the region of homology has sufficient homology to undergo binding with the corresponding genomic region.
  • sequence homology or identity when a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or specified portion of the length.
  • Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul et al, J. Mol. Biol.215:403- 410, 1990.
  • BLAST Basic Local Alignment Search Tool
  • a publicly available, internet interface, for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Additional known algorithms include those published in: Smith & Waterman, “Comparison of Biosequences”, Adv. Appl. Math.2:482, 1981; Needleman & Wunsch, WSGR Docket No.59761-775.601 “A general method applicable to the search for similarities in the amino acid sequence of two proteins” J. Mol.
  • Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277), and the GGSEARCH program https://fasta.bioch.virginia.edu/fasta_www2/, which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length.
  • polynucleotide or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules.
  • a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA.
  • a polynucleotide is double stranded, e.g., a double-stranded DNA in a gene. In some embodiments, a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.
  • Polynucleotides can have any three-dimensional structure.
  • a gene or gene fragment for example, a probe, primer, EST or SAGE tag
  • an exon an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated WSGR Docket No.59761-775.601 RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a tRNA-derived small RNA (tsRNA), an antisense RNA, an sh
  • a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof.
  • a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • 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.
  • the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).
  • a polynucleotide may be modified.
  • the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides.
  • modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide.
  • the modification may be on the internucleoside linkage (e.g., phosphate backbone).
  • multiple modifications are included in the modified nucleic acid molecule.
  • a single modification is included in the modified nucleic acid molecule.
  • complement refers to the ability of two polynucleotide molecules to base pair with each other.
  • Complementary polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • hydrogen bonding may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • an adenine on one polynucleotide molecule will base pair to a thymine or an uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to a guanine on a second polynucleotide molecule.
  • Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence.
  • first polynucleotide molecule comprising a first nucleotide sequence
  • second polynucleotide molecule comprising a second nucleotide sequence.
  • the two DNA molecules 5’-ATGC-3’ and 5’- GCAT-3’ are complementary, and the complement of the DNA molecule 5’-ATGC-3’ is 5’-GCAT- 3’.
  • a percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
  • Perfectly complementary means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule.
  • “Substantially complementary” as used herein refers to a degree of complementarity that can be 70%, 75%, 80%, WSGR Docket No.59761-775.601 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules.
  • the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides.
  • “Substantially complementary” can also refer to a 100% complementarity over a portion“or a region of two polynucl”otide molecules.
  • the portion or the region of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which polynucleotides, e.g., the transcribed mRNA, translated into peptides, polypeptides, or proteins.
  • expression may include splicing of the mRNA in a eukaryotic cell.
  • expression of a polynucleotide e.g., a gene or a DNA encoding a protein
  • expression of a polynucleotide is determined by the amount of the protein encoded by the gene after transcription and translation of the gene.
  • expression of a polynucleotide is determined by the amount of a functional form of the protein encoded by the gene after transcription and translation of the gene.
  • expression of a gene is determined by the amount of the mRNA, or transcript, that is encoded by the gene after transcription the gene.
  • expression of a polynucleotide e.g., an mRNA
  • expression of a polynucleotide is determined by the amount of the protein encoded by the mRNA after translation of the mRNA.
  • expression of a polynucleotide e.g., a mRNA or coding RNA, is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide.
  • sampling may comprise capillary sequencing, bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high- throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, or any combination thereof.
  • encode refers to a polynucleotide which is said to “encode” another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and/or translated to produce an amino acid, or a polypeptide or fragment thereof.
  • a WSGR Docket No.59761-775.601 polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid.
  • a polynucleotide comprises one or more codons that encode a polypeptide.
  • a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide.
  • the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide.
  • mutation refers to a change and/or alteration in an amino acid sequence of a protein or nucleic acid sequence of a polynucleotide. Such changes and/or alterations may comprise the substitution, insertion, deletion and/or truncation of one or more amino acids, in the case of an amino acid sequence, and/or nucleotides, in the case of nucleic acid sequence, compared to a reference amino acid or a reference nucleic acid sequence.
  • the reference sequence is a wild-type sequence.
  • a mutation in a nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide.
  • 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.
  • the term “subject” and its grammatical equivalents as used herein may refer to a human or a non-human.
  • a subject may be a mammal.
  • a subject is human.
  • a human subject may be male or female.
  • a human subject may be of any age.
  • a subject may be a human embryo.
  • a human subject may be a newborn, an infant, a child, an adolescent, or an adult.
  • a human subject may be in need of treatment for a genetic disease or disorder.
  • treatment may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder.
  • Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder.
  • Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder.
  • this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder.
  • Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder.
  • a condition may be pathological.
  • a treatment may not completely cure or prevent a disease, condition, or disorder. In some embodiments, a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder. In some embodiments, a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.
  • WSGR Docket No.59761-775.601 The term “ameliorate” and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. [0198]
  • the terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder.
  • a composition prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject.
  • effective amount or “therapeutically effective amount” refers to a quantity of a composition, for example a prime editing composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein.
  • An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo.
  • An effective amount can be the amount to induce, for example, at least about a 2-fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g., expression of a target CFTR gene to produce functional CFTR protein) observed relative to a negative control.
  • An effective amount or dose can induce, for example, about 2-fold increase, about 3-fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25-fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500-fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation (e.g., expression of a target CFTR gene to produce functional CFTR protein).
  • target gene modulation e.g., expression of a target CFTR gene to produce functional CFTR protein.
  • the amount of target gene modulation may be measured by any suitable method known in the art.
  • the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). [0200] In some embodiments, an effective amount can be an amount to induce, when administered to a population of cells, a certain percentage of the population of cells to have a correction of a mutation.
  • an effective amount can be the amount to induce, when administered to or introduced to a population of cells, installation of one or more intended nucleotide edits that correct a mutation in the target CFTR gene, in at least about 1%, 2%, 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the population of cells.
  • Prime Editing refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit (also referred to herein as a nucleotide change) into the target DNA through target-primed DNA synthesis.
  • an intended nucleotide edit also referred to herein as a nucleotide change
  • a target gene of prime editing may comprise a double stranded DNA molecule having two complementary strands: a first strand that may be referred to as a “target strand” or a “non-edit strand”, and a second strand that may be referred to as a “non-target strand,” or an “edit strand.”
  • a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a “search target sequence”.
  • the spacer sequence anneals with the target strand at the search target sequence.
  • the target strand may also be referred to as the “non-Protospacer Adjacent Motif (non-PAM strand).”
  • the non-target strand may also be referred to as the “PAM strand”.
  • the PAM strand comprises a protospacer sequence and optionally protospacer adjacent motif (PAM) sequence.
  • PAM sequence refers to a short DNA sequence immediately adjacent to the protospacer sequence on the PAM strand of the target gene.
  • a PAM sequence may be specifically recognized by a programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease.
  • a specific PAM is characteristic of a specific programmable DNA binding protein, e.g., a Cas nickase or a Cas nuclease.
  • a protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence.
  • a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T).
  • the double stranded target DNA comprises a nick site on the PAM strand (or non-target strand).
  • a “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target DNA.
  • the position of a nick site is determined relative to the position of a specific PAM sequence.
  • the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence.
  • the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA.
  • the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is upstream of a PAM sequence recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active RuvC domain and a nuclease inactive HNH domain. In some embodiments, the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P. lavamentivorans Cas9 nickase, a C.
  • the nick site is 3 base pairs upstream of the PAM sequence, and the PAM sequence is recognized by a Cas9 nickase, wherein the Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain.
  • the nick site is 2 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a S.
  • thermophilus Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive HNH domain.
  • upstream and downstream it is intended to define relevant positions at least two regions or upstream of a second sequence in a DNA molecule where the first sequence is positioned 5’ to the second sequence. Accordingly, the second sequence is downstream of the first sequence.
  • a “primer binding site” (also referred to as PBS or primer binding site sequence) is a single- stranded portion of the PEgRNA that comprises a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand).
  • the PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site.
  • the PEgRNA complexes with and directs a prime editor to bind the search target sequence on the target strand of the double stranded target DNA and generates a nick at the nick site on the non-target strand of the double stranded target DNA.
  • the PBS is complementary to or substantially complementary to, and primed DNA synthesis. [0204] the PBS and encodes a single strand of DNA.
  • the editing template may comprise a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand), and comprises one or more intended nucleotide edits compared to the endogenous sequence of the double stranded target DNA.
  • the editing template and the PBS are immediately adjacent to each other.
  • a PEgRNA in prime editing comprises a single-stranded portion that comprises the PBS and the editing template immediately adjacent to each other.
  • the single stranded portion of the PEgRNA comprising both the PBS and the editing template is complementary or substantially complementary to an endogenous sequence on the PAM strand (i.e., the non-target strand or the edit strand) of the double stranded target DNA except for one or more non-complementary nucleotides at the intended nucleotide edit position(s).
  • the editing template, and the relative positions as among elements of a PEgRNA are determined by double stranded target DNA that may have complementarity or identity to elements of the PEgRNA.
  • the editing template is complementary or substantially complementary to a WSGR Docket No.59761-775.601 sequence on the PAM strand that is immediately downstream of the nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions.
  • the endogenous, e.g., genomic, sequence that is complementary or substantially complementary to the editing template, except for the one or more non-complementary nucleotides at the position corresponding to the intended nucleotide edit may be referred to as an “editing target sequence”.
  • the editing template has identity or substantial identity to a sequence on the target strand that is complementary to, or having the same position in the genome as, the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions.
  • the editing template encodes a single stranded DNA, wherein the single stranded DNA has identity or substantial identity to the editing target sequence except for one or more insertions, deletions, or substitutions at the positions of the one or more intended nucleotide edits.
  • the editing template may encode the wild-type or non-disease associated gene sequence (or its complement if the edit strand is the antisense strand of a gene).
  • the editing template may encode the wild-type or non-disease associated protein, but contain one or more synonymous mutations relative to the wild-type or non-disease associated protein coding region.
  • Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence once the desired edit is installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer).
  • a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene.
  • the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene at the nick site.
  • a primer binding site (PBS) of the PEgRNA anneals with a free 3’ end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3’ end as a primer.
  • a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized.
  • the newly synthesized single-stranded DNA comprises one or more intended nucleotide edits compared to the endogenous target gene sequence.
  • the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template.
  • the endogenous, e.g., genomic, sequence that is partially complementary to the editing template may be referred to as an “editing target sequence”.
  • the newly synthesized single stranded DNA has identity or substantial identity to a sequence in the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions.
  • the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 nucleotides contiguous are located upstream of the 5’ most edit in the editing template.
  • the newly synthesized single-stranded DNA equilibrates with the editing target on the edit strand of the target gene for pairing with the target strand of the target gene.
  • the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FEN1.
  • the FEN is an endogenous FEN, for example, in a cell comprising the target gene.
  • the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided in trans.
  • the newly synthesized single stranded DNA which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the edit strand of the target gene.
  • the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene.
  • the newly synthesized single-stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the target strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch between the two otherwise complementary strands.
  • the mismatch is recognized by DNA repair machinery, e.g., an endogenous DNA repair machinery.
  • DNA repair through DNA repair, the intended nucleotide edit is incorporated into the target gene.
  • Prime Editor refers to the polypeptide or polypeptide components involved in prime editing.
  • a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity.
  • the prime editor further comprises a polypeptide domain having nuclease activity.
  • the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity.
  • the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease.
  • nickase refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target.
  • the prime editor comprises a polypeptide domain that is an inactive nuclease.
  • the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease.
  • 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.
  • the DNA polymerase is a reverse transcriptase.
  • the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having 5’ endonuclease activity, e.g., a 5’ endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation.
  • the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.
  • a prime editor may be engineered.
  • the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment.
  • the polypeptide components of a prime editor may be of different origins or from different organisms.
  • a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species.
  • a prime editor comprises a Cas polypeptide (DNA binding domain) and a reverse transcriptase polypeptide (DNA polymerase) that are derived from different species.
  • a prime editor may comprise a S.
  • polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein.
  • a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences.
  • a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g., a MS2 aptamer, which may be linked to a PEgRNA.
  • Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part.
  • a single polynucleotide, construct, or vector encodes the prime editor fusion protein.
  • multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein.
  • a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector.
  • Prime Editor Nucleotide Polymerase Domain [0210]
  • a prime editor comprises a nucleotide polymerase domain, e.g., a DNA polymerase domain.
  • the DNA polymerase domain may be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, or may be a functional mutant, a functional variant, or a functional fragment thereof.
  • the polymerase domain is a template dependent polymerase domain.
  • the DNA polymerase may rely on a template polynucleotide strand, e.g., the editing template sequence, for new strand DNA synthesis.
  • the prime editor comprises a DNA-dependent DNA polymerase.
  • a prime editor having a DNA-dependent DNA polymerase can synthesize a new single stranded DNA using a PEgRNA editing template that comprises a DNA sequence as a template.
  • the PEgRNA is a chimeric or hybrid PEgRNA, and comprising an extension arm comprising a DNA strand.
  • the chimeric or hybrid PEgRNA may comprise an RNA portion (including the spacer and the gRNA core) and a DNA portion (the extension arm comprising the editing template that includes a strand of DNA).
  • the DNA polymerases can be wild type polymerases from eukaryotic, prokaryotic, archaeal, or viral organisms, and/or the polymerases may be modified by genetic engineering, mutagenesis, or directed evolution-based processes.
  • the polymerases can be a T7 DNA polymerase, T5 DNA polymerase, T4 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III and the like.
  • the polymerases can be thermostable, and can include Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragment, VENT® and DEEPVENT® DNA polymerases, KOD, Tgo, JDF3, and mutants, variants and derivatives thereof.
  • the DNA polymerase is a bacteriophage polymerase, for example, a T4, T7, or phi29 DNA polymerase.
  • the DNA polymerase is an archaeal polymerase, for example, pol I type archaeal polymerase or a pol II type archaeal polymerase.
  • the DNA polymerase comprises a thermostable archaeal DNA polymerase.
  • the DNA polymerase comprises a eubacterial DNA polymerase, for example, Pol I, Pol II, or Pol III polymerase.
  • the DNA polymerase is a Pol I family DNA polymerase.
  • the DNA polymerase is a E.coli Pol I DNA polymerase.
  • the DNA polymerase is a Pol II family DNA polymerase.
  • the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase.
  • the DNA Polymerase is a Pol IV family DNA polymerase.
  • the DNA polymerase is a E.coli Pol IV DNA polymerase. In some embodiments, the DNA polymerase comprises a eukaryotic DNA polymerase. In some embodiments, the DNA polymerase is a Pol-beta DNA polymerase, a Pol- lambda DNA polymerase, a Pol-sigma DNA polymerase, or a Pol-mu DNA polymerase. In some embodiments, the DNA polymerase is a Pol-alpha DNA polymerase. In some embodiments, the DNA polymerase is a POLA1 DNA polymerase. In some embodiments, the DNA polymerase is a POLA2 DNA polymerase.
  • 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.
  • the DNA polymerase is a POLE1 DNA polymerase. In some embodiments, the DNA polymerase is a POLE2 DNA polymerase. In some embodiments, the DNA polymerase is a POLE3 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-eta (POLH) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-iota (POLI) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-kappa (POLK) DNA polymerase. In some embodiments, the DNA polymerase is a Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a human Rev1 DNA polymerase.
  • the DNA polymerase is a viral DNA-dependent DNA polymerase.
  • the DNA polymerase is a B family DNA polymerases.
  • the DNA polymerase is a herpes simplex virus (HSV) UL30 DNA polymerase.
  • the DNA polymerase is a cytomegalovirus (CMV) UL54 DNA polymerase.
  • the DNA polymerase is an archaeal polymerase.
  • the DNA polymerase is a Family B/pol I type DNA polymerase.
  • the DNA polymerase is a homolog of Pfu from Pyrococcus furiosus.
  • the DNA polymerase is a pol II type DNA polymerase.
  • the DNA polymerase is a homolog of P. furiosus DP1/DP22-subunit polymerase.
  • the derived from archaea with optimal growth temperatures that are similar to the desired assay temperatures.
  • the DNA polymerase comprises a thermostable archaeal DNA polymerase.
  • thermostable DNA polymerase is isolated or derived from Pyrococcus species (furiosus, species GB-D, woesii, abysii, horikoshii), Thermococcus species (kodakaraensis KOD1, litoralis, species 9 degrees North-7, species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobus fulgidus. [0215] Polymerases may also be from eubacterial species.
  • the DNA polymerase is a Pol I family DNA polymerase.
  • the DNA polymerase is an E.coli Pol I DNA polymerase.
  • the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcus furiosus (Pfu) Pol II DNA polymerase. In some embodiments, the DNA Polymerase is a Pol III family DNA polymerase. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is an E.coli Pol IV DNA polymerase. In some embodiments, the Pol I DNA activity.
  • thermostable pol I DNA polymerases can be isolated from a variety of thermophilic eubacteria, including Thermus species and Thermotoga maritima such as Thermus aquaticus (Taq), Thermus thermophilus (Tth) and Thermotoga maritima (Tma UlTma).
  • a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT).
  • RT reverse transcriptase
  • a RT or an RT domain may be a wild type RT domain, a full-length RT domain, or may be a functional mutant, a functional variant, or a functional fragment thereof.
  • An RT or an RT domain of a prime editor may comprise a wild-type RT, or may be engineered or evolved to contain specific amino acid substitutions, truncations, or variants.
  • An engineered RT may comprise sequences or amino acid changes different from a naturally occurring RT.
  • the engineered RT may have improved reverse transcription activity over a naturally occurring RT or RT domain.
  • the engineered RT may have WSGR Docket No.59761-775.601 improved features over a naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity.
  • a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a reference naturally occurring RT.
  • a prime editor comprises a virus RT, for example, a retrovirus RT.
  • virus RT include Moloney murine leukemia virus (M-MLV MMLVRT or M-MLV RT); human T-cell leukemia virus type 1 (HTLV-1) RT; bovine leukemia virus (BLV) RT; Rous Sarcoma Virus (RSV) RT; human immunodeficiency virus (HIV) RT, M-MFV RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) Helper
  • M-MLV MMLVRT human
  • a prime editor can comprise a wild-type M-MLV RT, a functional mutant, a functional variant, or a functional fragment thereof.
  • Table 1 provides sequences of illustrative M-MLV RTs suitable for use with compositions and methods of the disclosure.
  • a prime editor comprises a wild-type M-MLV RT as set forth in SEQ ID NO: 518.
  • a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 519.
  • a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 520.
  • a prime editor comprises a variant M-MLV RT as set forth in SEQ ID NO: 1209. [0221] Table 1. Illustrative M-MLV Sequences WSGR Docket No.59761-775.601 [0222]
  • the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions: H8X, P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X as compared to a reference M-MLV RT where X is any amino acid other than the original amino acid in the reference M-MLV RT.
  • the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions: H8Y, P51L, S67K, E69K, L139P, WSGR Docket No.59761-775.601 T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, or D653N as compared to a reference M- MLV RT.
  • the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 519. In some embodiments, the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 518.
  • a prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions D200N, T330P, L603W, T306K, or W313F as compared to a reference M- MLV RT. In some embodiments, the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 519.
  • the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 518.
  • a prime editor comprises a M-MLV RT comprising amino acid substitutions H8Y, D200N, T330P, L603W, T306K, and W313F as compared to a reference M- MMLV RT.
  • the reference M-MLV RT is a variant M-MLV RT as set forth in SEQ ID NO: 519.
  • the reference M-MLV RT is a WT M-MLV RT as set forth in SEQ ID NO: 518.
  • a prime editor comprises a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in in Table 1.
  • the prime editor comprises a M- MLV RT that comprises an amino acid sequence that is selected from the group consisting of: amino acid sequences provided in Table 1 or a variant or fragment thereof.
  • the prime editor comprises a variant M-MLV RT that comprises an amino acid sequence set forth in SEQ ID NO: 520.
  • an RT variant may be a functional fragment of a reference RT that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, up to 100, up to 200, up to 300, up to 400, or up to 500 or more amino acid changes compared to a reference RT.
  • the RT variant comprises a fragment of a reference RT, such that the fragment is about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the corresponding fragment of the reference RT.
  • the fragment is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identical, 96%, 97%, 98%, 99%, or 99.5% of the amino acid length of a corresponding reference RT (M-MLV reverse transcriptase).
  • a reference RT can be any one of the RTs shown in Table 1.
  • a functional RT fragment or variant is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or up to 600 or more amino acids in length. [0228] In still other embodiments, the functional RT variant is truncated at the N-terminus or the C- terminus, or both, by a certain number of amino acids which results in a truncated variant that retains sufficient DNA polymerase function.
  • the functional RT variant e.g., a functional MMLV RT variant
  • a prime editing composition or a prime editing system disclosed herein comprises a polynucleotide (e.g., a DNA, a RNA, e.g., a mRNA) that encodes a M-MLV RT.
  • the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in Table 1.
  • the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NOs: 518, 519, or 520.
  • the polynucleotide encodes a M-MLV RT that comprises an amino acid sequence that is selected from the group consisting of: the amino acid sequences provided in Table 1.
  • the polynucleotide encodes a variant M-MLV RT that comprises an amino acid sequence that is set forth in SEQ ID NO: 520.
  • a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT.
  • the prime editor comprises a Group II intron RT, for example, a Geobacillus stearothermophilus Group II Intron (GsI-IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT.
  • the prime editor comprises a retron RT.
  • a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT.
  • the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (GsI-IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT.
  • the prime editor comprises a retron RT.
  • Programmable DNA Binding Domain [0231]
  • the DNA-binding domain of a prime editor is a programmable DNA binding domain.
  • a programmable DNA binding domain refers to a protein domain that is designed to bind a specific nucleic acid sequence, e.g., a target DNA or a target RNA.
  • 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 WSGR Docket No.59761-775.601 sequence, e.g., a search target sequence in a target gene.
  • the DNA-binding domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated (Cas) protein.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • a Cas protein may comprise any Cas protein described herein or a functional fragment or functional variant thereof.
  • a DNA-binding domain may also comprise a zinc-finger protein domain.
  • a DNA-binding domain comprises a transcription activator-like effector domain (TALE).
  • TALE transcription activator-like effector domain
  • the DNA-binding domain comprises a DNA nuclease.
  • the DNA-binding domain of a prime editor may comprise an RNA-guided DNA endonuclease, e.g., a Cas protein.
  • the DNA-binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator like effector domain nuclease (TALEN), where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, e.g., a Fok I nuclease domain.
  • ZFN zinc finger nuclease
  • TALEN transcription activator like effector domain nuclease
  • the DNA-binding domain comprises a nuclease activity.
  • the DNA-binding domain of a prime editor comprises an endonuclease domain having single strand DNA cleavage activity.
  • the endonuclease domain may comprise a FokI nuclease domain.
  • the DNA-binding domain of a prime editor comprises a nuclease having full nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises a nuclease having modified or reduced nuclease activity as compared to a wild type endonuclease domain. For example, the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild type endonuclease domain. In some embodiments, the DNA- binding domain of a prime editor has nickase activity. In some embodiments, the DNA-binding domain of a prime editor comprises a Cas protein domain that is a nickase.
  • 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.
  • the Cas nickase comprises an amino acid substitution in a HNH domain.
  • the Cas nickase comprises an amino acid substitution in a RuvC domain.
  • the DNA-binding domain comprises a CRISPR associated protein (Cas protein) domain.
  • a Cas protein may be a Class 1 or a Class 2 Cas protein.
  • a Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or a type VI Cas protein.
  • Cas proteins include , Casl, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csx12), Cas10, CaslOd, Cas12a/Cpfl, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa
  • a Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides.
  • a Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms.
  • a Cas protein, e.g., Cas9 can be from any suitable organism.
  • the organism is Streptococcus pyogenes (S. pyogenes).
  • the organism is Staphylococcus aureus (S. aureus).
  • the organism is Streptococcus thermophilus (S. thermophilus).
  • the organism is Staphylococcus lugdunensis.
  • Non-limiting examples of suitable organism include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis rougevillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aerugi
  • 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).
  • a Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, WSGR Dock
  • Torquens Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp.
  • a Cas protein e.g., Cas9
  • a Cas protein can be a wild type or a modified form of a Cas protein.
  • a Cas protein e.g., Cas9
  • a Cas protein can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein.
  • a Cas protein e.g., Cas9
  • a Cas protein, e.g., Cas9 can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein.
  • a Cas protein e.g., Cas9
  • a Cas protein can be a polypeptide with at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein.
  • a Cas protein, e.g., Cas9 may comprise one or more domains.
  • Cas domains include, guide nucleic acid recognition and/or binding domain, nuclease domains (e.g., Dnase or Rnase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains.
  • a Cas protein comprises a guide nucleic acid recognition and/or binding domain can interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage.
  • a Cas protein e.g., Cas9, comprises one or more nuclease domains.
  • a Cas protein can comprise an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein.
  • a Cas protein comprises a single nuclease domain.
  • a Cpf1 may comprise a RuvC domain but lacks WSGR Docket No.59761-775.601 HNH domain.
  • a Cas protein comprises two nuclease domains, e.g., a Cas9 protein can comprise an HNH nuclease domain and a RuvC nuclease domain.
  • a prime editor comprises a Cas protein, e.g., Cas9, wherein all nuclease domains of the Cas protein are active.
  • a prime editor comprises a Cas protein having one or more inactive nuclease domains.
  • One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity.
  • a Cas protein e.g., Cas9
  • a Cas protein comprising mutations in a nuclease domain has reduced (e.g., nickase) or abolished nuclease activity while maintaining its ability to target a nucleic acid locus at a search target sequence when complexed with a guide nucleic acid, e.g., a PEgRNA.
  • a prime editor comprises a Cas nickase that can bind to the target gene in a sequence-specific manner and generate a single-strand break at a protospacer within double- stranded DNA in the target gene, but not a double-strand break.
  • a prime editor comprises a Cas nickase comprising two nuclease domains (e.g., Cas9), with one of the two nuclease domains modified to lack catalytic activity or deleted.
  • the Cas nickase of a prime editor comprises a nuclease inactive RuvC domain and a nuclease active HNH domain.
  • the Cas nickase of a prime editor comprises a nuclease inactive HNH domain and a nuclease active RuvC domain.
  • a prime editor comprises a Cas9 nickase having an amino acid substitution in the RuvC domain e.g., an amino acid substitution that reduces or abolishes nuclease activity of the RuvC domain.
  • the Cas9 nickase comprises a D10X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than D.
  • a prime editor comprises a Cas9 nickase having an amino acid substitution in the HNH domain e.g., an amino acid substitution that reduces or abolishes nuclease activity of the HNH domain.
  • the Cas9 nickase comprises a H840X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than H.
  • a prime editor comprises a Cas protein that can bind to the target gene in a sequence-specific manner but lacks or has abolished nuclease activity and may not cleave either strand of a double stranded DNA in a target gene.
  • Abolished activity or lacking activity can refer to an enzymatic activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type exemplary activity (e.g., wild-type Cas9 nuclease activity).
  • a Cas protein of a prime editor completely lacks nuclease activity.
  • a nuclease, e.g., Cas9, that lacks nuclease activity may be referred to as nuclease inactive or “nuclease dead” (abbreviated by “d”).
  • a nuclease dead Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide.
  • a dead Cas protein is a dead Cas9 protein.
  • a prime editor comprises a nuclease dead Cas protein wherein all of the nuclease domains (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpf1 protein) are mutated to lack catalytic activity or are deleted.
  • a Cas protein can be modified.
  • a Cas protein e.g., Cas9
  • Cas proteins can also be modified to change any other activity or property of the protein, such as stability.
  • one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of the Cas protein.
  • a Cas protein can be a fusion protein.
  • a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional regulation domain, or a polymerase domain.
  • a Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability.
  • the fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.
  • the Cas protein of a prime editor is a Class 2 Cas protein.
  • the Cas protein is a type II Cas protein.
  • the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, a Cas9 protein homolog, mutant, variant, or a functional fragment thereof.
  • a Cas9, Cas9 protein, Cas9 polypeptide or a Cas9 nuclease refers to an RNA guided nuclease comprising one or more Cas9 nuclease domains and a Cas9 gRNA binding domain having the ability to bind a guide polynucleotide, e.g., a PEgRNA.
  • a Cas9 protein may refer to a wild type Cas9 protein from any organism or a homolog, ortholog, or paralog from any organisms; any functional mutants or functional variants thereof; or any functional fragments or domains thereof.
  • a prime editor comprises a full-length Cas9 protein.
  • the Cas9 protein can generally comprises at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity to a wild type reference Cas9 protein (e.g., Cas9 from S. pyogenes).
  • the Cas9 comprises an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof as compared to a wild type reference Cas9 protein.
  • 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.
  • a Cas9 polypeptide is a SpCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP_038431314 or a fragment or variant thereof.
  • a Cas9 polypeptide is a SaCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No. J7RUA5 or a fragment or WSGR Docket No.59761-775.601 variant thereof.
  • a Cas9 polypeptide is a ScCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in Uniprot Accession No.
  • a Cas9 polypeptide is a StCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in NCBI Accession No. WP_007896501.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a SluCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_230580236.1 or WP_250638315.1 or WP_242234150.1, WP_241435384.1, WP_002460848.1, KAK58371.1, or a fragment or variant thereof.
  • a Cas9 polypeptide is a NmCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No. WP_002238326.1 or WP_061704949.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a CjCas9 polypeptide, e.g., comprising an amino acid sequence as set forth in any of NCBI Accession No.
  • a Cas9 polypeptide is a FnCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in Uniprot Accession No. A0Q5Y3 or a fragment or variant thereof.
  • a Cas9 polypeptide is a TdCas9 polypeptide, e.g., comprising the amino acid sequence as set forth in NCBI Accession No.
  • a Cas9 polypeptide is a chimera comprising domains from two or more of the organisms described herein or those known in the art.
  • a Cas9 polypeptide is a Cas9 polypeptide from Streptococcus macacae, e.g., comprising the amino acid sequence as set forth in NCBI Accession No. WP_003079701.1 or a fragment or variant thereof.
  • a Cas9 polypeptide is a Cas9 polypeptide generated by replacing a PAM interaction domain of a SpCas9 with that of a Streptococcus macacae Cas9 (Spy-mac Cas9).
  • Spy-mac Cas9 a Streptococcus macacae Cas9
  • Exemplary Cas9 and Cas9 nickase variants are provided in Table 2.
  • a prime editor comprises a DNA binding domain that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%,
  • the DNA binding domain comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 differences e.g., mutations e.g., deletions, substitutions and/or insertions compared to any one of the amino acid sequences set forth in Table 2.
  • a prime editor comprises a Cas9 protein that is a Cas9 nickase that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the nickase sequences set forth in Table 2.
  • a prime editor comprises a Cas9 protein that comprises an amino acid sequence that is selected from the group consisting of the sequences set forth in Table 2. In some embodiments, a prime editor comprises a Cas9 protein that comprises an amino acid sequence that lacks a N-terminus methionine relative to an amino acid sequence set forth in Table 2.
  • a prime editing compositions or prime editing systems disclosed herein comprises a polynucleotide (e.g., a DNA, or an RNA, e.g., an mRNA) that encodes a Cas9 protein that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in Table 2.
  • a polynucleotide e.g., a DNA, or an RNA, e.g., an mRNA
  • Cas9 protein that comprises an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%
  • a Cas9 protein comprises a Cas9 protein from Streptococcus pyogenes (Sp), e.g., as according to NC_002737.2:854751-858857 or the protein encoded by UniProt Q99ZW2, e.g., as according to SEQ ID NO: 521.
  • a prime editor comprises a Cas9 protein (e.g., a SpCas9) as according to any one of the sequences set forth in SEQ ID NOs: 521-524 or a variant thereof.
  • the Cas9 protein is a SpCas9.
  • a SpCas9 can be a wild type SpCas9, a SpCas9 variant, or a nickase SpCas9.
  • the SpCas9 lacks the N-terminus methionine relative to a corresponding SpCas9 (e.g., a wild type SpCas9, a SpCas9 variant or a nickase SpCas9).
  • a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 521, not including the N-terminus methionine.
  • a wild type SpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 521.
  • a prime editor comprises a Cas9 protein comprising one or WSGR Docket No.59761-775.601 more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding wild type Cas9 protein (e.g., a wild type SpCas9).
  • the Cas9 protein comprising one or more mutations relative to a wild type Cas9 (e.g., a wild type SpCas9) protein comprises an amino acid sequence set forth in SEQ ID NOs: 522, 523, or 524.
  • Exemplary Streptococcus pyogenes Cas9 (SpCas9) amino acid sequence useful in the prime editors disclosed herein are provided in Table 2.
  • a prime editor comprises a Cas9 protein (e.g., a SluCas9) as according to any one of the SEQ ID NOs: 525-527 or a variant thereof.
  • a prime editor comprises a Cas9 protein from Staphylococcus lugdunensis (SluCas9) e.g., as according to any one of the SEQ ID NO: 525, or a variant thereof.
  • the Cas9 protein is a SluCas9.
  • a SluCas9 can be a wild type SluCas9, a SluCas9 variant, or a nickase SluCas9.
  • the SluCas9 lacks the N-terminus methionine relative to a corresponding SluCas9 (e.g., a wild type SluCas9, a SluCas9 variant or a nickase SluCas9).
  • a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 525, not including the N-terminus methionine.
  • a wild type SluCas9 comprises an amino acid sequence set forth in SEQ ID NO: 525.
  • 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).
  • the Cas9 protein comprising one or mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NOs: 526 or 527.
  • a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as according to any of the SEQ ID NOs: 528-530, or a variant thereof.
  • a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as set forth in Table 2, or a variant thereof.
  • the Cas9 protein is a SaCas9.
  • a SaCas9 can be a wild type SaCas9, a SaCas9 variant, or a nickase SaCas9.
  • the SaCas9 lacks the N-terminus methionine relative to a corresponding SaCas9 (e.g., a wild type SaCas9, a SaCas9 variant or a nickase SaCas9).
  • a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 528, not including the N-terminus methionine.
  • a wild type SaCas9 comprises an amino acid sequence set forth in SEQ ID NO: 528.
  • 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).
  • the Cas9 protein comprising one or more mutations relative to a wild type Cas9 protein comprises an WSGR Docket No.59761-775.601 amino acid sequence set forth in SEQ ID NOs: 529 or 530.
  • Exemplary Staphylococcus aureus Cas9 (SaCas9) amino acid sequence useful in the prime editors disclosed herein are provided in Table 2.
  • a prime editor comprises a Cas9 protein as according to any one of the sequences set forth in SEQ ID NOs: 531-539, 546-548 or a variant thereof.
  • the Cas9 protein is a Cas9 variant, for example, a SpCas9 variant (e.g., SpCas9-NG, SpCas9-NGA, SpRY, or SpG).
  • a prime editor comprises a Cas9 protein that lacks the N- terminus methionine relative to a corresponding Cas9 protein (e.g., a Cas9 variant set forth in any one of SEQ ID NOs: 531-539, 546-548).
  • a prime editor comprises a Cas9 protein (e.g., a Cas9 variant), having an amino acid sequence as according to any one of SEQ ID NOs: 531- 539, 546-548 not including the N-terminus methionine.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in any one of SEQ ID NOs: 531-539, 546-548).
  • the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 532, 533, 535, 536, 538, 539, 547, or 548.
  • a Cas9 protein is a chimeric Cas9, e.g., modified Cas9, e.g., synthetic RNA-guided nucleases (sRGNs), e.g., modified by DNA family shuffling, e.g., sRGN3.1, sRGN3.3.
  • sRGNs synthetic RNA-guided nucleases
  • the DNA family shuffling comprises, fragmentation and reassembly of parental Cas9 genes, e.g., one or more of Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa).
  • a modified sluCas9 shows increased editing efficiency and/or specificity relative to a sluCas9 that is not modified.
  • a modified Cas9 e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in editing efficiency compared to a Cas9 that is not modified.
  • a Cas9 e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in specificity compared to a Cas9 that is not modified.
  • a Cas9 e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in cleavage activity compared to a Cas9 that is not modified.
  • a Cas9 e.g., a editor comprises a Cas9 protein (e.g., a chimeric Cas9), e.g., as according any one of the sequences WSGR Docket No.59761-775.601 set forth in SEQ ID NOs: 540-545, or a variant thereof.
  • Exemplary amino acid sequences of Cas9 protein (e.g., sRGN) useful in the prime editors disclosed herein are provided in Table 2.
  • a prime editor comprises a Cas9 protein, that lacks a N-terminus methionine relative to SEQ ID NO: 540 or SEQ ID NO: 543.
  • a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions) relative to a corresponding Cas9 protein (e.g., a Cas9 protein set forth in any one of SEQ ID NOs: 540, or 543).
  • the Cas9 protein comprising one or mutations relative to a corresponding Cas9 protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 541, 542, 544, or 545.
  • a Cas9 protein comprises a variant Cas9 protein containing one or more amino acid substitutions.
  • a wildtype Cas9 protein comprises a RuvC domain and an HNH domain.
  • a prime editor comprises a nuclease active Cas9 protein that may cleave both strands of a double stranded target DNA sequence.
  • the nuclease active Cas9 protein comprises a functional RuvC domain and a functional HNH domain.
  • a prime editor comprises a Cas9 nickase that can bind to a guide polynucleotide and recognize a target DNA, but can cleave only one strand of a double stranded target DNA.
  • the Cas9 nickase comprises only one functional RuvC domain or one functional HNH domain.
  • a prime editor comprises a Cas9 that has a non- functional HNH domain and a functional RuvC domain.
  • the prime editor can cleave the edit strand (i.e., the PAM strand), but not the non-edit strand of a double stranded target DNA sequence.
  • a prime editor comprises a Cas9 having a non-functional RuvC domain that can cleave the target strand (i.e., the non-PAM strand), but not the edit strand of a double stranded target DNA sequence.
  • a prime editor comprises a Cas9 that has neither a functional RuvC domain nor a functional HNH domain, which may not cleave any strand of a double stranded target DNA sequence.
  • a prime editor comprises a Cas9 having a mutation in the RuvC domain that reduces or abolishes the nuclease activity of the RuvC domain.
  • the Cas9 comprises a mutation at amino acid D10 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 comprises a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid D10, G12, and/or G17 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a D10A mutation, a G12A mutation, and/or a G17A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • 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.
  • the Cas9 polypeptide comprises a mutation at amino acid H840 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid E762, D839, H840, N854, N856, N863, H982, H983, A984, D986, and/or a A987 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a E762A, D839A, H840A, N854A, N856A, N863A, H982A, H983A, A984A, and/or a D986A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid residue R221, N394, and/or H840 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 521).
  • the Cas9 polypeptide comprises a R221K, N394L, and/or H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a mutation at amino acid residue R220, N393, and/or H839 as compared to a wild type SpCas9 (e.g., SEQ ID NO: 521) lacking a N-terminal methionine, or a corresponding mutation thereof.
  • the Cas9 polypeptide comprises a R220K, N393K, and/or H839A mutation as compared to a wild type SpCas9 (as set forth in SEQ ID NO: 521) lacking a N-terminal methionine, or a corresponding mutation thereof.
  • a prime editor comprises a Cas9 having one or more amino acid substitutions in both the HNH domain and the RuvC domain that reduce or abolish the nuclease activity of both the HNH domain and the RuvC domain.
  • the prime editor comprises a nuclease inactive Cas9, or a nuclease dead Cas9 (dCas9).
  • the dCas9 comprises a H840X substitution and a D10X mutation compared to a wild type SpCas9 as set forth in SEQ ID NO: 521 or corresponding mutations thereof, wherein X is any amino acid other than H for the H840X substitution and any amino acid other than D for the D10X substitution.
  • the dead Cas9 comprises a H840A and a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 521, or corresponding mutations thereof.
  • the N-terminal methionine is removed from the amino acid sequence of a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein.
  • methionine-minus (Met (-)) Cas9 nickases include any one of the sequences set forth in SEQ ID NOs: 523, 524, 527, 530, 533, 536, 539, 542, 545, 548, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
  • the Cas9 proteins used herein may also include other Cas9 variants having at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% sequence identity to any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or fragment Cas9, or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art.
  • a Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a reference Cas9, e.g., a wild type Cas9.
  • 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.
  • a reference Cas9 e.g., a gRNA binding domain or a DNA-cleavage domain
  • 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.
  • a Cas9 fragment is a functional fragment that retains one or more Cas9 activities.
  • the Cas9 fragment is at least 100 amino acids in length.
  • a prime editor comprises a Cas protein, e.g., a Cas9 variant, comprising modifications that allow altered PAM recognition.
  • Exemplary Cas9 protein amino acid sequence e.g., Cas9 variant with altered PAM recognition specificities
  • Table 2 Exemplary Cas9 protein amino acid sequence (e.g., Cas9 variant with altered PAM recognition specificities) that are useful in the Prime editors of the disclosure are provided in Table 2.
  • a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow altered PAM recognition.
  • a “protospacer adjacent motif (PAM)”, PAM sequence, or PAM-like motif may be used to refer to a short DNA sequence immediately following the protospacer sequence on the PAM strand of the target gene.
  • the PAM is recognized by the Cas nuclease in the prime editor during prime editing.
  • the PAM is required for target binding of the Cas protein.
  • the specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein.
  • a PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length. In some embodiments, the PAM can be a 5’ PAM (i.e., located upstream of the 5’ end of the protospacer). In WSGR Docket No.59761-775.601 other embodiments, the PAM can be a 3’ PAM (i.e., located downstream of the 5’ end of the protospacer). In some embodiments, the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5’-NGG-3’ PAM.
  • the Cas protein of a prime editor has altered or non-canonical PAM specificities.
  • Exemplary PAM sequences and corresponding Cas variants are described in Table 3. It should be appreciated that for each of the variants provided, the Cas protein comprises one or more of the amino acid substitutions as indicated compared to a wild type Cas protein sequence, for example, the Cas9 as set forth in SEQ ID NO: 521.
  • the PAM motifs as shown in Table 3 are in the order of 5’ to 3’.
  • the Cas proteins of the disclosure can also be used to direct transcriptional control of target sequences, for example silencing transcription by sequence-specific binding to target sequences.
  • a Cas protein described herein may have one or mutations in a PAM recognition motif. In some embodiments, a Cas protein described herein may have altered PAM specificity.
  • N refers to any one of nucleotides A, G, C, and T
  • R refers to nucleotide A or G
  • W refers to A or T
  • V refers to A, C, or G
  • Y refers to nucleotide C or T.
  • a prime editor comprises a Cas9 polypeptide comprising one or mutations selected from the group consisting of: A61R, L111R, D1135V, R221K, A262T, R324L, N394K, S409I, S409I, E427G, E480K, M495V, N497A, Y515N, K526E, F539S, E543D, R654L, R661A, R661L, R691A, N692A, M694A, M694I, Q695A, H698A, R753G, M763I, K848A, K890N, Q926A, K1003A, R1060A, L1111R, R1114G, D1135E, D1135L, D11
  • a prime editor comprises a SaCas9 polypeptide.
  • the SaCas9 polypeptide comprises one or more of mutations E782K, N968K, and R1015H as compared to a wild type SaCas9.
  • a prime editor comprises a FnCas9 polypeptide, for example, a wildtype FnCas9 polypeptide or a FnCas9 polypeptide comprising one or more of mutations E1369R, E1449H, or R1556A as compared to the wild type FnCas9.
  • a prime editor comprises a Sc Cas9, for example, a wild type ScCas9 or a ScCas9 polypeptide comprises one or more of mutations I367K, G368D, I369K, H371L, T375S, T376G, and T1227K as compared to the wild type ScCas9.
  • a prime editor comprises a St1 Cas9 polypeptide, a St3 Cas9 polypeptide, or a SluCas9 polypeptide.
  • a prime editor comprises a Cas polypeptide that comprises a circular permutant Cas variant.
  • a Cas9 polypeptide of a prime editor may be engineered such that the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein, or a Cas9 nickase) are topically rearranged to retain the ability to bind DNA when complexed with a guide RNA (gRNA).
  • a Cas9 protein e.g., a wild type Cas9 protein, or a Cas9 nickase
  • An exemplary circular permutant configuration may be N-terminus-[original C-terminus]- [original N-terminus]-C-terminus.
  • Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.
  • the circular permutants of a Cas protein may have the following structure: N-terminus–[original C-terminus]–[optional linker]–[original N-terminus]–C- terminus.
  • a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 521): [0271] N-terminus–[1268-1368]–[optional linker]–[1-1267]–C-terminus; WSGR Docket No.59761-775.601 [0272] N-terminus–[1168-1368]–[optional linker]–[1-1167]–C-terminus; [0273] N-terminus–[1068-1368]–[optional linker]–[1-1067]–C-terminus; [0274] N-terminus–[968-1368]–[optional linker]–[1-967]–C-terminus; [0275] N-terminus–[868-1368]–[optional linker]–[1-867]–C-terminus; [0276] N-terminus–[768-1368]–[optional linker]–[1-767]–C-terminus; [
  • a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 521– 1368 amino acids of UniProtKB – Q99ZW2: [0286] N-terminus–[102-1368]–[optional linker]–[1-101]–C-terminus; [0287] N-terminus–[1028-1368]–[optional linker]–[1-1027]–C-terminus; [0288] N-terminus–[1041-1368]–[optional linker]–[1-1043]–C-terminus; [0289] N-terminus–[1249-1368]–[optional linker]–[1-1248]–C-terminus; or [0290] N-terminus–[1300-1368]–[optional linker]–[1-1299]–C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variant
  • a circular permutant Cas9 comprises any one of the following structures (amino acid positions as set forth in SEQ ID NO: 521) [0292] N-terminus–[103-1368]–[optional linker]–[1-102]–C-terminus: [0293] N-terminus–[1029-1368]–[optional linker]–[1-1028]–C-terminus; [0294] N-terminus–[1042-1368]–[optional linker]–[1-1041]–C-terminus; [0295] N-terminus–[1250-1368]–[optional linker]–[1-1249]–C-terminus; or [0296] N-terminus–[1301-1368]–[optional linker]–[1-1300]–C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).
  • the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker.
  • thee C-terminal fragment may correspond to the 95% or more of the C-terminal amino acids of a Cas9 (e.g., amino acids about 1300-1368 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof), or the 90%, 85%, 80%, 75%, 70%, 65%, 60%, WSGR Docket No.59761-775.601 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the C-terminal amino acids of a Cas9 (e.g., SEQ ID NO: 521 or a ortholog or a variant thereof).
  • a Cas9 e.g., amino acids about 1300-1368 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof
  • the N-terminal portion may correspond to 95% or more of the N-terminal amino acids of a Cas9 (e.g., amino acids about 1-1300 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof), or 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the N terminal amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).
  • a Cas9 e.g., amino acids about 1-1300 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof
  • 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of the N terminal amino acids of a Cas9 e.g., as set forth in SEQ ID NO:
  • the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker.
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the –terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or 1% of the amino acids of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof.
  • the C-terminal fragment that is rearranged to the N-terminus includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).
  • the C-terminal portion that is rearranged to the N-terminus includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 ( e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof.
  • the C- terminal portion that is rearranged to the N-terminus includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof).
  • a Cas9 e.g., as set forth in SEQ ID NO: 521 or corresponding amino acid positions thereof.
  • circular permutant Cas9 variants may be a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S.
  • pyogenes Cas9 of SEQ ID NO: 521 (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N- terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to precede the original N-terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue.
  • CP circular permutant
  • the CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain.
  • the CP site may be located (as set forth in SEQ ID NO: 521 or corresponding amino acid WSGR Docket No.59761-775.601 positions thereof) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282.
  • Nomenclature of these CP-Cas9 proteins may be referred to as Cas9-CP 181 , Cas9-CP 199 , Cas9- CP 230 , Cas9-CP 270 , Cas9-CP 310 , Cas9-CP 1010 , Cas9-CP 1016 , Cas9-CP 1023 , Cas9-CP 1029 , Cas9-CP 1041 , Cas9-CP 1247 , Cas9-CP 1249 , and Cas9-CP 1282 , respectively.
  • a prime editor comprises a Cas9 functional variant that is of smaller molecular weight than a wild type SpCas9 protein.
  • a smaller-sized Cas9 functional variant may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery.
  • a smaller-sized Cas9 functional variant is a Class 2 Type II Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type V Cas protein. In certain embodiments, a smaller-sized Cas9 functional variant is a Class 2 Type VI Cas protein. [0301] In some embodiments, a prime editor comprises a SpCas9 that is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons.
  • a prime editor comprises a Cas9 functional variant or functional fragment that is less than 1300 amino acids, less than 1290 amino acids, than less than 1280 amino acids, less than 1270 amino acids, less than 1260 amino acid, less than 1250 amino acids, less than 1240 amino acids, less than 1230 amino acids, less than 1220 amino acids, less than 1210 amino acids, less than 1200 amino acids, less than 1190 amino acids, less than 1180 amino acids, less than 1170 amino acids, less than 1160 amino acids, less than 1150 amino acids, less than 1140 amino acids, less than 1130 amino acids, less than 1120 amino acids, less than 1110 amino acids, less than 1100 amino acids, less than 1050 amino acids, less than 1000 amino acids, less than 950 amino acids, less than 900 amino acids, less than 850 amino acids, less than 800 amino acids, less than 750 amino acids, less than 700 amino acids, less than 650 amino acids, less than 600 amino acids, less than 550 amino acids, or less than 500 amino acids, but at least larger than
  • 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 WSGR Docket No.59761-775.601 modified versions thereof
  • the napDNAbp can be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago) domain, or a functional variant or fragment thereof.
  • a Cas9 a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (Cas
  • Prime editors described herein may also comprise Cas proteins other than Cas9.
  • a prime editor as described herein may comprise a Cas12a (Cpf1) polypeptide or functional variants thereof.
  • the Cas12a polypeptide comprises a mutation that reduces or abolishes the endonuclease domain of the Cas12a polypeptide.
  • the Cas12a polypeptide is a Cas12a nickase.
  • the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12a polypeptide.
  • a prime editor comprises a Cas protein that is a Cas12b (C2c1) or a Cas12c (C2c3) polypeptide.
  • the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12b (C2c1) or Cas12c (C2c3) WSGR Docket No.59761-775.601 protein.
  • the Cas protein is a Cas12b nickase or a Cas12c nickase.
  • the Cas protein is a Cas12e, a Cas12d, a Cas13, Cas14a, Cas14b, Cas14c, Cas14d
  • protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally- occurring Cas12e, Cas12d, Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Nuclear Localization Sequences [0306]
  • a prime editor further comprises one or more nuclear localization sequence (NLS).
  • a prime editor comprises a fusion protein, e.g., a fusion protein comprising a DNA binding domain and a DNA polymerase, that comprises one or more NLSs.
  • one or more polypeptides of the prime editor are fused to or linked to one or more NLSs.
  • the prime editor comprises a DNA binding domain and a DNA polymerase domain that are provided in trans, wherein the DNA binding domain and/or the DNA polymerase domain is fused or linked to one or more NLSs.
  • a prime editor or prime editing complex comprises at least one NLS.
  • a prime editor or prime editing complex comprises at least two NLSs. In some embodiments, a prime editor or prime editing complex comprises at least three NLSs. In some embodiments, a prime editor or prime editing complex comprises more than 4, 5, 6, 7, 8, 9 or 10 NLSs. In embodiments with two or more NLSs, the NLSs can be the same NLS, or they can be different NLSs. In some embodiments, the one or more NLSs of a prime editor comprise bipartite NLSs. [0308] An NLS can be expressed as part of a prime editor or prime editing complex. In some embodiments, a NLS can be positioned anywhere in a protein’s amino acid sequence, and comprise a short sequence of three, four, or more amino acids.
  • the location of the NLS fusion can be at the N- terminus, the C-terminus, or positioned within a sequence of a prime editor or a component thereof (e.g., inserted between the DNA-binding domain and the DNA polymerase domain of a prime editor fusion protein, between the DNA binding domain and a linker sequence, between a DNA polymerase and a linker sequence, between two linker sequences of a prime editor fusion protein or a component thereof, in either N-terminus to C-terminus or C-terminus to N-terminus order).
  • a prime editor is a fusion protein that comprises an NLS at the N terminus.
  • a prime editor is a fusion protein that comprises an NLS at the C terminus.
  • a prime editor is a fusion protein that comprises at least one NLS at both the N WSGR Docket No.59761-775.601 terminus and the C terminus.
  • the prime editor is a fusion protein that comprises two NLSs at the N terminus and/or the C terminus.
  • Any NLSs that are known in the art are contemplated herein.
  • the NLSs may be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more mutations relative to a wild-type NLS).
  • a nuclear localization signal is predominantly basic.
  • the one or more NLSs of a prime editor are rich in lysine and arginine residues. In some embodiments, the one or more NLSs of a prime editor comprise proline residues.
  • NLS sequences suitable for use with methods and compositions of the disclosure are provided in Table 5.
  • a NLS comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence provided in Table 5.
  • a NLS comprises an amino acid sequence selected from the group consisting of: the amino acid sequences provided in Table 5.
  • a prime editing composition comprises a polynucleotide that encodes a NLS that comprises an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence provided in Table 5.
  • a prime editing composition comprises a polynucleotide that encodes a NLS that comprises any one of the amino acid sequences provided in Table 5.
  • Table 5 Exemplary nuclear localization sequences
  • a nuclear localization signal (NLS) comprises SEQ ID NO: 551.
  • a NLS is a monopartite NLS.
  • a NLS is a SV40 large T antigen NLS comprising the sequence SEQ ID NO: 549.
  • a NLS is a bipartite NLS.
  • a bipartite NLS comprises two basic domains separated by a spacer sequence comprising a variable number of amino acids.
  • a NLS is a bipartite NLS.
  • a bipartite NLS consists of two basic domains separated by a linker sequence comprising a variable number of amino acids.
  • the linker amino acid sequence comprises a Xenopus nucleoplasmin NLS sequence SEQ ID NO: 566.
  • the NLS comprises a nucleoplasmin NLS sequence SEQ ID NO: 565.
  • a NLS is a noncanonical sequence such as M9 of the hnRNP Al protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS.
  • the DNA binding domain and the DNA polymerase domain of a prime editor may be fused to form a fusion protein, or may be joined by a peptide or protein linker, in any order from the N terminus to the C terminus.
  • a prime editor comprises a DNA binding domain fused or linked to the C-terminal end of a DNA polymerase domain.
  • a prime editor comprises a DNA binding domain fused or linked to the N-terminal end of a DNA polymerase domain.
  • the prime editor comprises a fusion protein comprising the structure NH2-[DNA binding domain]-[polymerase]-COOH; or NH2-[polymerase]-[DNA binding domain]- COOH, wherein each instance of indicates the presence of an optional linker sequence.
  • a prime editor comprises a fusion protein and a DNA polymerase domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA binding domain]-[RNA-protein recruitment polypeptide]-COOH.
  • a prime editor comprises a fusion protein and a DNA binding domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA polymerase domain]-[RNA-protein recruitment polypeptide]-COOH.
  • a prime editor fusion protein, a polypeptide component of a prime editor, or a polynucleotide encoding the prime editor fusion protein or polypeptide component may be split into an N-terminal half and a C-terminal half or polypeptides that encode the N-terminal half and the C terminal half, and provided to a target DNA in a cell separately.
  • a prime editor fusion protein may be split into a N-terminal and a C-terminal half for separate delivery in AAV vectors, and subsequently translated and colocalized in a target cell to reform the complete polypeptide or prime editor protein.
  • a prime editor comprises a N-terminal half fused to an intein-N, and a C-terminal half fused to an intein-C, or polynucleotides or vectors (e.g., AAV vectors) encoding each thereof.
  • the intein-N and the intein-C can be excised via protein trans-splicing, resulting in a complete prime editor fusion protein in the target cell.
  • an exemplary protein described herein may lack a methionine residue at the N- terminus.
  • a prime editor fusion protein comprises a Cas9(H840A) nickase and a wild type M-MLV RT.
  • a prime editor fusion protein comprises a Cas9(H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT.
  • the amino acid sequence of an exemplary prime editor fusion protein and its individual components is shown in Table 6.
  • a prime editor fusion protein comprises a Cas9 (R221K N394K H840A) nickase and a M-MLV RT that comprises amino acid substitutions D200N, T330P, T306K, W313F, and L603W compared to a wild type M-MLV RT.
  • an exemplary prime editor protein may comprise an amino acid sequence as set forth in any of the SEQ ID NOs: 567 or 568.
  • a prime editor fusion protein comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to any of the prime editor fusion sequences described herein (e.g., PE2 or PE3; Table 6, Table 7) or known in the art.
  • a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA.
  • the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence SEQ ID NO: 567 (Table 6).
  • Sequence of an exemplary prime editor fusion protein comprising a DNA binding domain (e.g., Cas9(H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 6.
  • a DNA binding domain e.g., Cas9(H840A)
  • a reverse transcriptase e.g., a variant MMLV RT having the following structure: [NLS]- [Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and its components are shown in Table 6.
  • a prime editor comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the exemplary prime editor sequence in Table 6.
  • a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g., Cas9((R221K N394K H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]- [Cas9((R221K N394K H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA.
  • a DNA binding domain e.g., Cas9((R221K N394K H840A)
  • a reverse transcriptase e.g., a variant MMLV RT having the following structure: [NLS]- [Cas9((R221K N394K H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(
  • the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence SEQ ID NO: 568 .
  • a prime editor comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the exemplary prime editor sequence in Table 7.
  • Table 7 lists an exemplary prime editor and its components WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 [0325]
  • Polypeptides comprising components of a prime editor may be fused via peptide linkers or may be provided in trans relevant to each other.
  • a reverse transcriptase may be expressed, delivered, or otherwise provided as an individual component rather than as a part of a fusion protein with the DNA binding domain.
  • components of the prime editor may be associated through non-peptide linkages or co-localization functions.
  • a prime editor further comprises additional components capable of interacting with, associating with, or capable of recruiting other components of the prime editor or the prime editing system.
  • a prime editor may comprise an RNA-protein recruitment polypeptide that can associate with an RNA-protein recruitment RNA aptamer.
  • an RNA-protein recruitment polypeptide can recruit, or be recruited by, a specific RNA sequence.
  • Non limiting examples of RNA- protein recruitment polypeptide and RNA aptamer pairs include a MS2 coat protein and a MS2 RNA hairpin, a PCP polypeptide and a PP7 RNA hairpin, a Com polypeptide and a Com RNA hairpin, a Ku protein and a telomerase Ku binding RNA motif, and a Sm7 protein and a telomerase Sm7 binding RNA motif.
  • the prime editor comprises a DNA binding domain fused or linked to an RNA-protein recruitment polypeptide.
  • the prime editor comprises a DNA polymerase domain fused or linked to an RNA-protein recruitment polypeptide.
  • the DNA binding domain and the DNA polymerase domain fused to the RNA-protein recruitment polypeptide, or the DNA binding domain fused to the RNA-protein recruitment polypeptide and the DNA polymerase domain are co-localized by the corresponding RNA-protein recruitment RNA aptamer of the RNA-protein recruitment polypeptide.
  • an MS2 coat protein fused or linked to the DNA polymerase and a MS2 hairpin installed on the PEgRNA for co-localization of the DNA polymerase and the RNA-guided DNA binding domain e.g., a Cas9 nickase.
  • a prime editor comprises a polypeptide domain, an MS2 coat protein (MCP), that recognizes an MS2 hairpin.
  • MCP MS2 coat protein
  • the nucleotide sequence of the MS2 hairpin (or equivalently referred to as the “MS2 aptamer”) is a sequence provided in Table 8.
  • the amino acid sequence of the MCP is a sequence provided in Table 8.
  • a linker can be any chemical group or a molecule linking two molecules or moieties, e.g., a DNA binding domain and a polymerase domain of a prime editor.
  • a linker is an organic molecule, group, polymer, or chemical moiety.
  • the linker comprises a non-peptide moiety.
  • the linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length, for example, a polynucleotide sequence.
  • the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).
  • two or more components of a prime editor are linked to each other by a peptide linker.
  • a peptide linker is 5-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30- 35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length.
  • the peptide linker is 16 amino acids in length, 24 amino acids in length, 64 amino acids in length, or 96 amino acids in length. [0331] In some embodiments, a linker comprises 1-100 amino acids. [0332] Non-limiting examples of linkers are provided in Table 9. In some embodiments, a linker comprises any one of the amino acid sequences set forth in Table 9, or any combination thereof. [0333] Table 9. Illustrative Peptide Linker Sequences WSGR Docket No.59761-775.601 [0334] In certain embodiments, two or more components of a prime editor are linked to each other by a non-peptide linker. In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage.
  • the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker.
  • the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.).
  • the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid.
  • the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3- aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.).
  • the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker.
  • a nucleophile e.g., thiol, amino
  • any electrophile may be used as part of the linker.
  • exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.
  • PEgRNA for editing of CFTR gene refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA.
  • the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing.
  • Nucleotide edit or “intended nucleotide edit” refers to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene.
  • Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene, or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA that replaces the editing target sequence, as compared to the editing target sequence.
  • 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.
  • the PEgRNA comprises a gRNA core that associates with a DNA WSGR Docket No.59761-775.601 binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor.
  • the PEgRNA further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence may be referred to as an extension arm.
  • the extension arm comprises a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis.
  • PBS primer binding site sequence
  • the PBS is complementary or substantially complementary to a free 3’ end on the edit strand of the target gene at a nick site generated by the prime editor.
  • the extension arm further comprises an editing template that comprises one or more intended nucleotide edits to be incorporated in the target gene by prime editing.
  • the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the prime editor, for example, a reverse transcriptase domain.
  • the reverse transcriptase editing template may also be referred to herein as an RT template, or RTT.
  • the editing template comprises partial complementarity to an editing target sequence in the target gene, e.g., an CFTR gene. In some embodiments, the editing template comprises substantial or partial complementarity to the editing target sequence except at the position of the intended nucleotide edits to be incorporated into the target gene.
  • An exemplary architecture of a PEgRNA including its components is as demonstrated in Fig.2. [0337]
  • a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide.
  • a PEgRNA is a chimeric polynucleotide that includes both RNA and DNA nucleotides.
  • a PEgRNA can include DNA in the spacer sequence, the gRNA core, or the extension arm.
  • a PEgRNA comprises DNA in the spacer sequence.
  • the entire spacer sequence of a PEgRNA is a DNA sequence.
  • the PEgRNA comprises DNA in the gRNA core, for example, in a stem region of the gRNA core.
  • the PEgRNA comprises DNA in the extension arm, for example, in the editing template.
  • An editing template that comprises a DNA sequence may serve as a DNA synthesis template for a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase.
  • the PEgRNA may be a chimeric polynucleotide that comprises RNA in the spacer, gRNA core, and/or the PBS sequences and DNA in the editing template.
  • Components of a PEgRNA may be arranged in a modular fashion.
  • the spacer and the extension arm comprising a primer binding site sequence (PBS) and an editing template, e.g., a reverse transcriptase template (RTT), can be interchangeably located in the 5’ portion of the PEgRNA, the 3’ portion of the PEgRNA, or in the middle of the gRNA core.
  • a PEgRNA comprises a PBS and an editing template sequence in 5’ to 3’ order.
  • the gRNA core of a PEgRNA of this disclosure may be located in between a spacer and an extension arm of the PEgRNA. In some embodiments, the gRNA core of a PEgRNA may be located at the 3’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be WSGR Docket No.59761-775.601 located at the 5’ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 3’ end of an extension arm. In some embodiments, the gRNA core of a PEgRNA may be located at the 5’ end of an extension arm.
  • 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.
  • a PEgRNA comprises a single polynucleotide molecule that comprises the spacer sequence, the gRNA core, and the extension arm. In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules. In some embodiments, a PEgRNA comprise a first polynucleotide molecule that comprises the spacer and a portion of the gRNA core, and a second polynucleotide molecule that comprises the rest of the gRNA core and the extension arm.
  • the gRNA core portion in the first polynucleotide molecule and the gRNA core portion in the second polynucleotide molecule are at least partly complementary to each other.
  • the PEgRNA may comprise a first polynucleotide comprising the spacer and a first portion of a gRNA core comprising, which may also be referred to as a crRNA.
  • the PEgRNA comprise a second polynucleotide comprising a second portion of the gRNA core and the extension arm, wherein the second portion of the gRNA core may also be referred to as a trans-activating crRNA, or tracr RNA.
  • the crRNA portion and the tracr RNA portion of the gRNA core are at least partially complementary to each other.
  • the partially complementary portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem, as exemplified in FIG.3.
  • a spacer sequence comprises a region that has substantial complementarity to a search target sequence on the target strand of a double stranded target DNA, e.g., an CFTR gene.
  • the spacer sequence of a PEgRNA is identical or substantially identical to a protospacer sequence on the edit strand of the target gene (except that the protospacer sequence comprises thymine and the spacer sequence may comprise uracil). In some embodiments, the spacer sequence is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a search target sequence in the target gene. In some embodiments, the spacer comprises is substantially complementary to the search target sequence. [0341] In some embodiments, the length of the spacer varies from about 10 nucleotides to about 100 nucleotides.
  • the spacer is 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length. In some embodiments, the spacer is from 15 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, or WSGR Docket No.59761-775.601 20 to 30 nucleotides in length.
  • the spacer is 16 to 22 nucleotides in length, e.g., about 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, the spacer is 17 to 22 nucleotides in length, e.g., about 17, 18, 19, 20, 21, or 22 nucleotides in length.
  • a PEgRNA or a nick guide RNA sequence, or fragments thereof such as a spacer, PBS, or RTT sequence
  • the letter “T” or “thymine” indicates a nucleobase in a DNA sequence that encodes the PEgRNA or guide RNA sequence, and is intended to refer to a uracil (U) nucleobase of the PEgRNA or guide RNA or any chemically modified uracil nucleobase known in the art, such as 5-methoxyuracil.
  • the extension arm of a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g., an RTT).
  • the extension arm may be partially complementary to the spacer.
  • the editing template e.g., RTT
  • the editing template e.g., RTT
  • the editing template e.g., RTT
  • the primer binding site PBS
  • An extension arm of a PEgRNA may comprise a primer binding site sequence (PBS, or PBS sequence) that comprises complementarity to and can hybridize with a free 3’ end of a single stranded DNA in the target gene (e.g., the CFTR gene) generated by nicking with a prime editor at the nick site on the PAM strand.
  • PBS primer binding site sequence
  • the length of the PBS sequence may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA. [0346] In some embodiments, the PBS is about 3 to 19 nucleotides in length. In some embodiments, the PBS is about 3 to 17 nucleotides in length.
  • the PBS is about 4 to 16 nucleotides, about 6 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleotides in length.
  • the PBS is 8 to 17 nucleotides in length. In some embodiments, the PBS is 8 to 16 nucleotides in length. In some embodiments, the PBS is 8 to 15 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length.
  • the PBS is 8 to 13 nucleotides in length. In some embodiments, the PBS is 8 to 12 nucleotides in length. In some embodiments, the PBS is 8 to 11 nucleotides in length. In some embodiments, the PBS is 8 to 10 nucleotides in length. In some embodiments, the PBS is 8 or 9 nucleotides in length. In some embodiments, the PBS is 16 or 17 nucleotides in length. In some embodiments, the PBS is 15 to 17 nucleotides in length. In some embodiments, the PBS is 14 to 17 nucleotides in length. In some embodiments, the PBS is 13 to 17 nucleotides in length.
  • the PBS is 12 to 17 nucleotides in length. In some embodiments, the PBS is 11 to 17 nucleotides in length. In some embodiments, the PBS is 10 to 17 nucleotides in length. In some embodiments, the PBS is 9 to 17 nucleotides in length. In some embodiments, the PBS is about 7 to 15 nucleotides in length. In some embodiments, the PBS is 6, 7, WSGR Docket No.59761-775.601 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length. In some embodiments, the PBS is 9 to 14 nucleotides in length.
  • the PBS can be 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the PBS is 11 or 12 nucleotides in length. In some embodiments, the PBS is 11 to 13 nucleotides in length. In some embodiments, the PBS is 11 to 14 nucleotides in length. [0347]
  • the PBS may be complementary or substantially complementary to a DNA sequence in the edit strand of the target gene. By annealing with the edit strand at a free hydroxy group, e.g., a free 3’ end generated by prime editor nicking, the PBS may initiate synthesis of a new single stranded DNA encoded by the editing template at the nick site.
  • the PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the edit strand of the target gene (e.g., the CFTR gene). In some embodiments, the PBS is perfectly complementary, or 100% complementary, to a region of the edit strand of the target gene (e.g., the CFTR gene).
  • An extension arm of a PEgRNA may comprise an editing template that serves as a DNA synthesis template for the DNA polymerase in a prime editor during prime editing.
  • the length of an editing template may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA.
  • the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is referred to as a reverse transcription editing template (RTT).
  • the editing template e.g., RTT
  • RTT is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.
  • the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length. In some embodiments, the RTT is 10 to 110 nucleotides in length.
  • the RTT is 10 to 109, 10 to 108, 10 to 107, 10 to 106, 10 to 105, 10 to 104, 10 to 103, 10 to 102, or 10 to 101 nucleotides in length. In some embodiments, the RTT is at least 8 and no more than 50 nucleotides in length. In some embodiments, the RTT is at least 8 and no more than 25 nucleotides in length. In some embodiments, the RTT is about 10 to about 20 nucleotides in length. In some embodiments, the RTT is about 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the RTT is 11 to 17 nucleotides in length.
  • the RTT is 12 to 17 nucleotides in length. In some embodiments, the RTT is 12 to 16 nucleotides in length. In some embodiments, the RTT is 13 to 17 nucleotides in length. In some embodiments, the RTT is 11, 12, 13, 14, 15, 16, or 17 nucleotides in length. In some embodiments the RTT is 12 nucleotides in length. In some embodiments, the RTT is 16 nucleotides in length. In some embodiments the RTT is 17 WSGR Docket No.59761-775.601 nucleotides in length. In some embodiments, the RTT is about 20 to about 30 nucleotides in length.
  • the RTT is about 20 to about 25 nucleotides in length. In some embodiments, the RTT is about 20 to about 25 nucleotides in length. In some embodiments, the RTT is 21 to 24 nucleotides in length.
  • the editing template (e.g., RTT) sequence is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary to the editing target sequence on the edit strand of the target gene. In some embodiments, the editing template sequence (e.g., RTT) is substantially complementary to the editing target sequence.
  • the editing template sequence (e.g., RTT) is complementary to the editing target sequence except at positions of the intended nucleotide edits to be incorporated int the target gene.
  • the editing template comprises a nucleotide sequence comprising about 85% to about 95% complementarity to an editing target sequence in the edit strand in the target gene (e.g., the CFTR gene).
  • the editing template comprises about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementarity to an editing target sequence in the edit strand of the target gene (e.g., the CFTR gene).
  • An intended nucleotide edit in an editing template of a PEgRNA may comprise various types of alterations as compared to the target gene sequence.
  • the nucleotide edit is a single nucleotide substitution as compared to the target gene sequence.
  • the nucleotide edit is a deletion as compared to the target gene sequence.
  • the nucleotide edit is an insertion as compared to the target gene sequence.
  • the editing template comprises one to ten intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises one or more intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises two or more intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises three or more intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises four or more, five or more, or six or more intended nucleotide edits as compared to the target gene sequence.
  • the editing template comprises two single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises three single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises four, five, or six single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, a nucleotide substitution comprises an adenine (A)-to-thymine (T) substitution. In some embodiments, a nucleotide substitution comprises an A-to-guanine (G) substitution.
  • a nucleotide substitution comprises an A-to-cytosine (C) substitution.
  • a nucleotide WSGR Docket No.59761-775.601 substitution comprises a T-A substitution.
  • a nucleotide substitution comprises a T-G substitution.
  • a nucleotide substitution comprises a T-C substitution.
  • a nucleotide substitution comprises a G-to-A substitution.
  • a nucleotide substitution comprises a G-to-T substitution.
  • a nucleotide substitution comprises a G-to-C substitution.
  • a nucleotide substitution comprises a C-to-A substitution. In some embodiments, a nucleotide substitution comprises a C-to-T substitution. In some embodiments, a nucleotide substitution comprises a C-to-G substitution.
  • a nucleotide insertion is at least 1, at least 2, at least 3, at least 4, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length.
  • a nucleotide insertion is from 1 to 2 nucleotides, from 1 to 3 nucleotides, from 1 to 4 nucleotides, from 1 to 5 nucleotides, form 2 to 5 nucleotides, from 3 to 5 nucleotides, from 3 to 6 nucleotides, from 3 to 8 nucleotides, from 4 to 9 nucleotides, from 5 to 10 nucleotides, from 6 to 11 nucleotides, from 7 to 12 nucleotides, from 8 to 13 nucleotides, from 9 to 14 nucleotides, from 10 to 15 nucleotides, from 11 to 16 nucleotides, from 12 to 17 nucleotides, from 13 to 18 nucleotides, from 14 to 19 nucleotides, from 15 to 20 nucleotides in length.
  • a nucleotide insertion is a single nucleotide insertion. In some embodiments, a nucleotide insertion comprises insertion of two nucleotides.
  • the editing template of a PEgRNA may comprise one or more intended nucleotide edits, compared to the CFTR gene to be edited. Position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to particular nucleotides (e.g., mutations) in the CFTR target gene may vary. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to or homologous to the protospacer sequence.
  • the nucleotide edit is in a region of the PEgRNA corresponding to a region of the CFTR gene outside of the protospacer sequence.
  • the position of a nucleotide edit incorporation in the target gene may be referred to relative to position of the nick site.
  • position of an intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides apart from the nick site.
  • position of an intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides downstream of the nick site on the PAM strand (or the non-target strand, or the edit strand) of the double stranded target DNA.
  • position of the intended nucleotide edit in the editing template may be referred to by aligning the WSGR Docket No.59761-775.601 editing template with the partially complementary editing target sequence on the edit strand, and referring to nucleotide positions on the editing strand where the intended nucleotide edit is incorporated.
  • a nucleotide edit in an editing template is at a position corresponding to a position about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 nucleotides apart from the nick site.
  • a nucleotide edit in an editing template is at a position corresponding to a position about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18
  • a nucleotide edit in an editing template is at a position corresponding to a position about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, , 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleot
  • the relative positions of the intended nucleotide edit(s) and nick site may be referred to by numbers.
  • the nucleotide immediately downstream of the nick site on a PAM strand may be referred to as at position 0.
  • the nucleotide immediately upstream of the nick site on the PAM strand (or the non-target strand, or the edit strand) may be referred to as at position -1.
  • nucleotides downstream of position 0 on the PAM strand may be referred to as at positions +1, +2, +3, +4, ... +n, and the nucleotides upstream of position -1 on the PAM strand may be referred to as at positions -2, -3, -4, ..., -n.
  • the nucleotide in the editing template that corresponds to position 0 when the editing template is aligned with the partially complementary editing target sequence by complementarity may also be referred to as position 0 in the editing template
  • the nucleotides in the editing template corresponding to the nucleotides at positions +1, +2, +3, +4, ..., +n on the PAM strand of the double stranded target DNA may also be referred to as at positions +1, +2, +3, +4, ..., +n in the editing template
  • the nucleotides in the editing template corresponding to the nucleotides at positions -1, - 2, -3, -4, ..., -n on the PAM strand on the double stranded target DNA may also be referred to as at positions -1, -2, -3, -4, ..., -n on the editing template, even though when the PEgRNA is viewed as a is at position +n of the editing template relative to position 0.
  • the intended nucleotide edit may be incorporated at position +n of the PAM strand of the double stranded target DNA (and subsequently, the target strand of the double stranded target DNA) by prime editing.
  • the corresponding positions of the intended nucleotide edit incorporated in the CFTR gene may also be referred to based on the nicking position generated by a prime editor based on sequence homology and complementarity.
  • the distance between the nucleotide edit to be incorporated into the CFTR gene and the nick site may be determined by the position of the nick site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence.
  • the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand).
  • the distance between the nick site and the nucleotide edit refers to the 5’ most position of the nucleotide edit for a nick that creates a 3’ free end on the edit strand (i.e., the “near position” of the nucleotide edit to the nick site).
  • the nick-to-edit distance is 2 to 106 nucleotides.
  • the WSGR Docket No.59761-775.601 nick-to-edit distance is 2 to 105, 2 to 104, 2 to 103, 2 to 102, 2 to 101, 2 to 100, 2 to 99, 2 to 98, or 2 to 97 nucleotides.
  • the nick-to-edit distance is 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 nucleotides.
  • the nick-to-edit distance is 2 to 25, 2 to 20, 2 to 15, or 2 to 10 nucleotides.
  • the nick-to-edit distance is 2, 3, 4, 5, 6, or 7 nucleotides in length.
  • the nick-to-edit distance is 28 nucleotides. In some embodiments, the nick-to-edit distance is 22 nucleotides. In some embodiments, the nick-to-edit distance is 21 nucleotides. In some embodiments, the nick-to-edit distance is 17 nucleotides. In some embodiments, the nick-to-edit distance is 16 nucleotides. In some embodiments, the nick-to-edit distance is 4 nucleotides. [0357] The RTT length and the nick-to-edit distance relate to the length of the portion of the RTT that is upstream of (i.e.5’ to) the 5’-most edit in the RTT and is complementary to the edit strand.
  • the editing template comprises at least 4 contiguous nucleotides of complementarity with the edit strand wherein the at least 4 nucleotides contiguous are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more contiguous nucleotides of complementarity with the edit strand wherein the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more contiguous nucleotides are located upstream of the 5’ most edit in the editing template.
  • the editing template comprises 20-25, 25-30, 30-35, 35-40, 45-45, or 45-50 contiguous nucleotides of complementarity with the edit strand wherein the 20-25, 25-30, 30- 35, 35-40, 45-45, or 45-50 or more contiguous nucleotides are located upstream of the 5’ most edit in the editing template.
  • the editing template comprises 9-14 contiguous nucleotides of complementarity with the edit strand wherein the 9-14 contiguous nucleotides are located upstream of the 5’ most edit in the editing template.
  • the editing template comprises 6-10 contiguous nucleotides of complementarity with the edit strand wherein the 6-10 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 10 contiguous nucleotides of complementarity with the edit strand wherein the 10 contiguous nucleotides are located upstream of the 5’ most edit in the editing template. In some embodiments, the editing template comprises 9 contiguous nucleotides of complementarity with the edit strand wherein the 9 contiguous nucleotides are located upstream of the 5’ most edit in the editing template.
  • positions of the one or more intended nucleotide edits may be referred to relevant to components of the PEgRNA.
  • an intended nucleotide edit may be 5’ or 3’ to the PBS.
  • a PEgRNA comprises the structure, from 5’ to 3’: a spacer, a gRNA core, an editing template, and a PBS.
  • the intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides upstream to the 5’ most nucleotide of the WSGR Docket No.59761-775.601 PBS.
  • the intended nucleotide edit is 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 4 nucleo
  • the corresponding positions of the intended nucleotide edit incorporated in the target gene may also be referred to based on the nicking position generated by a prime editor based on sequence homology and complementarity.
  • the distance between the nucleotide edit to be incorporated into the target CFTR gene and the nick site (also referred to as the “nick to edit distance”) may be determined by the position of the nick site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence.
  • the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand) generated by the prime editor, such that the distance between the nick site and the intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the position of the nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the position of the nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides upstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides downstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0 base pair from the nick site on the edit strand, that is, the editing position is at the same position as the nick site.
  • the distance between the nick site and the nucleotide edit refers to the 5’ most position of the nucleotide edit for a nick that creates a 3’ free end on the edit strand (i.e., the “near position” of the nucleotide edit to the nick site).
  • the distance between WSGR Docket No.59761-775.601 the nick site and a PAM position edit refers to the 5’ most position of the nucleotide edit and the 5’ most position of the PAM sequence.
  • the editing template extends beyond a nucleotide edit to be incorporated to the target CFTR gene sequence.
  • the editing template comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.
  • the editing template can comprise a second edit relative to a target sequence.
  • the second edit can be designed to mutate or otherwise silence a PAM sequence such that a corresponding nucleic acid guided nuclease or CRISPR nuclease is no longer able to cleave the target sequence (such edits referred to as “PAM silencing edits).
  • PAM silencing edits may prevent the Cas, e.g., Cas9, nickase, from re-nicking the edit strand before the edit is incorporated in the target strand, therefore improving prime editing efficiency.
  • a PAM silencing edit is a synonymous edit that does not alter the amino acid sequence encoded by the CFTR gene after incorporation of the edit.
  • a PAM silencing edit is at a position corresponding to a coding region, e.g., an exon, of a CFTR gene. In some embodiments, a PAM silencing edit is at a position corresponding to a non-coding region, e.g., an intron, of a CFTR gene. In some embodiments, the edits in an intron of a CFTR gene is not at a position that corresponds to intron-exon junction and the edit does not affect transcript splicing.
  • the length of the editing template is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides longer than the nick to edit distance.
  • the nick to edit distance is 8 nucleotides
  • the editing template is 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, or 10 to 80 nucleotides in length.
  • the nick to edit distance is 22 nucleotides
  • the editing template is 24 to 28, 24 to 30, 24 to 32, 24 to 34, 24 to 36, 24 to 37, 24 to 38, 24 to 40, 24 to 45, 24 to 50, 24 to 55, 24 to 60, 24 to 65, 24 to 70, 24 to 75, 24 to 80, 24 to 85, 24 to 90, 24 to 95, 24 to 100, 24 to 105, 24 to 100, 24 to 105, or 24 to 110 nucleotides in length.
  • the editing template comprises at least 4 to 30 base pairs 3’ to the nucleotide edit to be incorporated to the target CFTR gene sequence.
  • the editing template comprises at least 4 to 25 base pairs 3’ to the nucleotide edit to be incorporated to the WSGR Docket No.59761-775.601 target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 20 base pairs 3’ to the nucleotide edit to be incorporated to the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 30 base pairs 5’ to the nucleotide edit to be incorporated to the target CFTR gene sequence. In some embodiments, the editing template comprises at least 4 to 25 base pairs 5’ to the nucleotide edit to be incorporated to the target CFTR gene sequence.
  • the editing template comprises at least 4 to 20 base pairs 5’ to the nucleotide edit to be incorporated to the target CFTR gene sequence.
  • the editing template comprises an adenine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template comprises a guanine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template comprises an uracil at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template comprises a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template does not comprise a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5’-spacer-gRNA core-RTT-PBS-3’ orientation, the 5’ most nucleobase is the “first base”).
  • the editing template of a PEgRNA may encode a new single stranded DNA (e.g., by reverse transcription) to replace an editing target sequence in the target gene.
  • the editing target sequence in the edit strand of the target gene is replaced by the newly synthesized strand, and the nucleotide edit(s) are incorporated in the region of the target gene.
  • the target gene is an CFTR gene.
  • the editing template of the PEgRNA encodes a newly synthesized single stranded DNA that comprises a wild type CFTR gene sequence.
  • the newly synthesized DNA strand replaces the editing target sequence in the target CFTR gene, wherein the editing target sequence (or the endogenous sequence complementary to the editing target sequence on the target strand of the CFTR gene) comprises a mutation or a nucleotide alteration compared to a wild type CFTR gene.
  • the mutation is associated with cystic fibrosis.
  • the newly synthesized single stranded DNA encoded by the editing template replaces the editing target sequence and corrects the mutation in the editing target sequence of the target CFTR gene.
  • the editing target sequence comprises position 117587778 in human chromosome 7.
  • the editing target sequence comprises a mutation as compared to a wild type CFTR gene, wherein the mutation is a nucleotide insertion, a nucleotide deletion, a WSGR Docket No.59761-775.601 nucleotide substitution, two or more nucleotide substitutions, or any combination thereof.
  • the mutation results in a premature stop codon in a mRNA encoded by the CFTR gene.
  • the mutation results in an amino acid alteration in the CFTR protein encoded by the CFTR gene.
  • the mutation results in an amino acid substitution in the CFTR protein encoded by the CFTR gene. In some embodiments, the mutation results in a truncated CFTR polypeptide encoded by the CFTR gene as compared to a wild type CFTR polypeptide. In some embodiments, the mutation results in an aberrant CFTR polypeptide encoded by the CFTR gene. In some embodiments, the mutation results in a CFTR polypeptide encoded by the CFTR gene that has reduced biological activity as compared to a wild type CFTR polypeptide. In some embodiments, the mutation results in a CFTR polypeptide encoded by the CFTR gene that has abolished biological activity as compared to a wild type CFTR polypeptide.
  • the editing target sequence comprises a mutation corresponding to position 1624 of the coding sequence of the CFTR protein. In some embodiments, the editing target sequence comprises a c.1624G->T mutation (editing target sequence on the sense strand) or a corresponding C->A mutation (editing target sequence on the antisense strand) at position 1624 of the coding sequence of the CFTR protein.
  • the editing template comprises one or more intended nucleotide edits compared to the sequence on the target strand of the CFTR gene that is complementary to the editing target sequence. In some embodiments, the editing template encodes a single stranded DNA that comprises one or more intended nucleotide edits compared to the editing target sequence.
  • the single stranded DNA replaces the editing target sequence by prime editing, thereby incorporating the one or more intended nucleotide edits.
  • the one or more intended nucleotide edits encodes a T->G substitution at a position corresponding to position 1624 of the coding sequence of the CFTR protein compared to the editing target sequence (editing target sequence on the sense strand).
  • the one or more intended nucleotide edits encodes a A->C substitution at a position corresponding to position 1624 of the coding sequence of the CFTR protein compared to the editing target sequence (editing target sequence on the antisense strand).
  • incorporation of the one or more intended nucleotide edits corrects the mutation in the editing target sequence to wild type nucleotides at corresponding positions in the target CFTR gene.
  • “correcting” a mutation means restoring a wild type sequence at the place of the mutation in the double stranded target DNA e.g. target gene, by prime editing.
  • incorporation of the one or more nucleotide edits can correct any mutations in the CFTR gene that are in the portion of the gene that shares homology with the editing template. [0370]
  • incorporation of the one or more intended nucleotide edits results in expression of a functional CFTR protein.
  • incorporation of the one or more intended nucleotide edits results in a nucleotide substitution, insertion, or deletion that results in a codon that encodes a wild type amino acid as compared to a wild type CFTR polypeptide, WSGR Docket No.59761-775.601 while the codon is not the same as the wild type nucleotide at the corresponding position.
  • the editing template comprises and/or encodes a wild type CFTR gene sequence.
  • incorporation of the one or more intended nucleotide edits does not correct the mutation in the editing target sequence to wild type sequence, but allows for expression of a functional CFTR protein encoded by the CFTR gene.
  • incorporation of the one or more intended nucleotide edits results in one or more codons that are different from a wild type codon but encode one or more amino acids same as the wild type CFTR protein.
  • incorporation of the one or more intended nucleotide edits results in one or more codons that encode one or more amino acids different from the wild type CFTR protein, but allows for expression of a functional CFTR protein.
  • Exemplary amino acid sequence of wild type CFTR protein is provided in SEQ ID NO: 751.
  • a guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor.
  • the gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a DNA nickase of the prime editor.
  • a prime editor such as a DNA nickase of the prime editor.
  • the gRNA core is capable of binding to a Cas9-based prime editor.
  • the gRNA core is capable of binding to a Cpf1-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cas12b-based prime editor. [0374] In some embodiments, the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins. For example, in a Cas9 based prime editing system, the gRNA core of a PEgRNA may comprise one or more regions of a base paired “lower stem” adjacent to the spacer sequence and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs.
  • the gRNA core may further comprise a “nexus” distal from the spacer sequence, followed by a hairpin structure, e.g., at the 3’ end, as exemplified in FIG.3.
  • the gRNA core comprises modified nucleotides as compared to a wild type gRNA core in the lower stem, upper stem, and/or the hairpin.
  • nucleotides in the lower stem, upper stem, an/or the hairpin regions may be modified, deleted, or replaced.
  • RNA nucleotides in the lower stem, upper stem, an/or the hairpin regions may be replaced with one or more DNA sequences.
  • the gRNA core comprises unmodified or wild type RNA sequences in the nexus and/or the bulge regions. In some embodiments, the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU-AAAAC pairing element.
  • a prime editing system comprises a prime WSGR Docket No.59761-775.601 editor and a PEgRNA, wherein the prime editor comprises a SpCas9 nickase or a variant thereof, and the gRNA core of the PEgRNA comprises a sequence capable of binding to a SpCas9.
  • gRNA core sequences known in the art are also contemplated in the prime editing compositions described herein.
  • the PEgRNA and/or ngRNA comprises a universal gRNA core.
  • a universal gRNA core can be used in a PEgRNA or ngRNA that comprises any spacer that has a PAM sequence compatible with the Cas9 protein capable of binding to the gRNA core, and any PBS and RTT sequences designed to incorporate the intended nucleotide edit(s) based on the spacer.
  • the PEgRNA and/or ngRNA comprises a universal gRNA core that comprises a nucleic acid sequence selected from the Table 10.
  • the PEgRNA and/or ngRNA comprises a gRNA core that comprises a nucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences on Table 10.
  • Table 10 Exemplary nucleic acid sequences of universal gRNA core (also referred to herein as gRNA scaffold) for PEgRNAs compatible with SpCas9 prime editors. The sequences in Table 10 are annotated with SEQ ID NO as required by ST.26 standard.
  • a PEgRNA comprises a sequence specific gRNA core.
  • a sequence specific gRNA core may be designed to form optimal secondary or tertiary structure with other components of the PEgRNA, for example, the spacer, RTT, and/or PBS.
  • a prime editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA).
  • ngRNA nick guide RNA
  • a ngRNA comprises a spacer (referred to as a ngRNA spacer or ng spacer) and a gRNA core, wherein the spacer of the ngRNA comprises a region of complementarity to the edit strand, and wherein the gRNA core can interact with a Cas, e.g., Cas9, of a prime editor.
  • a Cas e.g., Cas9
  • an ngRNA may bind to the edit strand and direct Cas nickase to generate a nick on the non- edit strand (or target strand).
  • the nick on the non-edit strand directs endogenous DNA repair machinery to use the edit strand as a template for repair of the non-edit strand, which may increase efficiency of prime editing.
  • the non-edit strand is nicked by a prime editor localized to the non-edit strand by the ngRNA. Accordingly, also provided herein are PEgRNA systems comprising at least one PEgRNA and at least one ngRNA.
  • a prime editing system comprising a PEgRNA (or one or more polynucleotide encoding the PEgRNA) and a prime editor protein (or one or more polynucleotides encoding the prime editor), may be referred to as a PE2 prime editing system and the corresponding editing approach referred to as PE2 approach or PE2 strategy.
  • a PE2 system does not contain a ngRNA.
  • a prime editing system comprising a PEgRNA (or one or more polynucleotide encoding the PEgRNA), a prime editor protein (or one or more polynucleotides encoding the prime editor), and a ngRNA (or one or more polynucleotides encoding the ngRNA) may be referred to as a “PE3” prime editing system.
  • an ng spacer sequence is complementary to, and may hybridize with the second search target sequence only after an intended nucleotide edit has been incorporated on the edit strand, by the editing template of a PEgRNA.
  • Such ngRNA may be referred to a “PE3b” ngRNA, and the prime editing system a PE3b prime editing system.
  • the ng search target sequence is located on the non-target strand, within 10 base pairs to 100 base pairs of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the ng target search target sequence is within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 WSGR Docket No.59761-775.601 bp, or 100 bp of an intended nucleotide edit incorporated by the PEgRNA on the edit strand.
  • the 5’ ends of the ng search target sequence and the PEgRNA search target sequence are within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bp apart from each other. In some embodiments, the 5’ ends of the ng search target sequence and the PEgRNA search target sequence are within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp apart from each other.
  • the gRNA core of a PEgRNA or ngRNA can be any gRNA scaffold sequence that is capable of interacting with a Cas protein that recognizes the corresponding PAM of the PEgRNA or ngRNA.
  • gRNA core of a PEgRNA or a ngRNA comprises a sequence selected from from the sequences in Table 10.
  • the PEgRNA and/or ngRNA comprises a 3’ motif.
  • the PEgRNA and/or ngRNA comprises a 3’ motif comprising a nucleic acid sequence that has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the sequences provided in Table 11.
  • the PEgRNA and/or ngRNA comprises a 3’ motif comprising a nucleic acid sequence selected from the group consisting of: the sequences provided in Table 11. [0384]
  • Table 11 Illustrative nucleic acid sequences for 3’ motif (e.g., universal 3’ motif). The sequences in Table 11 are annotated with SEQ ID NO as required by ST.26 standard.
  • a PEgRNA further comprises a nucleotide linker.
  • the secondary structure is linked to one or more other component of a PEgRNA via a linker.
  • a secondary structure or a 3’ motif of a PEgRNA is linked to one or more other components of a PEgRNA via a linker.
  • the secondary structure is at the 3’ end of the PEgRNA (e.g., a RTT, or a PBS) and is linked to the 3’ end of a PBS via a linker.
  • a 3’ motif is at the 3’ end of the PEgRNA and is linked to the 3’ end of a PEgRNA (e.g., a RTT or a PBS) via a linker.
  • 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.
  • the linker is a nucleotide linker that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the WSGR Docket No.59761-775.601 linker is 5 to 10 nucleotides in length.
  • the linker is 10 to 20 nucleotides in length.
  • the linker is 15 to 25 nucleotides in length.
  • the linker is 8 nucleotides in length.
  • the linker is designed to minimize base pairing between the linker and another component of the PEgRNA. In some embodiments, the linker is designed to minimize base pairing between the linker and the spacer. In some embodiments, the linker is designed to minimize base pairing between the linker and the PBS. In some embodiments, the linker is designed to minimize base pairing between the linker and the editing template. In some embodiments, the linker is designed to minimize base pairing between the linker and the sequence of the RNA secondary structure. In some embodiments, the linker is optimized to minimize base pairing between the linker and another component of the PEgRNA, in order of the following priority: spacer, PBS, editing template and then scaffold.
  • base paring probability is calculated using ViennaRNA 2.0 ,as described in Lorenz, R. et al. ViennaRNA package 2.0. Algorithms Mol. Biol.6, incorporated by reference in its entirety herein, under standard parameters (37 °C, 1 M NaCl, 0.05 M MgCl2).
  • the PEgRNA comprises secondary RNA structure, such as, but not limited to, aptamers, hairpins, stem/loops, toeloops, and/or RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein).
  • a PEgRNA (or ngRNA) comprises an additional secondary structure at the 5’ end.
  • a PEgRNA (or ngRNA) comprises an additional secondary structure at the 3’ end.
  • the secondary structure comprises a pseudoknot.
  • the secondary structure comprises a pseudoknot derived from a virus.
  • the secondary structure comprises a pseudoknot of a Moloney murine leukemia virus (M-MLV) genome (a mpknot).
  • M-MLV Moloney murine leukemia virus
  • the secondary structure comprises a nucleotide sequence selected from the group consisting of sequences provided in Table 12, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence provided in Table 12.
  • the secondary structure comprises a quadruplex.
  • the secondary structure comprises a G-quadruplex.
  • the secondary structure comprises a riboswitch aptamer.
  • the secondary structure comprises a riboswitch aptamer derived from a prequeosine-1 riboswitch aptamer. In some embodiments, the secondary structure comprises a modified prequeosine-1 riboswitch aptamer.
  • the PEgRNA comprises a toeloop element having the sequence 5’-GAAANNNNN-3’, wherein N is any nucleobase.
  • the secondary RNA structure is positioned within the spacer. In some embodiments, the secondary structure is positioned within the extension arm. In some embodiments, the secondary structure is positioned within the gRNA core.
  • the secondary structure is positioned between the spacer and the gRNA core, between the gRNA core and WSGR Docket No.59761-775.601 the extension arm, or between the spacer and the extension arm. In some embodiments, the secondary structure is positioned between the PBS and the editing template. In some embodiments the secondary structure is positioned at the 3’ end or at the 5’ end of the PEgRNA.
  • the PEgRNA comprises a RNA secondary structure and/or a linker disclosed in Nelson et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. (2021), the entirety of which is incorporated herein by reference. [0389] Exemplary secondary structure sequences are provided in Table 12.
  • the PEgRNA comprises a self-cleaving element.
  • the self-cleaving element improves transcription and/or processing of the PEgRNA when transcribed form the nucleotide encoding the PEgRNA.
  • the PEgRNA comprises a hairpin or a RNA quadruplex.
  • the PEgRNA comprises a self- cleaving ribozyme element, for example, a hammerhead, a pistol, a hatchet, a hairpin, a VS, a twister, or a twister sister ribozyme.
  • the PEgRNA comprises a HDV ribozyme.
  • the PEgRNA comprises a hairpin recognized by Csy4.
  • the PEgRNA comprises an ENE motif.
  • the PEgRNA comprises an element for nuclear expression (ENE) from MALAT1 lnc RNA.
  • the PEgRNA comprises an ENE element from Kaposi’s sarcoma-associated herpesvirus (KSHV).
  • the PEgRNA comprises a 3’ box of a U1 snRNA. In some embodiments, the PEgRNA forms a circular RNA. [0392] In some embodiments, the PEgRNA comprises a RNA secondary structure or a motif that improves binding to the DNA-RNA duple or enhances PEgRNA activity. In some embodiments, the PEgRNA comprises a sequence derived from a native nucleotide element involved in reverse transcription, e.g., initiation of retroviral transcription. In some embodiments, the PEgRNA comprises a sequence of, or derived from, a primer binding site of a substrate of a reverse transcriptase, a polypurine tract (PPT), or a kissing loop.
  • PPT polypurine tract
  • the PEgRNA comprises a dimerization motif, a kissing loop, or a GNRA tetraloop – tetraloop receptor pair that results in circularization of the PEgRNA.
  • the PEgRNA comprises a RNA secondary structure of a motif that results in physical separation of the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity.
  • the PEgRNA comprises a secondary structure or motif, e.g., a 5’ or 3’ extension in the spacer region that form a toehold or hairpin, wherein the secondary structure or motif competes favorably against annealing between the spacer and the PBS of the PEgRNA, thereby prevents occlusion of the spacer and improves PEgRNA activity.
  • a PEgRNA additionally comprises a sequence provided in Table 13.
  • a PEgRNA comprises the sequence of SEQ ID NO: 735 at the 3’ end.
  • a PEgRNA comprises the structure [spacer]-[gRNA core]-[editing template]- [PBS]-[3’ motif or secondary structure selected from Tables 11-13] or [spacer]-[gRNA core]-[editing template]-[PBS]- [3’ motif or secondary structure selected from Tables 11-13]. [0395] In some embodiments, the PEgRNA comprises the sequence of SEQ ID NO: 737 at the 5’ end and/or the sequence UGGGAGACGUCCCACC (SEQ ID NO: 738) at the 3’ end.
  • the PEgRNA comprises the following structure (M-MLV kissing loop): GGUGGGAGACGUCCCACC (SEQ ID NO: 737)-[spacer]-[gRNA core]-[editing template]-[PBS]- UGGGAGACGUCCCACC (SEQ ID NO: 738), or GGUGGGAGACGUCCCACC (SEQ ID NO: 737)- [spacer]-[gRNA core]-[editing template]-[PBS]-UGGGAGACGUCCCACC-(U)n (SEQ ID NO: 739), wherein n is an integer between 3 and 7.
  • the kissing loop structure is italicized.
  • the PEgRNA comprises the sequence of SEQ ID NO: 740 at the 5’ end and/or the sequence SEQ ID NO: 747 at the 3’ end.
  • the PEgRNA comprises the following structure (VS ribozyme kissing loop): GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 740)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG (SEQ ID NO: 747), or GAGCAGCAUGGCGUCGCUGCUCAC (SEQ ID NO: 740)-[spacer]-[gRNA core]-[editing template]-[PBS]- CCAUCAGUUGACACCCUGAGG-(U)n (SEQ ID NO: 742), wherein n is an integer between 3 and 7.
  • the PEgRNA comprises the sequence of SEQ ID NO: 743 at the 5’ end and/or the sequence of SEQ ID NO: 744 at the 3’ end.
  • the PEgRNA comprises the following structure (tetraloop and receptor): GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 743)-[spacer]-[gRNA core]-[editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG (SEQ ID NO: 744), or GCAGACCUAAGUGGUGACAUAUGGUCUG (SEQ ID NO: 743)-[spacer]-[gRNA core]-[editing template]-[PBS]- CAUGCGAUUAGAAAUAAUCGCAUG-(U)n (SEQ ID NO: 745), wherein n is an integer between 3 and 7.
  • the PEgRNA comprises the sequence GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGG CGAAUGGGAC (SEQ ID NO: 735) or UCUGCCAUCAAAGCUGCGACCGUGCUCAGUCUGGUGGGAGACGUCCCACCGGCCGGCA UGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAUGGCGAAUGGG AC (SEQ ID NO: 746).
  • Example sequences of components within PEgRNA are provided in Table 13. [0400] Table 13. Exemplary sequences of components within PEgRNA.
  • a PEgRNA may comprise one or more linkers.
  • a PEgRNA comprises a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase.
  • the chemical linker may function to prevent reverse transcription of the gRNA core.
  • a secondary structure or a 3’ motif is directly connected to the 3’ end of a PBS.
  • a secondary structure or a 3’ motif is directly connected to the 3’ end of a PBS via a nucleotide linker.
  • the nucleotide linker may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the nucleotide linker is about 4-12 nucleotides in length. In some embodiments, the nucleotide linker is about 4 nucleotides in length. In some embodiments, the nucleotide linker is a universal linker, e.g., as set forth in AACATTGA (SEQ ID NO: 706). In some embodiments, the nucleotide linker is a sequence specific linker, for example, designed to optimize the secondary or tertiary structure of the PEgRNA.
  • a PEgRNA may also comprise optional modifiers, e.g. end modifier region.
  • a PEgRNA comprises at least one nucleotide that is not part of a spacer, a gRNA core, or an extension arm.
  • the optional sequence modifiers could be [0405]
  • a PEgRNA comprises a short stretch of uracil at the 5’ end or the 3’ end.
  • a PEgRNA comprising a 3’ extension arm comprises a “UUU” sequence at the 3’ end of the extension arm.
  • a PEgRNA is transcribed from a nucleotide encoding the PEgRNA, for example, a DNA plasmid encoding the PEgRNA.
  • the PEgRNA comprises [0407]
  • a PEgRNA and/or an ngRNA of this disclosure may include modified nucleotides, e.g., chemically modified DNA or RNA nucleobases, and may include one or more nucleobase analogs (e.g., modifications which might add functionality, such as temperature resilience).
  • PEgRNAs and/or ngRNAs as described herein may be chemically modified.
  • the phrase “chemical modifications,” as used herein, can include modifications which WSGR Docket No.59761-775.601 introduce chemistries which differ from those seen in naturally occurring DNA or RNAs, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in DNA or RNA molecules).
  • the PEgRNAs provided in the disclosure may further comprise nucleotides added to the 5’ of the PEgRNAs.
  • the PEgRNA further comprises 1, 2, or 3 additional nucleotides added to the 5’ end.
  • the additional nucleotides can be guanine, cytosine, adenine, or uracil.
  • the additional nucleotide at the 5’ end of the PEgRNA is a guanine or cytosine.
  • the additional nucleotides can be chemically or biologically modified.
  • the PEgRNAs provided in the disclosure may further comprise nucleotides to the 3’ of the PEgRNAs.
  • the PEgRNA further comprises 1, 2, or 3 additional nucleotides to the 3’ end.
  • the additional nucleotides can be guanine, cytosine, adenine, or uracil.
  • the additional nucleotides at the 3’ end of the PEgRNA is a polynucleotide comprising at least 1 uracil. In some embodiments, the additional nucleotides can be chemically or biologically modified.
  • a PEgRNA or ngRNA is produced by transcription from a template nucleotide, for example, a template plasmid.
  • a polynucleotide encoding the PEgRNA or ngRNA is appended with one or more additional nucleotides that improves PEgRNA or ngRNA function or expression, e.g., expression from a plasmid that encodes the PEgRNA or ngRNA.
  • a polynucleotide encoding a PEgRNA or ngRNA is appended with one or more additional nucleotides at the 5’ end or at the 3’ end.
  • the polynucleotide encoding the PEgRNA or ngRNA is appended with a guanine at the 5’ end, for example, if the first nucleotide at the 5’ end of the spacer is not a guanine.
  • a polynucleotide encoding the PEgRNA or ngRNA is appended with nucleotide sequence CACC at the 5’ end.
  • the polynucleotide encoding the PEgRNA or ngRNA is appended with an additional nucleotide adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS is a Thymine.
  • the polynucleotide encoding the PEgRNA or ngRNA is appended with additional nucleotide sequence TTTTTT, TTTTTTT, TTTTT, or TTTT at the 3’ end.
  • the PEgRNA or ngRNA comprises the appended nucleotides from the transcription template.
  • the PEgRNA or ngRNA further comprises one or more nucleotides at the 5’ end or the 3’ end in addition to spacer, PBS, and RTT sequences.
  • the PEgRNA or ngRNA further comprises a guanine at the 5’ end, for example, when the first nucleotide at the 5’ end of the spacer is not a guanine.
  • the PEgRNA or ngRNA further comprises nucleotide sequence CACC at the 5’ end.
  • the PEgRNA or ngRNA further comprises an adenine at the 3’ end, for example, if the last nucleotide at the 3’ end of the PBS WSGR Docket No.59761-775.601 is a thymine.
  • the PEgRNA or ngRNA further comprises nucleotide sequence UUUUUU, UUUUU, UUUUU, or UUUU at the 3’ end.
  • a PEgRNA or a nick guide RNA can be chemically synthesized, or can be assembled or cloned and transcribed from a DNA sequence, e.g., a plasmid DNA sequence, or by any RNA oligonucleotide synthesis method known in the art.
  • DNA sequence that encodes a PEgRNA (or ngRNA) may be designed to append one or to enhance PEgRNA transcription. For example, in some embodiments, a DNA sequence that encodes end.
  • the PEgRNA may comprise an (or nick guide RNA) may be designed to append a sequence that enhances transcription, e.g., a Kozak
  • the PEgRNA (or nick guide RNA) may comprise an appended sequence CACC guide RNA) may be designed to append the sequence TTT, TTTT, TTTTT, TTTTTT, TTTTTTT at end.
  • a PEgRNA or a ngRNA comprises the sequence TTTTTTT (sequence sequence (e.g., TTTT; sequence number 707) at the 3’ end.
  • a PEgRNA or a ngRNA comprises a transcription adaptation sequence (e.g., TTTTTTT sequence number 708) at the 3’ end.
  • the sequences in sequence number 707, and sequence number 708 are annotated with a sequence number as required by ST.26 standard.
  • the sequences set forth in sequence number 707, and sequence number 708 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard.
  • the PEgRNAs and/or ngRNAs provided in this disclosure may have undergone a chemical or biological modifications.
  • Modifications may be made at any position within a PEgRNA or ngRNA and may include modification to a nucleobase or to a phosphate backbone of the PEgRNA or ngRNA.
  • chemical modifications can be a structure guided modifications.
  • a chemical modification is at the 5’ end and/or the 3’ end of a PEgRNA.
  • a chemical modification is at the 5’ end and/or the 3’ end of a ngRNA.
  • a chemical modification may be within the spacer sequence, the extension arm, the editing template sequence, or the primer binding site of a PEgRNA.
  • a chemical modification may be within the spacer sequence or the gRNA core of a PEgRNA or a ngRNA. In some embodiments, a chemical modification may be within the 3’ most nucleotides of a PEgRNA or ngRNA. In some embodiments, a chemical modification may be within the 3’ most end of a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modification may be within the 5’ most end of a PEgRNA or ngRNA. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 more chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 or more chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 more chemically modified nucleotides at the 5’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 5’ end.
  • a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3’ end.
  • a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 5’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more contiguous chemically modified nucleotides near the 3’ end.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is not modified, and the 1, 2, 3, 4, 5, or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3’ order.
  • a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides near the 3’ end, where the 3’ most nucleotide is WSGR Docket No.59761-775.601 not modified, and the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides precede the 3’ most nucleotide in a 5’-to-3’ order.
  • a PEgRNA or ngRNA comprises one or more chemical modified nucleotides in the gRNA core.
  • the gRNA core of a PEgRNA may comprise one or more regions of a base paired lower stem, a base paired upper stem, where the lower stem and upper stem may be connected by a bulge comprising unpaired RNAs.
  • the gRNA core may further comprise a nexus distal from the spacer sequence.
  • the gRNA core comprises one or more chemically modified nucleotides in the lower stem, upper stem, and/or the hairpin regions.
  • all of the nucleotides in the lower stem, upper stem, and/or the hairpin regions are chemically modified.
  • phosphorothioate bond modification any other chemical modifications known in the art, or any combination thereof.
  • a chemical modification may also include, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the PEgRNA and/or ngRNA (e.g., modifications to one or both of the 3’ and 5’ ends of a guide RNA molecule).
  • Prime Editing Compositions can include the addition of bases to an RNA sequence, complexing the RNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).
  • agent e.g., a protein or a complementary nucleic acid molecule
  • elements which change the structure of an RNA molecule e.g., which form secondary structures.
  • Prime Editing Compositions Disclosed herein, in some embodiments, are compositions, systems, and methods using a prime editing composition.
  • the term “prime editing composition” or “prime editing system” refers to compositions involved in the method of prime editing as described herein.
  • a prime editing composition may include a prime editor, e.g., a prime editor fusion protein, and a PEgRNA.
  • a prime editing composition may further comprise additional elements, such as second strand nicking ngRNAs.
  • a prime editing composition comprises a prime editor fusion protein complexed with a PEgRNA and optionally complexed with a ngRNA.
  • the prime editing composition comprises a prime editor comprising a DNA binding domain and a DNA polymerase domain associated with each other through a PEgRNA.
  • the prime editing composition may comprise a prime editor comprising a DNA binding domain and a DNA polymerase domain linked to each other by an WSGR Docket No.59761-775.601 RNA-protein recruitment aptamer RNA sequence, which is linked to a PEgRNA.
  • a prime editing composition comprises a PEgRNA and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein.
  • a prime editing composition comprises a PEgRNA, a ngRNA, and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein.
  • a prime editing composition comprises multiple polynucleotides, polynucleotide constructs, or vectors, each of which encodes one or more prime editing composition components.
  • the PEgRNA of a prime editing composition is associated with the DNA binding domain, e.g., a Cas9 nickase, of the prime editor. In some embodiments, the PEgRNA of a prime editing composition complexes with the DNA binding domain of a prime editor and directs the prime editor to the target DNA.
  • a prime editing composition comprises one or more polynucleotides that encode prime editor components and/or PEgRNA or ngRNAs. In some embodiments, a prime editing composition comprises a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain.
  • a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, and (ii) a PEgRNA or a polynucleotide encoding the PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iii) an ngRNA or a polynucleotide encoding the ngRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, and (iii) a PEgRNA or a polynucleotide encoding the PEgRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iv) an ngRNA or a polynucleotide encoding the ngRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEgRNA or a
  • a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N and (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C.
  • a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) an ngRNA or a WSGR Docket No.59761-775.601 polynucleotide encoding the ngRNA.
  • a prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain.
  • the DNA binding domain is a Cas protein domain, e.g., a Cas9 nickase.
  • the prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) a ngRNA or a polynucleotide encoding the ngRNA.
  • a prime editing system comprises one or more polynucleotides encoding one or more prime editor polypeptides, wherein activity of the prime editing system can be temporally regulated by controlling the timing in which the vectors are delivered.
  • a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA can be delivered simultaneously.
  • a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA can be delivered sequentially.
  • a polynucleotide encoding a component of a prime editing system can further comprise an element that is capable of modifying the intracellular half-life of the polynucleotide and/or modulating translational control.
  • the polynucleotide is a RNA, for example, an mRNA.
  • the half-life of the polynucleotide, e.g., the RNA may be increased.
  • the half-life of the polynucleotide, e.g., the RNA may be decreased.
  • the element may be capable of increasing the stability of the polynucleotide, e.g., the RNA.
  • the element may be capable of decreasing the UTR of the RNA.
  • the element may include a polyadenylation signal (PA).
  • the element may include a cap, e.g., an upstream mRNA or PEgRNA end.
  • the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription.
  • the element may include at least one AU-rich element (ARE).
  • the AREs may be bound by ARE binding proteins (ARE-BPs) in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment.
  • the destabilizing element may promote RNA decay, affect RNA stability, or activate translation.
  • the ARE may comprise 50 to 150 nucleotides in length. In some embodiments, the ARE may comprise at least one copy of the sequence AUUUA. In some embodiments, at least one ARE may be Virus Posttranscriptional Regulatory Element (WPRE). In further embodiments, the element is a modified and/or truncated WPRE sequence that is capable of enhancing expression from the WSGR Docket No.59761-775.601 In some embodiments, the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts.
  • WPRE Virus Posttranscriptional Regulatory Element
  • the polynucleotide e.g., a vector, encoding the PE or the PEgRNA may be self-destroyed via cleavage of a target sequence present on the polynucleotide, e.g., a vector. The cleavage may prevent continued transcription of a PE or a PEgRNA.
  • Polynucleotides encoding prime editing composition components can be DNA, RNA, or any combination thereof.
  • a polynucleotide encoding a prime editing composition component is an expression construct.
  • a polynucleotide encoding a prime editing composition component is a vector.
  • the vector is a DNA vector.
  • the vector is a plasmid.
  • the vector is a virus vector, e.g., a retroviral vector, adenoviral vector, lentiviral vector, herpesvirus vector, or an adeno-associated virus vector (AAV).
  • AAV adeno-associated virus vector
  • polynucleotides encoding polypeptide components of a prime editing composition are codon optimized by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • a polynucleotide encoding a polypeptide component of a prime editing composition are operably linked to one or more expression regulatory elements, for example, a promoter, a 3’ UTR, a 5’ UTR, or any combination thereof.
  • a polynucleotide encoding a prime editing composition component is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA comprises a Cap at the 5’ end and/or a poly A tail at the 3’ end.
  • PEgRNA Prime Editing guide RNA
  • Tables 18-20 Each of Tables 18-20 contains three columns. The left column is the sequence number. The middle column provides the sequence of the component, labeled with a SEQ ID NO where allowed by the ST.26 standard. Although all the sequences provided in Tables 18-20 are RNA sequences, “T” is used instead of a “U” in the sequences for consistency with the ST.26 standard. The right column contains a description of the sequence.
  • All of the PEgRNAs disclosed in Tables 18-20 are designed to correct a c.1624G->T mutation in the CFTR gene that results in a G542x nonsense mutation (x indicates a premature stop codon; also referred to as G542ter) associated with cystic fibrosis.
  • the PEgRNA disclosed in Tables 18-20 are also WSGR Docket No.59761-775.601 capable of correcting any other mutations in the CFTR gene that are in the portion of the gene that shares homology or complementarity with the editing template/RTT.
  • the PEgRNAs exemplified in Tables 18-20 comprise: (a) a spacer comprising at its 3’ end a sequence corresponding to a listed PEgRNA spacer; (b) a gRNA core capable of complexing with a Cas9 protein; and (c) an extension arm comprising: (i) an editing template comprising at its 3’ end any RTT sequence from the same table as the PEgRNA spacer, and (ii) a primer binding site (PBS) comprising at its 5’ end any PBS sequence from the same table as the PEgRNA spacer.
  • the PEgRNA spacer can be, for example, 17-22 nucleotides in length.
  • the PEgRNA spacers in Tables 18-20 are annotated in column 3 according to their associated PAM sequence, enabling selection of a prime editor comprising an appropriate Cas9 protein.
  • a prime editor comprising a SpCas9 (H840A) nickase can be used for prime editing with a spacer sequence adjacent to a NGG PAM sequence
  • a prime editor comprising a SpCas9 (H840A, D1135V, G1218R, R1335Q, T1337R) nickase variant (the SpCas9 “VRQR” variant) can be used for prime editing with a spacer sequence adjacent to an NGA PAM sequence, wherein N is any nucleotide selected from A, G, C, and T.
  • the editing template can be referred to as a reverse transcription template (RTT).
  • the editing template can encode a wildtype CFTR gene sequence and are annotated in column 3 of Tables 18-20 as simply “RTT”.
  • the editing template can encode one or more synonymous mutations relative to the wildtype CFTR gene.
  • the editing template can be designed to encode one or more PAM silencing mutations besides the nucleotide edit(s) designed to correct the G542X mutation.
  • the PBS can be, for example, 5 to 19 nucleotides in length. In some embodiments, the PBS is 8 to 15 nucleotides in length. In some embodiments, the PBS is 7-15 nucleotides in length. In some embodiments, the PBS is 10-15 nucleotides in length. In some embodiments, the PBS is 8 nucleotides in length.
  • the PBS is 11 nucleotides in length. In some embodiments, the PBS is 7, 9, 11, 13, or 15 nucleotides in length.
  • the gRNA core can be any gRNA core capable of binding to a Cas9 protein.
  • the gRNA core can be a canonical SpCas9 guide RNA or a variant thereof. Exemplary gRNA cores can be found in Table 10.
  • the PEgRNA provided in Tables 18-20 can comprise, from 5’ to 3’, the spacer, the gRNA core, the edit template, and the PBS. The 3’ end of the editing template can be contiguous with the 5’ end of the PBS.
  • the PEgRNA can comprise multiple RNA molecules or can be a single RNA WSGR Docket No.59761-775.601 molecule.
  • Any PEgRNA exemplified in Tables 18-20 may comprise, or further comprise, a 3’ motif at the 3’ end of the extension arm, such as a universal motif, a sequence specific motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides.
  • the PEgRNA comprises 4 U nucleotides at its 3’ end; without being bound by theory, this is believed to increase PEgRNA stability.
  • the PEgRNA comprises a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used.
  • exemplary 3’ motifs can be found in Tables 11 and 12.
  • a sequence specific motif is used that is designed to hybridize with a portion of the RTT while not covering the PBS. Whether a universal or sequence specific motif is used, it can be connected to the 3’ end of the PBS via a linker sequence. Exemplary linker sequences can be found in Table 14. Alternatively, the 3’ motif can be directly connected to the 3’ end of the PBS without a linker sequence.
  • PEgRNA sequences exemplified in Tables 18-20 may include adaptations for transcription from a nucleic acid template (e.g., with a U6 promoter).
  • Such transcription adaptations can include the addition of a 5’ terminal G if the spacer of the PEgRNA begins with another nucleotide, the addition of 6 or 7 U nucleotides at the 3’ end of the extension arm, or both.
  • the 3’ terminal U series may serve as a transcription stop signal; the actual transcribed PEgRNA may therefore contain from 1 to 7 (e.g., 4) 3’ U nucleotides.
  • Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the PBS and the 3’ terminal U series.
  • the PEgRNA may include an additional A nucleotide between the PBS and the 3’ terminal U series.
  • the expression adaptations e.g., a 5’ terminal G, are annotated in Tables 18-20.
  • such adaptation nucleotide(s) may be removed from the PEgRNA sequence.
  • a chemically synthesized PEgRNA sequence may comprise at its 5’ end nucleotides 2-n of a PEgRNA sequence provided in Tables 18-20.
  • the PEgRNA sequences exemplified in Tables 18-20 may be chemically synthesized.
  • Such chemically synthesized PEgRNA may comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof.
  • the PEgRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • the chemically synthesized PEgRNA comprises an additional 4 U nucleotides on its 3’ end and the chemical modifications, if included, would comprise 3’ mU*mU*mU*U and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • Any of the PEgRNAs of Tables 18-20 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA).
  • ngRNA can comprise a spacer and a gRNA core WSGR Docket No.59761-775.601 capable of complexing with a Cas9 protein.
  • the ngRNA spacer can be, for example, 17-22 nucleotides in length. In some embodiments, the ngRNA spacer comprises at its 5’ end nucleotides 4- 20 of an ngRNA spacer listed in the same table as the PEgRNA.
  • the ngRNA can comprise multiple RNA molecules (e.g., a crRNA containing the ngRNA spacer and a tracrRNA) or can be a single gRNA molecule.
  • the ngRNA and ngRNA spacers in Tables 18-20 are annotated with their corresponding PAM sequences. It can be advantageous to select a ngRNA spacer that has a PAM sequence compatible with the Cas9 protein used in the Prime Editor, thus avoiding the need to use two different Cas9 proteins.
  • the ngRNA is capable of directing a complexed Cas9 protein to bind the edit strand of the CFTR gene; thus, a complexed Cas9 nickase containing a nuclease inactivating mutation in the HNH domain will nick the non-edit strand.
  • a PE3 ngRNA spacer has perfect complementarity to the edit strand both pre- and post-edit; a PE3b ngRNA spacer has perfect complementarity to the edit strand post-edit.
  • the PE3b ngRNAs annotated with a * followed by a number code in Tables 18-20 have perfect complementarity to the edit strand post-edit with a PEgRNA containing an RTT from the same Table and annotated with the same number code.
  • any ngRNA exemplified in Tables 18-20 may comprise, or further comprise, a 3’ motif at the 3’ end of the scaffold, such as a universal motif, or a series of 1, 2, 3, 4, 5, 6, 7 or more U nucleotides.
  • the ngRNA comprises 4 U nucleotides at its 3’ end; without being bound by theory, this is believed to increase ngRNA stability.
  • the ngRNA comprises a universal or structural 3’ motif that is capable of forming a tertiary structure on its own such as a hairpin, a pseudoknot, or other RNA structure is used.
  • ngRNA sequences exemplified in Tables 18-20 may include adaptations for transcription from a nucleic acid template (e.g., with a U6 promoter). Such transcription adaptations can include the addition of a 5’ terminal G if the spacer of the ngRNA begins with another nucleotide, the addition of 6 or 7 U nucleotides at the 3’ end of the ngRNA, or both.
  • the 3’ terminal U series may serve as a transcription stop signal; the actual transcribed ngRNA may therefore contain from 1 to 7 (e.g., 4) 3’ U nucleotides.
  • Such transcription-adapted sequences may further comprise a universal or sequence specific motif between the gRNA core and the 3’ terminal U series.
  • the ngRNA sequences exemplified in Tables 18-20 may be chemically synthesized.
  • Such chemically synthesized ngRNA may comprise one or more chemical modifications, such as phosphorothioate (PS) bond(s), 2’-O-methylated (2’-Ome) nucleotides, or a combination thereof.
  • PS phosphorothioate
  • the ngRNA comprise 3’ mN*mN*mN*N and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence WSGR Docket No.59761-775.601 of a phosphorothioate bond.
  • the chemically synthesized ngRNA comprises an additional 4 U nucleotides on its 3’ end and the chemical modifications, if included, would comprise 3’ mU*mU*mU*U and 5’mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates the presence of a phosphorothioate bond.
  • compositions comprising any of the prime editing composition components, for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, ngRNAs, and/or prime editing complexes described herein.
  • pharmaceutical composition refers to a composition formulated for pharmaceutical use.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.
  • a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).
  • a pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.)
  • Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • compositions can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Methods of Editing [0440] The methods and compositions disclosed herein can be used to edit a target gene of interest by prime editing.
  • the prime editing method comprises contacting a target gene, e.g., a CFTR gene,
  • the target gene is double stranded, and comprises two strands of DNA complementary to each other.
  • the contacting with a PEgRNA and the contacting with a prime editor are performed sequentially.
  • the contacting with a prime editor is performed after the contacting with a PEgRNA.
  • the contacting with a PEgRNA is performed after the contacting with a prime editor.
  • the contacting with a PEgRNA, and the contacting with a prime editor are performed simultaneously.
  • the PEgRNA and the prime editor are associated in a complex prior to contacting a target gene.
  • contacting the target gene with the prime editing composition results in binding of the PEgRNA to a target strand of the target gene, e.g., a CFTR gene. In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a search target sequence on the target strand of the target gene upon contacting with the PEgRNA. In some embodiments, contacting the target gene with the prime editing composition results in binding of a spacer sequence of the PEgRNA to a search target sequence with the search target sequence on the target strand of the target gene upon said contacting of the PEgRNA.
  • contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, e.g. the target CFTR gene, upon the contacting of the PE composition with the target gene.
  • the DNA binding domain of the PE associates with the PEgRNA.
  • the PE binds the target gene, e.g. a CFTR gene, directed by the PEgRNA. Accordingly, in some embodiments, the contacting of the target gene result in binding of a DNA binding domain of a prime editor of the target CFTR gene directed by the PEgRNA.
  • contacting the target gene with the prime editing composition results in a nick in an edit strand of the target gene, e.g., a CFTR gene by the prime editor upon contacting with the target gene, thereby generating a nicked on the edit strand of the target gene.
  • contacting the target gene with the prime editing composition results in a single- stranded DNA comprising a free 3 ⁇ end at the nick site of the edit strand of the target gene.
  • contacting the target gene with the prime editing composition results in a nick in the edit strand of the target gene by a DNA binding domain of the prime editor, thereby generating a single-stranded DNA comprising a free 3 ⁇ end at the nick site.
  • the DNA binding domain of the prime editor is a Cas domain.
  • the DNA binding domain of the prime editor is a Cas9.
  • the DNA binding domain of the prime editor is a Cas9 nickase.
  • contacting the target gene with the prime editing composition results in hybridization of the PEgRNA with the 3’ end of the nicked single-stranded DNA, thereby priming DNA polymerization by a DNA polymerase domain of the prime editor.
  • the free 3’ end of the single-stranded DNA generated at the nick site hybridizes to a primer binding site sequence (PBS) of the contacted PEgRNA, thereby priming DNA polymerization.
  • PBS primer binding site sequence
  • the DNA polymerization is reverse transcription catalyzed by a reverse transcriptase domain of the prime editor.
  • the method comprises contacting the target gene with a DNA polymerase, e.g., a reverse transcriptase, as a part of a prime editor fusion protein or prime editing complex (in cis), or as a separate protein (in trans).
  • a DNA polymerase e.g., a reverse transcriptase
  • contacting the target gene with the prime editing composition generates an edited single stranded DNA that is coded by the editing template of the PEgRNA by DNA polymerase mediated polymerization from the 3’ free end of the single-stranded DNA at the nick site.
  • the editing template of the PEgRNA comprises one or more intended nucleotide edits compared to endogenous sequence of the target gene, e.g., a CFTR gene.
  • the intended nucleotide edits are incorporated in the target gene, by excision of the 5’ single stranded DNA of the edit strand of the target gene generated at the nick site and DNA repair.
  • the intended nucleotide edits are incorporated in the target gene by excision of the editing target sequence and DNA repair.
  • excision of the 5’ single stranded DNA of the edit strand generated at the nick site is by a flap endonuclease.
  • the flap nuclease is FEN1.
  • the method further comprises contacting the target gene with a flap endonuclease.
  • the flap endonuclease is provided as a part of a prime editor fusion protein.
  • the flap endonuclease is provided in trans. [0447] In some embodiments, contacting the target gene with the prime editing composition generates a mismatched heteroduplex comprising the edit strand of the target gene that comprises the edited single stranded DNA, and the unedited target strand of the target gene.
  • the endogenous DNA repair and replication may resolve the mismatched edited DNA to incorporate the nucleotide change(s) to form the desired edited target gene.
  • the method further comprises contacting the target gene, e.g., a CFTR gene, with a nick guide (ngRNA) disclosed herein.
  • the ngRNA comprises a spacer that binds a second search target sequence on the edit strand of the target gene.
  • the contacted ngRNA directs the PE to introduce a nick in the target strand of the target gene.
  • the nick on the target strand results in endogenous DNA repair machinery to use the edit strand to repair the non-edit strand, thereby incorporating the intended nucleotide edit in both strand of the target gene and modifying the target gene.
  • the ngRNA comprises a spacer sequence that is complementary to, and may hybridize with, the second search target sequence on the edit strand only after the intended nucleotide edit(s) are incorporated in the edit strand of the target gene.
  • the target gene is contacted by the ngRNA, the PEgRNA, and the PE simultaneously.
  • the ngRNA, the PEgRNA, and the PE form a complex when they contact the target gene.
  • the target gene is contacted with the ngRNA, the PEgRNA, and the prime editor sequentially.
  • the target gene is contacted with the ngRNA and/or the PEgRNA after contacting the target gene with the PE.
  • the target gene is contacted with the ngRNA and/or the PEgRNA before contacting the target gene with the prime editor.
  • the target gene e.g., a CFTR gene, is in a cell.
  • the prime editing method comprises introducing a PEgRNA, a prime editor, and/or a ngRNA into the cell that has the target gene.
  • the prime editing method comprises introducing into the cell that has the target gene with a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA.
  • the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex prior to the introduction into the cell.
  • the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex after the introduction into the cell.
  • the prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, including ribonucleoprotein (RNPs), lipid nanoparticles (LNPs), viral vectors, non-viral vectors, mRNA delivery, and physical techniques such as cell membrane disruption by a microfluidics device.
  • RNPs ribonucleoprotein
  • LNPs lipid nanoparticles
  • viral vectors non-viral vectors
  • mRNA delivery mRNA delivery
  • physical techniques such as cell membrane disruption by a microfluidics device.
  • the prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell simultaneously or sequentially.
  • the prime editing method comprises introducing into the cell a PEgRNA or a polynucleotide encoding the PEgRNA, a prime editor polynucleotide encoding a prime editor polypeptide, and optionally an ngRNA or a polynucleotide encoding the ngRNA.
  • the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell simultaneously.
  • the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell sequentially. In some embodiments, the method comprises introducing the polynucleotide encoding the prime editor polypeptide into the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA.
  • the polynucleotide encoding the prime editor polypeptide is introduced into and expressed in the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into the cell after the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA are introduced into the cell.
  • the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, for example, by RNPs, LNPs, viral vectors, non-viral vectors, mRNA delivery, and physical delivery.
  • the WSGR Docket No.59761-775.601 polynucleotide is a DNA polynucleotide.
  • the polynucleotide is a RNA polynucleotide, e.g., mRNA polynucleotide.
  • the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA integrate into the genome of the prime editing target cell after being introduced into the cell.
  • the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA are introduced into the cell for transient expression.
  • the cell is a eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a non-human primate cell, a rodent cell, a bovine cell, or a porcine cell.
  • the cell is a human cell.
  • the cell is a stem cell.
  • the cell is a progenitor cell.
  • the cell is a pluripotent stem cell.
  • the cell is an embryonic stem cell.
  • the cell is a mesenchymal stem cell.
  • a cell is a bronchioalveolar stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, a cell is a lung progenitor cell. [0456] In some embodiments, the cell is derived from a stem cell. In some embodiments, the cell is a primary cell. As used herein, the term primary cell can refer to a cell isolated from a subject, which is then grown in tissue culture (i.e., in vitro) for the first time before subdivision and subsequently transferred to a subculture. [0457] In some embodiments, the cell is a part of or derived from a tissue, an organ, or a cell type.
  • a cell is in, a part of, or derived from a respiratory organ system of a subject.
  • the respiratory system can include the nose, mouth, throat, voice box, windpipe, or lungs.
  • a cell is in, part of, or derived from a lung tissue of a subject.
  • the cell is a part of an organoid, e.g., an intestinal organoid.
  • the cell is a somatic cell.
  • the cell can be an epithelial cell (e.g., a mammary epithelial cell, intestinal epithelial cell, a hepatocyte), a fibroblast, a keratinocyte, an endothelial cell, a glial cell, a neural cell, a muscle cell (e.g., a cardiac muscle cell, a smooth muscle cell, a myosatellite cell), a formed element of the blood (e.g., a lymphocyte, a bone marrow cell), or a precursor of any of these somatic cell types.
  • the cell is an epithelial cell.
  • the cell is an airway epithelial cell, a bronchial epithelial cell, a pancreatic epithelial cell, a pancreatic ductal epithelial cell, a pancreatic acinar cell, a kidney epithelial cell, an intestine epithelial cell, or a reproductive tissue epithelial cell (e.g., a sperm canal epithelial cell).
  • the cell is a tuft cell.
  • the cell is a neuroendocrine cell.
  • the cell is a goblet cell.
  • a cell is a basal cell.
  • a cell is a basal cell from the respiratory epithelium, e.g., from the bronchioles or WSGR Docket No.59761-775.601 alveoli of the lung.
  • a cell is a club cell.
  • a cell is a ciliated cell.
  • a cell is an ionocyte.
  • the target gene edited by prime editing is in a chromosome of the cell.
  • the intended nucleotide edits incorporate in the chromosome of the cell and are inheritable by progeny cells.
  • the intended nucleotide edits introduced to the cell by the prime editing compositions and methods are such that the cell and progeny of the cell also include the intended nucleotide edits.
  • the cell is autologous, allogeneic, or xenogeneic to a subject.
  • the cell is from or derived from a subject.
  • the cell is from or derived from a human subject.
  • the cell is introduced back into the subject, e.g., a human subject, after incorporation of the intended nucleotide edits by prime editing.
  • the method provided herein comprises introducing the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA into a plurality or a population of cells that comprise the target gene.
  • the population of cells is of the same cell type.
  • the population of cells is of the same tissue or organ.
  • the population of cells is heterogeneous.
  • the population of cells is homogeneous.
  • the population of cells is from a single tissue or organ, and the cells are heterogeneous.
  • the introduction into the population of cells is ex vivo.
  • the introduction into the population of cells is in vivo, e.g., into a human subject.
  • the target gene is in a genome of each cell of the population.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of one or more intended nucleotide edits in the target gene in at least one of the cells in the population of cells.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide Edits in the target gene in a plurality of the population of cells.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in each cell of the population of cells.
  • introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or WSGR Docket No.59761-775.601 the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in sufficient number of cells such that the disease or disorder is treated, prevented or ameliorated.
  • editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition.
  • the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition.
  • editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition.
  • the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition.
  • the population of cells introduced with the prime editing composition is ex vivo. In some embodiments, the population of cells introduced with the prime editing composition is in vitro. In some embodiments, the population of cells introduced with the prime editing composition is in vivo. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 25% relative to a suitable control.
  • the prime editing methods disclosed herein have an editing efficiency of at least 35% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein has an editing efficiency of at least 30% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 45% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 50% relative to a suitable control. In some embodiments, editing efficiency of prime the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells after in vivo engraftment of the edited cells.
  • the editing efficiency is determined after 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks of engraftment. In some embodiments, the editing efficiency is determined after 8 or 16 weeks of engraftment. In some embodiments, prime editing is able to maintain in edited cells at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more than 95% of editing efficiency after 8 or 16 weeks post engraftment.
  • the methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a primary cell (as measured in a population of primary cells) relative to a suitable control.
  • the methods disclosed herein have an editing efficiency of at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a cell as disclosed herein.
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits without generating a significant proportion of indels.
  • Indel(s) refers to the insertion or deletion of a nucleotide base within a polynucleotide, for example, a target gene. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene.
  • Indel frequency of editing can be calculated by methods known in the art. In some embodiments, indel frequency can be calculated based on sequence alignment such as the CRISPResso 2 algorithm as described in Clement et al., Nat. Biotechnol. 37(3): 224-226 (2019), which is incorporated herein in its entirety.
  • the prime editing methods disclosed herein can have an indel frequency of less than 30%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, or less than 1%.
  • any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a CFTR gene within the genome of a cell
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits efficiently without generating a significant proportion of indels in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a target cell. [0468] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 5% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods WSGR Docket No.59761-775.601 disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a target cell. [0469] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a target cell. [0470] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a target cell. [0471] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a target cell. [0473] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a target cell. In some embodiments, the prime editing methods WSGR Docket No.59761-775.601 disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a target cell. [0474] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a target cell. [0475] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a target cell.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a target cell. [0476] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% in a target cell. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% in a target cell.
  • the prime editing compositions provided herein are capable of incorporating one or more intended nucleotide edits efficiently without generating a significant proportion of indels in a population of cells.
  • the prime editing methods WSGR Docket No.59761-775.601 disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a population of target cells. [0478] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a population of target cells. [0479] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about WSGR Docket No.59761-775.601 10% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a population of target cells. [0480] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 2.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than WSGR Docket No.59761-775.601 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a population of target cells. [0482] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a population of target cells. [0483] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 2.5% in population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a population of target cells. [0484] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an WSGR Docket No.59761-775.601 indel frequency of less than 5% in population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 2.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a population of target cells. [0486] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed WSGR Docket No.59761-775.601 herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a population of target cells. [0487] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 7.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a population of target cells. [0488] In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 10% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime WSGR Docket No.59761-775.601 editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 2.5% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% as measured in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 10% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 7.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 2.5% in a population of target cells. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a population of target cells.
  • the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a population of target cells.
  • the population of target cells can be a population of any of the cells disclosed herein.
  • any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a CFTR gene within the genome of a cell
  • the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a CFTR gene within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a CFTR gene within the genome of a cell
  • the prime editing composition described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% off-target editing in a chromosome that includes the target gene.
  • off-target editing is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, WSGR Docket No.59761-775.601 at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a nucleic acid within the genome of a cell) to a prime editing composition.
  • a target gene e.g., a nucleic acid within the genome of a cell
  • the prime editing methods described herein result in less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 4% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 3%large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 2% large deletion in edited cells. In some embodiments, the prime editing methods described herein result in less than 1% large deletion in edited cells.
  • the prime editing methods described herein does not result in detectable level of large deletion in edited cells.
  • the prime editing methods disclosed herein can be used to edit a target CFTR gene.
  • the target CFTR gene comprises a mutation compared to a wild type CFTR gene.
  • the mutation is associated with cystic fibrosis.
  • the target CFTR gene comprises an editing target sequence that contains the mutation associated with cystic fibrosis.
  • the mutation is in exon 12 of the target CFTR gene.
  • the mutation is a c.1624G->T substitution in the sequence encoding a CFTR protein and results in a G542x nonsense mutation (x indicates a premature stop codon; also referred to as G542ter) and a truncated CFTR protein, which may be associated with cystic fibrosis.
  • the prime editing method comprises contacting a target CFTR gene with a prime editing composition comprising a prime editor, a PEgRNA, and/or a ngRNA. In some embodiments, contacting the target CFTR gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target CFTR gene.
  • incorporation of the one more intended nucleotide edits results in correction of the c.1624G->T mutation in the target CFTR gene. In some embodiments, incorporation of the one or more intended nucleotide edits results in correction of one or more other mutations in the target CFTR gene that are in the portion of the gene that shares homology with the editing template of the PEgRNA. In some embodiments, incorporation of the one or more intended nucleotide edits results in restoration of wild type expression of a CFTR protein, i.e. with a full length CFTR protein having a Glycine at position 542.
  • Exemplary wild type CFTR protein MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELAS KKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGL CLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFD EGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRA WSGR Docket No.59761-775.601 GKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGF FVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQE YKTLEYNLTTTEVV
  • a method of editing a target cell comprising a target CFTR gene that encodes a polypeptide that comprises one or more mutations relative to a wild type CFTR gene.
  • the methods of the present disclosure comprise introducing a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA into the target cell that has the target CFTR gene to edit the target CFTR gene, thereby generating an edited cell.
  • restoration of the CFTR expression and/or function of the CFTR protein may be measured when expressed in a target cell.
  • incorporation of the one or more intended nucleotide edits in the target CFTR gene comprising one or more mutations lead to a fold change in a level of CFTR gene expression, CFTR protein expression, or a combination thereof.
  • a change in the level of CFTR protein expression can comprise a fold change of, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or greater as compared to expression in a suitable control cell not introduced with a prime editing composition described herein.
  • incorporation of the one or more intended nucleotide edits in the target CFTR gene that comprises one or more mutations restores wild type expression of CFTR protein by at WSGR Docket No.59761-775.601 least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, o99% or more as compared to wild type expression of the CFTR protein in a suitable control cell that comprises a wild type CFTR gene.
  • a CFTR protein expression increase can be measured by a CFTR functional assay.
  • protein expression can be measured using a protein assay.
  • protein expression can be measured using antibody testing.
  • an antibody can comprise anti-CFTR.
  • protein expression can be measured using ELISA, mass spectrometry, Western blot, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), electrophoresis, or any combination thereof.
  • a protein assay can comprise SDS-PAGE and densitometric analysis of a Coomassie Blue-stained gel.
  • biological activity of a CFTR protein can be measured by any assay known in the art, for example, a chloride efflux assay.
  • methods for treatment of a subject diagnosed with a disease associated with or caused by one or more pathogenic mutations are methods for treatment of a subject diagnosed with a disease associated with or caused by one or more pathogenic mutations.
  • methods for treatment of a subject diagnosed with a disease associated with or caused by one or more pathogenic mutations that can be corrected by prime editing.
  • methods of treatment provided herein comprises editing one or more genes other than the gene that harbors the one or more pathogenic mutations.
  • methods for treating Cystic Fibrosis that comprise administering to a subject a therapeutically effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein.
  • administration of the prime editing composition results in incorporation of one or more intended nucleotide edits in the target gene in the subject.
  • administration of the prime editing composition results in correction of one or more pathogenic mutations, e.g., point mutations, insertions, or deletions, associated with Cystic Fibrosis in the subject.
  • the target gene comprise an editing target sequence that contains the pathogenic mutation.
  • administration of the prime editing composition results in incorporation of one or more intended nucleotide edits in the target gene that corrects the pathogenic mutation in the editing target sequence (or a double stranded region comprising the editing target sequence and the complementary sequence to the editing target sequence on a target strand) of the target gene in the subject.
  • the method provided herein comprises administering to a subject an effective amount of a prime editing composition, for example, a PEgRNA, a prime editor, and/or a ngRNA.
  • the method comprises administering to the subject an effective amount of a prime editing composition described herein, for example, polynucleotides, vectors, or constructs that encode prime editing composition components, or RNPs, LNPs, and/or polypeptides WSGR Docket No.59761-775.601 comprising prime editing composition components.
  • Prime editing compositions can be administered to target the CFTR gene in a subject, e.g., a human subject, suffering from, having, susceptible to, or at risk for Cystic Fibrosis.
  • Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).
  • the subject has Cystic Fibrosis.
  • the subject has been diagnosed with Cystic Fibrosis by sequencing of a CFTR gene in the subject.
  • the subject comprises at least a copy of the CFTR gene that comprises one or more mutations compared to a wild type CFTR gene.
  • the subject comprises at least a copy of the CFTR gene that encodes a G542X amino acid substitution in the CFTR protein compared to a wildtype CFTR protein.
  • the subject comprises at least a copy of the CFTR gene that comprises a c.1624G->T mutation compared to a wild type CFTR gene.
  • administration of the prime editing composition described herein results in incorporation of one or more intended nucleotide edits in the CFTR gene, thereby editing the CFTR gene and treating CF.
  • incorporation of the one or more intended nucleotide edits results in wild type expression of the CFTR protein.
  • incorporation of the one or more intended nucleotide edits corrects the one or more mutations to wild type nucleotides at corresponding positions in the CFTR gene.) In some embodiments, incorporation of the one or more intended nucleotide edits introduces in the CFTR gene one or more synonymous mutations compared to a wildtype CFTR gene.
  • the method comprises directly administering prime editing compositions provided herein to a subject.
  • the prime editing compositions described herein can be delivered with in any form as described herein, e.g., as LNPs, RNPs, polynucleotide vectors such as viral vectors, or mRNAs.
  • the prime editing compositions can be formulated with any pharmaceutically acceptable carrier described herein or known in the art for administering directly to a subject.
  • Components of a prime editing composition or a pharmaceutical composition thereof may be administered to the subject simultaneously or sequentially.
  • the method comprises administering a prime editing composition, or pharmaceutical composition thereof, comprising a complex that comprises a prime editor fusion protein and a PEgRNA and/or a ngRNA, to a subject.
  • the method comprises administering a polynucleotide or vector encoding a prime editor to a subject simultaneously with a PEgRNA and/or a ngRNA.
  • the method comprises administering a polynucleotide or vector encoding a prime editor to a subject before administration with a PEgRNA and/or a ngRNA.
  • a population of patients each having one or more mutations in the CFTR gene may be treated with a prime editing composition (e.g., a PEgRNA, a prime editor, and optionally an ngRNA as described herein) disclosed herein.
  • a patient with multiple mutations in the CFTR gene can be treated with a prime editing composition (e.g., a PEgRNAs, a prime editor, and optionally an ngRNA as described herein).
  • a prime editing composition e.g., a PEgRNAs, a prime editor, and optionally an ngRNA as described herein.
  • a subject may comprise two copies of the gene, each comprising one or more different mutations.
  • a patient with one or more different mutations in the target gene can be treated with a prime editing composition comprising a PEgRNAs, a prime editor, and optionally an ngRNA.
  • the editing template may comprise one or more synonymous mutations relative to the wild-type CFTR gene.
  • Such synonymous mutations may include, for example, mutations that decrease the ability of a PEgRNA to rebind to the same target sequence once the desired edit is installed in the genome (e.g., synonymous mutations that silence the endogenous PAM sequence or that edit the endogenous protospacer). Accordingly, one or more synonymous mutations may include a PAM silencing edit.
  • Suitable routes of administrating the prime editing compositions to a subject include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
  • the compositions described are administered intraperitoneally, intravenously, or by direct injection or direct infusion.
  • the compositions described are administered by direct injection or infusion into a subject.
  • the compositions described herein are administered by direct injection or infusion into an affected organ or tissue of a subject, e.g., the lungs or pancreas. In some embodiments, the compositions described herein are administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant. [0506] In some embodiments, the method comprises administering cells edited with a prime editing composition described herein to a subject. In some embodiments, the cells are allogeneic. In some embodiments, allogeneic cells are or have been contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are introduced into a human subject in need thereof. In some embodiments, the cells are autologous to the subject.
  • cells are removed from a subject and contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are re-introduced into the subject.
  • cells are contacted ex vivo with one or more components of a prime editing composition.
  • the ex vivo-contacted cells are introduced into the subject, and the subject is administered in vivo with one or more components of a prime editing composition.
  • cells are contacted ex vivo with a prime editor and introduced into a subject.
  • the subject is then administered with a PEgRNA and/or a ngRNA, or a polynucleotide encoding the PEgRNA and/or the ngRNA.
  • cells contacted with the prime editing composition are determined for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject.
  • the cells are enriched for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject.
  • the prime editing composition or components thereof may be introduced into a cell by any delivery approaches as described herein, including LNP administration, RNP administration, electroporation, nucleofection, transfection, viral transduction, microinjection, cell membrane disruption and diffusion, or any other approach known in the art.
  • the cells edited with prime editing can be introduced into the subject by any route known in the art.
  • the edited cells are administered to a subject by direct infusion.
  • the edited cells are administered to a subject by intravenous infusion.
  • the edited cells are administered to a subject as implants.
  • the pharmaceutical compositions, prime editing compositions, and cells, as described herein can be administered in effective amounts.
  • the effective amount depends upon the mode of administration.
  • the effective amount depends upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner.
  • the specific dose administered can be a uniform dose for each subject.
  • a subject’s dose can be tailored to the approximate body weight of the subject. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient.
  • the time between sequential administration can be at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days.
  • a method of monitoring treatment progress is provided.
  • the method includes the step of determining a level of diagnostic marker, for example, correction of a mutation in CFTR gene, or diagnostic measurement associated with Cystic Fibrosis, (e.g., chloride efflux assay) in a subject suffering from Cystic Fibrosis symptoms and has been administered an effective amount of a prime editing composition described herein.
  • the level of the diagnostic marker determined in the method can be compared to known levels of the marker in either healthy normal controls or in other afflicted subjects to establish the subject’s disease status.
  • Delivery [0515] Prime editing compositions described herein can be delivered to a cellular environment with any approach known in the art.
  • Components of a prime editing composition can be delivered to a cell WSGR Docket No.59761-775.601 by the same mode or different modes.
  • a prime editor can be delivered as a polypeptide or a polynucleotide (DNA or RNA) encoding the polypeptide.
  • a PEgRNA can be delivered directly as an RNA or as a DNA encoding the PEgRNA.
  • a prime editing composition component is encoded by a polynucleotide, a vector, or a construct.
  • a prime editor polypeptide, a PEgRNA and/or a ngRNA is encoded by a polynucleotide.
  • the polynucleotide encodes a prime editor fusion protein comprising a DNA binding domain and a DNA polymerase domain.
  • the polynucleotide encodes a DNA polymerase domain of a prime editor.
  • the polynucleotide encodes a DNA polymerase domain of a prime editor.
  • the polynucleotide encodes a portion of a prime editor protein, for example, a N- terminal portion of a prime editor fusion protein connected to an intein-N.
  • the polynucleotide encodes a portion of a prime editor protein, for example, a C-terminal portion of a prime editor fusion protein connected to an intein-C. In some embodiments, the polynucleotide encodes a PEgRNA and/or a ngRNA. In some embodiments, the polypeptide encodes two or more components of a prime editing composition, for example, a prime editor fusion protein and a PEgRNA. [0517] In some embodiments, the polynucleotide encoding one or more prime editing composition components is delivered to a target cell is integrated into the genome of the cell for long-term expression, for example, by a retroviral vector.
  • the polynucleotide delivered to a target cell is expressed transiently.
  • the polynucleotide may be delivered in the form of a mRNA, or a non-integrating vector (non-integrating virus, plasmids, minicircle DNAs) for episomal expression.
  • a polynucleotide encoding one or more prime editing system components can be operably linked to a regulatory element, e.g., a transcriptional control element, such as a promoter.
  • the polynucleotide is operably linked to multiple control elements.
  • the polynucleotide encoding one or more prime editing composition components is a part of, or is encoded by, a vector.
  • the vector is a viral vector.
  • the vector is a non-viral vector.
  • Non-viral vector delivery systems can include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • RNA e.g., a transcript of a vector described herein
  • the polynucleotide is provided as an RNA, e.g., a mRNA or a transcript.
  • Any RNA of the prime editing systems for example a guide RNA or a base editor- WSGR Docket No.59761-775.601 encoding mRNA, can be delivered in the form of RNA.
  • one or more components of the prime editing system that are RNAs is produced by direct chemical synthesis or may be transcribed in vitro from a DNA.
  • a mRNA that encodes a prime editor polypeptide is generated using in vitro transcription.
  • Guide polynucleotides e.g., PEgRNA or ngRNA
  • PEgRNA or ngRNA can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence “GG”, and guide polynucleotide sequence.
  • the prime editor encoding mRNA, PEgRNA, and/or ngRNA are synthesized in vitro using an RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, the RNA can directly contact a target CFTR gene or can be introduced into a cell using any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection).
  • the prime editor-coding sequences, the PEgRNAs, and/or the ngRNAs are modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.
  • Methods of non-viral delivery of nucleic acids can include lipofection, electroporation, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, nanoparticles, cell penetrating peptides and associated conjugated molecules and chemistry, naked DNA, artificial virions, cell membrane disruption by a microfluidics device, and agent-enhanced uptake of DNA.
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used.
  • Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell. RNA or DNA viral based systems can be used to target specific cells and trafficking the viral payload to an organelle of the cell. Viral vectors can be administered directly (in vivo) or they can be used to treat cells in vitro, and the modified cells can optionally be administered after delivery (ex vivo).
  • the viral vector is a retroviral, lentiviral, adenoviral, adeno-associated viral or herpes simplex viral vector.
  • Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.
  • the retroviral vector is a lentiviral vector.
  • the retroviral vector is a gamma retroviral vector.
  • the viral vector is an adenoviral vector.
  • the viral vector is an adeno-associated virus (“AAV”) vector.
  • AAV adeno-associated virus
  • polynucleotides encoding one or more prime editing composition components are packaged in a virus particle.
  • Packaging cells can be used to form virus particles that can infect a target cell.
  • Such cells can include 293 cells, (e.g., for packaging adenovirus), and .psi.2 WSGR Docket No.59761-775.601 cells or PA317 cells (e.g., for packaging retrovirus).
  • Viral vectors can be generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors can contain the minimal viral sequences required for packaging and subsequent integration into a host.
  • the vectors can contain other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed.
  • the missing viral functions can be supplied in trans by the packaging cell line.
  • AAV vectors can comprise ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • the polynucleotides are a DNA polynucleotide.
  • the polynucleotides are an RNA polynucleotide; e.g., an mRNA polynucleotide.
  • the AAV vector is selected for tropism to a particular cell, tissue, organism.
  • the AAV vector is pseudotyped, e.g., AAV5/8.
  • polynucleotides encoding one or more prime editing composition components are packaged in a first AAV and a second AAV.
  • the polynucleotides encoding one or more prime editing composition components are packaged in a first rAAV and a second rAAV.
  • dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5’ and 3’ ends that encode N-terminal portion and C- terminal portion of, e.g., a prime editor polypeptide), where each half of the cassette is no more than 5kb in length, optionally no more than 4.7 kb in length, and is packaged in a single AAV vector.
  • the full-length transgene expression cassette is reassembled upon co-infection of the same cell by both dual AAV vectors.
  • a portion or fragment of a prime editor polypeptide e.g., a Cas9 nickase, is fused to an intein.
  • the portion or fragment of the polypeptide can be fused to the N-terminus or the C-terminus of the intein.
  • a N-terminal portion of the polypeptide is fused to an intein-N, and a C-terminal portion of the polypeptide is separately fused to an intein-C.
  • a portion or fragment of a prime editor fusion protein is fused to an intein and fused to an AAV capsid protein.
  • intein-N may be fused to the N-terminal portion of a first domain described herein
  • intein-C may be fused to the C-terminal portion of a second domain described herein for the joining of the N- terminal portion to the C-terminal portion, thereby joining the first and second domains.
  • the first and second domains are each independently chosen from a DNA binding domain or a DNA polymerase domain.
  • the intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.).
  • a polynucleotide encoding a prime editor fusion protein is split in two separate halves, each encoding a portion of the prime editor fusion protein and separately fused to an intein.
  • each of the two halves of the polynucleotide is packaged in an individual AAV vector of a dual AAV vector system.
  • each of the two halves of the polynucleotide is no more than 5kb in length, optionally no more than 4.7 kb in length.
  • the full-length prime editor fusion protein is reassembled upon co-infection of the same cell by both dual AAV vectors, expression of both halves of the prime editor fusion protein, and self- excision of the inteins.
  • the in vivo use of dual AAV vectors results in the expression of full- length full-length prime editor fusion proteins.
  • the use of the dual AAV vector platform allows viable delivery of transgenes of greater than about 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 kb in size.
  • an intein is inserted at a splice site within a Cas protein. In some embodiments, insertion of an intein disrupts a Cas activity.
  • intein refers to a self- splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined).
  • an intein may comprise a polypeptide that is able to excise itself and join exteins with a peptide bond (e.g., protein splicing).
  • an intein of a precursor gene comes from two genes (e.g., split intein).
  • an intein may be a synthetic intein.
  • Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: dnaE-n and dnaE-c. a 4-hydroxytamoxifen (4-HT)-responsive intein, an iCas molecule, a Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, Cfa DnaE intein, Ssp GyrB intein, and Rma DnaB intein.
  • intein fragments may be fused to the N terminal and C-terminal portion of a split Cas protein respectively for joining the fragments of split Cas9.
  • the split Cas9 system may be used in general to bypass the packing limit of the viral delivery vehicles.
  • a split Cas9 may be a Type II CRISPR system Cas9.
  • a first nucleic acid encodes a first portion of the Cas9 protein having a first split-intein and wherein the second nucleic acid encodes a second portion of the Cas9 protein having a second split-intein complementary to the first split-intein and wherein the first portion of the Cas9 protein and the second portion of the Cas9 protein are joined together to form the Cas9 protein.
  • the first portion of the Cas9 protein is the N-terminal fragment of the Cas9 protein and the second portion of the Cas9 protein is the C-terminal fragment of the Cas9 protein.
  • a split site may be selected which are surface exposed due to the sterical need for protein splicing.
  • a Cas protein may be split into two fragments at any C, T, A, or S.
  • a Cas9 may be intein split at residues 203-204, 280-292, 292-364, 311-325, 417- 438, 445-483, 468-469, 481-502, 513-520, 522-530, 565-637, 696-707, 713-714, 795-804, 803-810, 878-887, and 1153-1154.
  • protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574.
  • a functional Cas9 protein may be WSGR Docket No.59761-775.601 reconstituted from two inactive split-Cas9 peptides in the presence of gRNA by using a split-intein protein splicing strategy.
  • the split Cas9 fragments are fused to either a N- terminal intein fragment or a C-terminal intein fragment, which can associate with each other and catalytically splice the two split Cas9 fragments into a functional reconstituted Cas9 protein.
  • a split-Cas9 can be packaged into self-complementary AAV.
  • a split-Cas9 comprises a 2.5 kb and a 2.2 kb fragment of S. pyogenes Cas9 coding sequences.
  • a split-Cas9 architecture reduces the length and/or size of the coding sequences of a viral vector, e.g., AAV.
  • a target cell can be transiently or non-transiently transfected with one or more vectors described herein.
  • a cell can be transfected as it naturally occurs in a subject.
  • a cell can be taken or derived from a subject and transfected.
  • a cell can be derived from cells taken from a subject, such as a cell line.
  • a cell transfected with one or more vectors described herein can be used to establish a new cell line comprising one or more vector-derived sequences.
  • a cell transiently transfected with the compositions of the disclosure (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editor, can be used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
  • Any suitable vector compatible with the host cell can be used with the methods of the disclosure.
  • Non-limiting examples of vectors include pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40.
  • a prime editor protein can be provided to cells as a polypeptide.
  • the prime editor protein is fused to a polypeptide domain that increases solubility of the protein.
  • the prime editor protein is formulated to improve solubility of the protein.
  • a prime editor polypeptide is fused to a polypeptide permeant domain to promote uptake by the cell.
  • the permeant domain is a including peptide, a peptidomimetic, or a non-peptide carrier.
  • a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 748).
  • the permeant peptide can comprise the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein.
  • Other permeant domains can include poly-arginine motifs, for example, the region of amino acids 34- 56 of HIV-1 rev protein, nona-arginine (SEQ ID NO: 749), and octa-arginine (SEQ ID NO: 750).
  • the nona-arginine (R9) sequence (SEQ ID NO: 749) can be used.
  • the site at which the fusion can be made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide.
  • WSGR Docket No.59761-775.601 [0535]
  • a prime editor polypeptide is produced in vitro or by host cells, and it may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc. and may be further refolded.
  • a prime editor polypeptide is prepared by in vitro synthesis.
  • Various commercial synthetic apparatuses can be used. By using synthesizers, naturally occurring amino acids can be substituted with unnatural amino acids.
  • a prime editor polypeptide is isolated and purified in accordance with recombinant synthesis methods, for example, by expression in a host cell and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
  • a prime editing composition for example, prime editor polypeptide components and PEgRNA/ngRNA are introduced to a target cell by nanoparticles.
  • the prime editor polypeptide components and the PEgRNA and/or ngRNA form a complex in the nanoparticle. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components.
  • the nanoparticle is inorganic. In some embodiments, the nanoparticle is organic. In some embodiments, a prime editing composition is delivered to a target cell in an organic nanoparticle, e.g. a lipid nanoparticle (LNP) or polymer nanoparticle.
  • LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof. In some embodiments, neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance transfection activity and nanoparticle stability. In some embodiments, LNPs are formulated with hydrophobic lipids, hydrophilic lipids, or combinations thereof.
  • Lipids may be formulated in a wide range of molar ratios to produce an LNP. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Exemplary lipids used to produce LNPs are provided in Table 15. [0538]
  • components of a prime editing composition form a complex prior to delivery to a target cell.
  • a prime editor fusion protein, a PEgRNA, and/or a ngRNA can form a complex prior to delivery to the target cell.
  • a prime editing polypeptide e.g. a prime editor fusion protein
  • a guide polynucleotide e.g.
  • a PEgRNA or ngRNA form a ribonucleoprotein (RNP) for delivery to a target cell.
  • the RNP comprises a prime editor fusion protein in complex with a PEgRNA.
  • RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, or any other approaches known in the art.
  • delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell.
  • the RNP comprising the prime editing complex is degraded over time in the target cell. Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 15.
  • Table 15 Exemplary lipids for nanoparticle formulation or gene transfer WSGR Docket No.59761-775.601
  • Exemplary polymers for use in nanoparticle formulations and/or gene transfer are shown in Table 16.
  • Table 16 Exemplary lipids for nanoparticle formulation or gene transfer WSGR Docket No.59761-775.601
  • Exemplary delivery methods for polynucleotides encoding prime editing composition components are shown in Table 17.
  • the prime editing compositions of the disclosure can be provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days.
  • compositions may be provided to the subject cells one or more times, e.g., one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount WSGR Docket No.59761-775.601 of time following each contacting event e.g., 16-24 hours.
  • the compositions may be delivered simultaneously (e.g., as two polypeptides and/or nucleic acids).
  • the prime editing compositions and pharmaceutical compositions of the disclosure can be administered to subjects in need thereof for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days.
  • compositions may be provided to the subject one or more times, e.g., one time, twice, three times, or more than three times.
  • two or more different prime editing system components e.g. two different polynucleotide constructs are administered to the subject (e.g., different components of the same prime editing system, or two different guide nucleic acids that are complementary to different sequences within the same or different target genes)
  • the compositions may be administered simultaneously (e.g., as two polypeptides and/or nucleic acids).
  • they may be provided sequentially, e.g., one composition being provided first, followed by a second composition.
  • PEgRNA libraries may be assembled by any method known in the art. In a first method, pooled synthesized DNA oligos encoding the PEgRNA and flanking U6 expression plasmid homology regions may be cloned into U6 expression plasmids via Gibson cloning and sequencing of bacterial colonies via Sanger or Next-generation sequencing. In a second method, double-stranded linear DNA fragments encoding PEgRNA and homology sequences as above may be individually Gibson-cloned into U6 expression plasmids.
  • PEgRNA extension PBS and RTT
  • PBS and RTT PEgRNA extension
  • HEK cell culture and transfection HEK 293T cells may be propagated in DMEM with 10% FBS. Prior to transfection, cells may be seeded in 96-well plates and then transfected with Lipofectamine 2000 or MessengerMax according to the manufacturer’s directions with DNA or mRNA encoding a prime editor and PEgRNA (and ngRNA for PE3 experiments). Three days after transfection, gDNA may be harvested in lysis buffer for high throughput sequencing and may be sequenced using MiSeq.
  • iPSC cell culture and transfection iPSC cells may be propagated in mTeSR Plus (STEMCELL Technologies) or E8 (Life Technologies) with 10uM ROCK inhibitor Y27632 (STEMCELL Technologies) with daily media changes. Prior to transfection, cells may be seeded in 24-well plates (Corning) coated with Vitronectin (Gibco) or Matrigel Growth Factor Reduced Basement Membrane Matrix LDEV-free (Corning) and may be transfected with Lipofectamine STEM (Invitrogen) with DNA or mRNA encoding a prime editor, PEgRNA or ngRNA. Three days after transfection, gDNA may be harvested for high throughput sequencing and sequenced using MiSeq.
  • Endoderm cell differentiated from iPSCs and transfection Endoderm cells may be derived from iPSCs using the STEMdiff Definitive Endoderm Kit (STEMCELL Technologies). Prior to transfection, cells may be seeded in 24-well plates coated with Matrigel Growth Factor Reduced Basement Membrane Matrix LDEV-free (Corning) and then may be transfected with Lipofectamine 2000 or MessengerMax with DNA or mRNA encoding a prime editor, PEgRNA or ngRNA. Three days after transfection, gDNA may be harvested in lysis buffer for high throughput sequencing and may be sequenced using MiSeq.
  • iPSC cells may be propagated in mTeSR Plus (STEMCELL Technologies) or E8 (Life Technologies).
  • iPSCs may be plated at 1 million cells/well on 6-well tissue culture plates (Corning) coated with Matrigel Growth Factor Reduced Basement Membrane Matrix LDEV-free (Corning) in mTeSR Plus with 10uM ROCK inhibitor Y27632 (STEMCELL Technologies).
  • the media On the day of differentiation, the media may be exchanged with 3mL/well STEMdiff DE media (STEMCELL Technologies).
  • the media On the following days 1 and 2 of differentiation, the media may be exchanged with 2mL/well DE media.
  • the cells may be dissociated with Gentle Cell Dissociation Reagent (STEMCELL Technologies) and re-suspended in STEMdiff Mid-/Hindgut Induction media (STEMCELL Technologies) with 10uM ROCK inhibitor Y27632 (STEMCELL Technologies).
  • the cells may be plated on an Anti-Adherence solution (STEMCELL Technologies) pre-treated AggreWell 400 Microwell culture plate (STEMCELL Technologies) at a density of 300,000-500,000 WSGR Docket No.59761-775.601 cells per well.
  • the plate may be centrifuged at 200-300g for 5 minutes to settle the cells into the microwells, the cells may then self-assembled into organoids.
  • the organoids may be collected from the Aggrewells using a P1000 and pelleted.
  • the pellet may be re-suspended in Matrigel Growth Factor Reduced Basement Membrane Matrix LDEV-free (Corning) or Matrigel hESC-Qualified Matrix LDEV-free (Corning) and 20-50uL of the suspension may be plated into each well of a 96-well, 48-well or 24-well plate. The plate may be incubated at 37C for 10-15 minutes to solidify the Matrigel.
  • the organoids may be maintained in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) and propagated using the same media. For transfection, the organoids may be dissociated using Accutase (STEMCELL Technologies).
  • the single cell suspension may be plated in a 24-well ultra-low attachment plate (Corning) at a density of about 100,000 cells/well in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) with 0-2uM ROCK inhibitor.
  • the cells may be transfected with Lipofectamine MessengerMax according to the manufacturer’s directions with DNA or mRNA encoding a prime editor, PEgRNA (and ngRNA for PE3 experiments).
  • the cell suspensions may be incubated on a shaker plate at 250rpm for 1-4 hours at 37C with 5% CO2.
  • the cells may be pelleted and may be re-suspended in 20-50uL/well Matrigel Growth Factor Reduced Basement Membrane Matrix LDEV-free (Corning) or Matrigel hESC- Qualified Matrix LDEV-free (Corning) and may be plated on a 96-well plate, 48-well plate or 24-well plate (Corning).
  • the organoids may be maintained in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) with 10uM ROCK inhibitor Y27632 for the first day.
  • Organoids may be derived from a mouse model with the G542X mutation installed in both copies of the endogenous gene and cultured as described in McHugh et. al. PLoS One.2018 Jun 20;13(6):e0199573. Organoids may be transfected using Lipofectamine MessengerMax according to the manufacturer’s directions with DNA or mRNA encoding a prime editor, PEgRNA (and ngRNA for PE3 experiments). Electroporation may be used for transfection; organoids may be cultured for 2 days prior to electroporation.
  • Media may then be removed from wells and organoids, collected using PBS and dissociated with a pipette and pelleted after centrifugation at 100G for 5 minutes.
  • Pellets may be resuspended in Accutase (Stemcell Technologies) and incubated at 37C with intermittent vortexing.
  • the single cell suspension may be resuspended in Complete Media (Stemcell Technologies) and aliquoted at a density of 75,000- 100,000 per tube.
  • Human bronchial epithelial cell culture and transfection Human bronchial epithelial cell line UNCCF13T possessing mutations in both copies of the endogenous G542X may be propagated as WSGR Docket No.59761-775.601 described in Lee et. al. The Journal of Clinical Investigation.2022;132(18):e154571.
  • cells may be suspended in PneumaCult Ex-Plus medium (STEMCELL Technologies) supplemented with 10 uM Y-27632 (Stemcell Technologies, 72304), 1 uM A 83-01 (R&D Systems, 2939/10) and 1 uM DMH-1 (R&D Systems, 4126/10) and may be plated on collagen 1-coated 96-well plates prior to transfection with DNA or mRNA using Lipofectamine LTX (Thermo Fisher) utilized according to the manufacturer’s instructions. Alternately lipid nanoparticles may be used to deliver DNA or mRNA into the cell line.
  • G542X mutations by prime editing Generation of cell lines carrying the G542X mutation in the endogenous CFTR gene: PEgRNAs and ngRNAs may be designed to incorporate the G542X mutation in the wild type endogenous CFTR gene in HEK293T cells, iPSCs, endoderm cells, and/or intestinal organoids.
  • Cells may be transfected with a nucleic acid encoding a prime editor and a G542X mutation installation PEgRNA-ngRNA pair in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • cells Prior to transfection, cells may be seeded in 96- well plates and then transfected with Lipofectamine 2000 according to the manufacturer’s directions.
  • gDNA Three days after transfection, gDNA may be harvested in lysis buffer for high throughput sequencing, which may be sequenced using MiSeq. After the installation rates in the cell population are confirmed, single cell colonies may be generated and their genotype at the CFTR Exon 12 locus may be assessed using MiSeq. Cells with successful incorporation of the G542X mutation may used for screening Prime Editors designed to correct this mutation.
  • G542X mutation correction with PE2 system may be expanded, and transiently transfected with a PEgRNA and one or more nucleic acids encoding a prime editor in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNA may be any PEgRNA disclosed herein.
  • the prime editor may be any prime editor comprising a Cas9 protein capable of recognizing the PAM associated with the spacer of the PEgRNA.
  • G542X mutation correction with PE3 system a nick guide RNA (“ngRNA”) that can direct the prime editor to generate a nick on the opposite strand compared to the PEgRNA (i.e., on the non- edit strand) may be included in the transfection mixture referenced above. Addition of a ngRNA may improve efficiency and/or fidelity of prime editing as discussed herein.
  • the ngRNA may be any ngRNA disclosed herein.
  • Preferred ngRNA include those that include a spacer associated with a PAM that is recognized by the Cas9 protein of the prime editor.
  • EXAMPLE 2 Screening of PEgRNA for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0559]
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will comprise from 1-7 additional uridine nucleotides, e.g., 4 Us.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70% confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9-VRQR.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • EXAMPLE 3 Screening of PEgRNA with scaffold variations for editing the c.1624G- >T (G542X) associated with Cystic Fibrosis [0565] A screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. An additional adenine nucleotide was inserted between the PBS and the poly-T terminator because many of the tested PBSs contain one or more 3’ terminal uridine nucleotides. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will comprise from 1-7 additional uridine nucleotides, e.g., 4 Us.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent. The cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9-VRQR. Three days post transfection, genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler.
  • QuickExtract Solution (Lucigen).
  • the plate was WSGR Docket No.59761-775.601 incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • 60 PEgRNAs were tested, each of which were designed to restore wild-type CFTR sequence. The results are shown in Table 22.
  • Table 22 Prime Editing to correct the c.1624G->T (G542X) mutation in the endogenous CFTR gene of HEK293T cells with a PEgRNA (PE2 system) WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 1.
  • EXAMPLE 4 Screening of PEgRNA with scaffold variations for editing of the c.1624G->T (G542X) associated with Cystic Fibrosis [0571] A screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were chemically synthesized by Integrated DNA A, and four U nucleotides were added to the 3’ end, and the three consecutive nucleotides that designed to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected with MessengerMax transfection cocktail containing a test PEgRNA and an mRNA encoding a Prime Editing fusion protein containing a Cas9-VRQR.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler WSGR Docket No.59761-775.601 at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • EXAMPLE 5 Screening of ngRNA scaffolds for PE3 editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0577]
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • PEgRNAs and ngRNA used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA and ngRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 Us. [0579] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid, a ngRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9-VRQR.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel adfdadafdafdsafdafdvalues are determined based on the percentage of sequencing reads.
  • PEgRNA and ngRNA used experimentally were expressed from a U6 promoter and include an additional A between the PBS and the poly-T terminator of the expression cassette; the expressed PEgRNA and ngRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end.
  • PEgRNA Sequence Number 373 includes a linker having Sequence Number 763 linking the PBS to a 3’ structural motif having Sequence Number 607.
  • An additional A nucleotide was added between the PBS and the poly-T terminator signal in the expression cassette for PEgRNA Sequence Numbers 143 and 163.
  • NA means that no ngRNA was included in the test condition.
  • EXAMPLE 6 Screening of ngRNA scaffolds for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- WSGR Docket No.59761-775.601 carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNA and ngRNA used in this experiment were chemically synthesized by Integrated nucleotides. One A nucleotide and four U nucleotides were added to the 3’ end, and the three methyl nucleotides. Each was designed to be capable of correcting the c.1624G->T (G542X) mutation. [0585] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected with MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein containing a Cas9- VRQR.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’- O-Me modificationand a * indicates a phosphorothioate bond. All PEgRNAs use scaffold Seq. # 593.
  • EXAMPLE 7 Screening of PEgRNA RTTs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 Us. [0591] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9-VRQR.
  • WSGR Docket No.59761-775.601 Three days post transfection, genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes.
  • Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • 50 PEgRNAs were tested, each of which were designed to restore wild-type CFTR sequence. The results are shown in Table 26.
  • Table 26 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR gene of HEK293T cells with a PEgRNA (PE2 system) WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 1.
  • PEgRNA used experimentally each contained a scaffold having Sequence Number 593 and were expressed from a U6 promoter; the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end.
  • EXAMPLE 8 – PE2 screening of PEgRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0595] A screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 Us.
  • Some of the PegRNA expression cassettes included an additional adenine nucleotide between the 3’ end of the PBS and the poly-T terminator.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent. The cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein. Three days post transfection, genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler.
  • QuickExtract Solution (Lucigen).
  • the plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels WSGR Docket No.59761-775.601 for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads. [0598] In total, 135 PEgRNAs were tested, each of which were designed to restore wild-type CFTR sequence. The results are shown in Table 27.
  • Table 27 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR gene of HEK293T cells with a PEgRNA (PE2 system) WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 1.
  • the PEgRNA used experimentally were expressed from a U6 promoter; the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end. 2.
  • NA means that the tested PEgRNA did not have the linker and 3’ motif.
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 Us. [0603] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid, a test ngRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9-VRQR.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA used experimentally contained a scaffold having Sequence Number 593 and were expressed from a U6 promoter and include an additional A between the PBS and the poly-T terminator of the expression cassette; the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end.
  • EXAMPLE 10 PE2 screening of PEgRNA for editing of the c.1624G->T(G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 Us. Some of the expression cassettes contained an A nucleotide between the 3’ of the PBS and the poly-T terminator. [0609] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9-VRQR.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 WSGR Docket No.59761-775.601 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA used experimentally were expressed from a U6 promoter and include an additional A between the PBS and the poly-T terminator of the expression cassette; the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end. 2. NA indicates that the tested PEgRNA did not have the linker and 3’ motif.
  • EXAMPLE 11 PE3 screening of PEgRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0613] A screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene. The mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNA and ngRNA used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator.
  • An additional adenine nucleotide was inserted between the PBS and the poly-T terminator in the PEgRNA expression cassettes because many of the tested PBSs contain one or more 3’ terminal uridine nucleotides.
  • the 3’ end of the expressed PEgRNA and ngRNA will comprise from 1-7 additional uridine nucleotides, e.g., 4 Us. Each was designed to be capable of correcting the c.1624G- >T (G542X) mutation.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent. The cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid, a ngRNA plasmid and a plasmid encoding a Prime Editing fusion protein. Three days post transfection, genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler.
  • QuickExtract Solution (Lucigen).
  • the plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • Table 30 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR gene of HEK293T cells with a PEgRNA and ngRNA (PE3 system) WSGR Docket No.59761-775.601 1.
  • the PEgRNA and ngRNA used experimentally were expressed from a U6 promoter.
  • the expressed PEgRNA and ngRNA will contain from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, being added to the 3’ end.
  • ngRNAs have the scaffold listed in the PEgRNA Scaffold Seq. # column of the same row.
  • Example 12 PE3 screening of PEgRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • PEgRNAs and ngRNA used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator.
  • Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA and ngRNA will comprise from 1-7 additional WSGR Docket No.59761-775.601 uridine nucleotides, e.g., 4 Us. Each was designed to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well.
  • cells were approximately 60-70 confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid, a test ngRNA plasmid and a plasmid encoding a Prime Editing fusion protein.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes.
  • Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads. [0622] In total, 7 PEgRNAs and 6 ngRNAs were tested together for a total of 45 combinations including controls without ngRNAs, each combination was designed to restore wild-type CFTR sequence. The results are shown in Table 31.
  • Table 31 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR gene of HEK293T cells with a PEgRNA and ngRNA (PE3 system) WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 1.
  • the PEgRNA and ngRNA used experimentally were expressed from a U6 promoter.
  • the expressed PEgRNA and ngRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end. 2.
  • Example 13 PE2 screening of PEgRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0625] A screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator. Those of skill in the art will recognize that the 3’ end of the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 Us.Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well.
  • cells were approximately 60-70 confluent.
  • the cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler WSGR Docket No.59761-775.601 at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • Table 32 Prime Editing to correct the c.1624G->T (G542X) mutation in the endogenous CFTR gene of HEK293T cells with a PEgRNA (PE2 system) WSGR Docket No.59761-775.601 1.
  • the PEgRNA used experimentally each contained a scaffold having Sequence Number 593 and a 3’ motif having Sequence Number 607. They were expressed from a U6 promoter; the expressed PEgRNA will have contained from 1-7 additional uridine nucleotides, e.g., 4 nucleotides, at the 3’ end.
  • Example 14 PE2 screening of PEgRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were chemically synthesized by Integrated DNA of correcting the c.1624G->T (G542X) mutation.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent. The cells were transfected with MessengerMax transfection cocktail containing a test PEgRNA and an mRNA encoding a Prime Editing fusion protein. Three days post transfection, genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: WSGR Docket No.59761-775.601 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • Table 33 Prime Editing to correct the c.1624G->T (G542X) mutation in the endogenous CFTR gene of HEK293T cells with a PEgRNA (PE2 system) WSGR Docket No.59761-775.601 1.
  • Example 15 PE3 screening of PEgRNAs and ngRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in the endogenous CFTR gene.
  • the mutation- carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by Integrated DNA Technologies (IDT).
  • PEgRNA and ngRNA were modified at methyl nucleotides. Four U nucleotides were added to the 3’ end, and the three consecutive nucleotides. These were designed to be used in combination to be capable of correcting the c.1624G- >T (G542X) mutation.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent. The cells were transfected with MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA WSGR Docket No.59761-775.601 was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • EXAMPLE 16 PE3 screening of PEgRNAs in iPSCs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0642] A screen was performed in iPSCs edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the mutation- carrying cell line was transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 24-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by added between the PBS and the four U nucleotides. These were designed to be used in combination to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant iPSC were plated in a 24-well plate (Corning) coated with Vitronectin (Gibco) at a density of about 86,000 cells per well (or about 45,000 cells/cm2) in E8 media (Life Technologies) with 10uM ROCK inhibitor Y27632 (STEMCELL Technologies). The media was exchanged with E8 prior to transfection.
  • the cells were transfected with a Lipofectamine STEM (Invitrogen) transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in three different doses.
  • the cell culture media was exchanged daily using E8.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were lysed in this solution and transferred to a new 96- well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes.
  • Short term storage of gDNA was at 4°C and long- term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads. [0645] In total, 5 PEgRNAs and 1 ngRNA were tested together at the three different dosages for a total of 15 combinations with each combination designed to restore wild-type CFTR amino acid sequence. Each combination was tested in two independent transfections and the results are shown in Table 35.
  • Table 35 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR gene of iPSC cells with a PEgRNA and ngRNA (PE3 system) WSGR Docket No.59761-775.601
  • EXAMPLE 17 PE3 screening of PEgRNAs in iPSC-derived endoderm cells for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis WSGR Docket No.59761-775.601
  • a screen was performed in iPSC-derived endoderm cells edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the mutation-carrying cells were transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 24-well plates for assessment of editing by high- throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by added between the PBS and the four U nucleotides. These were designed to be used in combination to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant endoderm cells were dissociated with Gentle Cell Dissociation Reagent (STEMCELL Technologies) and re-suspended in STEMdiff Definitive Endoderm media (STEMCELL Technologies) with 10uM ROCK inhibitor Y27632 (STEMCELL Technologies).
  • the cells were plated on an AggreWell 400 Microwell culture plate (STEMCELL Technologies) at a density of 300,000 cells per well.
  • the microwell plate had previously been treated with Anti-Adherence Solution (STEMCELL Technologies) according to the manufacturer’s protocol.
  • the cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in three different doses.
  • two dosing schemes were used a low dose consisting of 750 ng mRNA, 250 ng pegRNA and 83.3 ng ngRNA and a high dose consisting of 1500 ng mRNA, 500 ng pegRNA and 166.7 ng ngRNA.
  • the plate was spun at 250g for 5 minutes to settle the cells into the microwells. Each microwell contained 250 cells which self-assembled into organoids.
  • the media on the plate was exchanged daily with Mid-/Hindgut media (STEMCELL Technologies).
  • genomic DNA was harvested by collecting a subset of the organoids from each well and pelleting by centrifugation. The pellets were resuspended in QuickExtract Solution (Lucigen) and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 WSGR Docket No.59761-775.601 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me WSGR Docket No.59761-775.601 modification and a * indicates a phosphorothioate bond; all pegRNAs and ngRNAs used a scaffold with sequence number 593.
  • EXAMPLE 18 PE3 screening of PEgRNAs in iPSC-derived intestinal organoids for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in intestinal organoid cells edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the mutation-carrying cells were cultured and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 24-well plates for assessment of editing by high- throughput sequencing.
  • mutant intestinal organoids were differentiated from mutant iPSCs using STEMdiff Intestinal Organoid Kit (STEMCELL Technologies). On the day of transfection, the organoids were dissociated using Accutase (STEMCELL Technologies). The single cell suspension was plated in a 24-well ultra-low attachment plate (Corning) at a density of about 100,000 cells/well in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies).
  • the cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in three different doses.
  • two dosing schemes were used a low dose consisting of 750 ng PE mRNA, 250 ng pegRNA and 83.3 ng ngRNA and high dose consisting of 1500 ng PE mRNA, 500 ng pegRNA and 166.7 ng ngRNA.
  • the cell suspensions were incubated on a shaker plate at 250rpm for 3.5 hours at 37C with 5% CO2.
  • the cells were then pelleted and re-suspended in 50uL/well Matrigel hESC-Qualified Matrix LDEV-free (Corning) and plated on a 24-well plate (Corning).
  • the organoids were maintained in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) with 10uM ROCK inhibitor Y27632 for the first day. Eleven days post transfection, genomic DNA was harvested by incubating the organoids in Cell Recovery Solution (Corning), triturating them, and pelleting by centrifugation. The pellets were resuspended in QuickExtract Solution (Lucigen) and transferred to a new 96-well PCR plate compatible with the thermocycler.
  • QuickExtract Solution (Lucigen)
  • the plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC WSGR Docket No.59761-775.601 A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • Forward primer- ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC WSGR Docket No.59761-775.601 A (SEQ ID NO: 775)
  • reverse primer- TG
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • 3 PEgRNAs and 1 ngRNA were tested together at the two different dosages of PEgRNA, ngRNA and prime editor mRNA for a total of 6 combinations with each combination designed to restore wild-type CFTR amino acid sequence. The results are shown in Table 37.
  • Table 37 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR genes of intestinal organoids with a PEgRNA and ngRNA (PE3 system) 1.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs and ngRNAs used a scaffold with sequence number 593.
  • EXAMPLE 19 PE3 screening of PEgRNAs in humanized mice derived intestinal organoids for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in intestinal organoids derived from humanized mice in collaboration with Case Western Reserve University’s Cystic Fibrosis Mouse Models Core in accordance with the methods detailed in Example 1.
  • the mutation-carrying organoids were cultured and transiently transfected with a PEgRNA, a ngRNA and an mRNA encoding a Prime Editor fusion protein for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by added between the PBS and the four U nucleotides. These were designed to be used in combination to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant intestinal organoids were plated in a 24-well plate at a density of 400-500 organoids per well. The cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in two different doses.
  • a low dose consisting of 750 ng PE mRNA, 250 ng pegRNA and 83.3 ng ngRNA and a high dose consisting of 1500 ng PE mRNA, 500 ng pegRNA and 166.7 ng ngRNA.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at - 20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs and ngRNAs used a scaffold with sequence number 593.
  • EXAMPLE 20 Screening of PEgRNA with 3’ motif variations for editing the c.1624G- >T (G542X) associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were chemically synthesized by Integrated DNA Four U nucleotides were added to the 3’ end, and the three consecutive nucleotides that preceded the between the PBS and the four terminal U nucleotides in those PEgRNAs lacking a 3’ linker and structural motif. Each was designed to be capable of correcting the c.1624G->T (G542X) mutation [0668] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70% confluent.
  • the cells were transfected with MessengerMax transfection cocktail containing a test PEgRNA and an mRNA encoding a Prime Editing fusion protein.
  • WSGR Docket No.59761-775.601 genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. Two transfections were carried out to generate biological replicates and the percent correction and percent indel values were determined based on the percentage of sequencing reads.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were chemically synthesized by Integrated DNA Four U nucleotides were added to the 3’ end, and the three consecutive nucleotides that preceded the between the PBS and the four terminal U nucleotides in those PEgRNAs lacking a 3’ linker and structural motif. Each was designed to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70% confluent. The cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9 nickase compatible with the PAM associated with the tested PEgRNAs. Three days post transfection, genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen).
  • QuickExtract Solution (Lucigen).
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long- term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC WSGR Docket No.59761-775.601 A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. Two transfections were carried out to generate biological replicates and the percent correction and percent indel values were determined based on the percentage of sequencing reads.
  • the PEgRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs used a scaffold with sequence number 593.
  • NA indicates that no linker or 3’ motif was present in the tested PEgRNA.
  • R1 indicates this is the first replicate out of 2 replicates.
  • R2 indicates this is the second replicate out of 2 replicates.
  • EXAMPLE 22 PE3 screening of PEgRNA and ngRNA combinations in iPSC-derived intestinal organoid cells for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis [0678] A screen was performed in intestinal organoid cells edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the mutation-carrying cells were cultured and transiently transfected with a PEgRNA, ngRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 24-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by the PBS and the four terminal U nucleotides in the PEgRNAs. These were designed to be used in combination to be able to correct the c.1624G->T (G542X) mutation.
  • mutant intestinal organoids were differentiated from mutant iPSCs using STEMdiff Intestinal Organoid Kit (STEMCELL Technologies).
  • the organoids were dissociated using Accutase (STEMCELL Technologies).
  • the single cell suspension was plated in a 24-well ultra-low attachment plate (Corning) at a density of about 100,000 cells/well in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies).
  • the cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in three different doses.
  • the organoids were maintained in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) with 10 uM ROCK inhibitor Y27632 for the first day.
  • the forskolin-induced swelling assay was run.
  • differentiated organoids were maintained in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) supplemented with 10 ⁇ M of forskolin (Sigma Aldrich) for 24h at 370C and 5% CO2. Control wells were incubated with 0.1% DMSO as a vehicle control.
  • the pellets were resuspended in QuickExtract Solution (Lucigen) and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • Three doses low: 250 ng PE mRNA, 83.3 ng PEgRNA and 27.8 ng ngRNA, medium: 500ng mRNA, 166.7 ng PEgRNA and 55.6 ng of ngRNA and high: 750 ng PE mRNA, 250 ng pegRNA and 83.3 ng ngRNA 2.
  • the PEgRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs used a scaffold with sequence number 593.
  • EXAMPLE 23 Screening of PEgRNA and PE2 editor mRNA dosages for editing the c.1624G->T (G542X) associated with Cystic Fibrosis
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were chemically synthesized by Integrated DNA Four U nucleotides were added to the 3’ end, and the three consecutive nucleotides that preceded the between the PBS and the four terminal U nucleotides in those PEgRNAs lacking a 3’ linker and structural motif. Each was designed to be capable of correcting the c.1624G->T (G542X) mutation [0688] Briefly, one day prior to transfection, mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70 confluent.
  • the cells were transfected with MessengerMax transfection cocktail containing a test PEgRNA in various dosages and an mRNA encoding a Prime Editing fusion protein also in various dosages.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. Two transfections were carried out to generate biological replicates and the percent correction and percent indel values were determined based on the percentage of sequencing reads.
  • the PEgRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all PEgRNAs utilized a spacer having sequence number 11, an RTT having sequence number 87, and a PBS having sequence number 41. 2. NA indicates that no linker or 3’ motif was present in the tested PEgRNA.
  • EXAMPLE 24 PE3 screening of PEgRNA and ngRNA combinations in humanized mouse derived intestinal organoids for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in intestinal organoids derived from humanized mice in collaboration with Case Western Reserve University’s Cystic Fibrosis Mouse Models Core in accordance with the methods detailed in Example 1.
  • the mutation-carrying organoids were cultured and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein for assessment of editing by high-throughput sequencing.
  • PEgRNAs and ngRNAs used in this experiment were chemically synthesized by the PBS and the four terminal U nucleotides in those PEgRNAs lacking a 3’ linker and structural motif. These were designed to be used in combination to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant intestinal organoids were plated in a 24-well plate at a density of 400-500 organoids per well.
  • the cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA WSGR Docket No.59761-775.601 encoding a Prime Editing fusion protein in multiple doses.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at - 20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; the PEgRNA having sequence number 828 contained a 3’ linker having sequence number 634 and a structural motif having sequence number 734 between the PBS and 4 terminal U nucleotides.
  • EXAMPLE 25 PE3 screening of PEgRNA and ngRNA combinations using humanized mice derived intestinal organoids for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in intestinal organoids derived from humanized mice in collaboration with Case Western Reserve University’s Cystic Fibrosis Mouse Models Core in accordance with the methods detailed in Example 1.
  • the mutation-carrying organoids were cultured and transiently transfected with a PEgRNA, a ngRNA and an mRNA encoding a Prime Editor fusion protein for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by the PBS and the four terminal U nucleotides in the PEgRNAs. These were designed to be used in combination to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant intestinal organoids were plated in a 24-well plate at a density of 400-500 organoids per well. The cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in multiple doses.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long- term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: WSGR Docket No.59761-775.601 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C. Following the first PCR reaction, a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification. The second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing. NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA sequences used experimentally had scaffolds having sequence number 593 and contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond. 3. NA indicates that sequencing failure prevented assessment.
  • EXAMPLE 26 PE3 screening of PEgRNA and ngRNA combinations using humanized mice derived intestinal organoids using electroporation delivery for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis WSGR Docket No.59761-775.601
  • a screen was performed in intestinal organoids derived from humanized mice in collaboration with Case Western Reserve University’s Cystic Fibrosis Mouse Models Core in accordance with the methods detailed in Example 1.
  • the mutation-carrying organoids were cultured and transiently transfected with a PEgRNA, a ngRNA and an mRNA encoding a Prime Editor fusion protein for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by the PBS and the four terminal U nucleotides in the PEgRNAs. These were designed to be used in combination to be capable of correcting the c.1624G->T (G542X) mutation.
  • mutant intestinal organoid were plated in a 24-well plate at a density of 400-500 organoids per well. Cells were transfected using electroporation with a transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in various dosages. Three days post transfection, genomic DNA was harvested by removing the media from the cells.
  • the cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene. The percent correction and percent indel values are determined based on the percentage of sequencing reads.
  • the PEgRNA and ngRNA sequences used experimentally had scaffolds having sequence number 593 and contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond. 3. NA indicates that sequencing failure prevented assessment.
  • EXAMPLE 27 PE3 screening of PEgRNA and ngRNA combinations in iPSC-derived intestinal organoid cells for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in intestinal organoid cells edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the mutation-carrying cells were cultured and transiently transfected with a PEgRNA, ngRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 24-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by WSGR Docket No.59761-775.601 the PBS and the four terminal U nucleotides in the PEgRNA. These were designed to be used in combination to be able to correct the c.1624G->T (G542X) mutation.
  • mutant intestinal organoids were differentiated from mutant iPSCs using STEMdiff Intestinal Organoid Kit (STEMCELL Technologies). On the day of transfection, the organoids were dissociated using Accutase (STEMCELL Technologies).
  • the single cell suspension was plated in a 24-well ultra-low attachment plate (Corning) at a density of about 100,000 cells/well in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies).
  • the cells were transfected with a Lipofectamine MessengerMax transfection cocktail containing a test PEgRNA, a test ngRNA and an mRNA encoding a Prime Editing fusion protein in three different doses.
  • various dosages of total mRNA were utilized with a 3:1:3 mass ratio of PEgRNA: ngRNA: PE2 mRNA respectively.
  • the cell suspensions were incubated on a shaker plate at 250rpm for 3.5 hours at 37C with 5% CO2.
  • the cells were then pelleted and re-suspended in 50uL/well Matrigel hESC-Qualified Matrix LDEV-free (Corning) and plated on a 24-well plate (Corning).
  • the organoids were maintained in STEMdiff Intestinal Organoid Growth Media (STEMCELL Technologies) with 10uM ROCK inhibitor Y27632 for the first day. Eleven days post transfection, genomic DNA was harvested by incubating the organoids in Cell Recovery Solution (Corning), triturating them, and pelleting by centrifugation. The pellets were resuspended in QuickExtract Solution (Lucigen) and transferred to a new 96-well PCR plate compatible with the thermocycler.
  • QuickExtract Solution (Lucigen)
  • the plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long-term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification. The DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene.
  • 1 PEgRNA and 1 ngRNA were tested together at different dosages of PEgRNA, ngRNA and prime editor mRNA for a total of 5 combinations with each combination designed to restore wild-type WSGR Docket No.59761-775.601 CFTR amino acid sequence. Each combination was tested in two independent transfections and the results are shown in Table 46.
  • Table 46 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR genes of iPSC-derived intestinal organoid cells with a PEgRNA and ngRNA (PE3 system) 1.
  • Dosages represent total delivered RNA with a mass ratio of 3:1:3 of PEgRNA: ngRNA :mRNA respectively 2.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs and ngRNAs used a scaffold with sequence number 593. 3.
  • R1 indicates this is the first replicate out of 2 replicates.
  • R2 indicates this is the second replicate out of 2 replicates.
  • EXAMPLE 28 PE3 screening of PEgRNA and ngRNA combinations in human bronchial epithelial cells for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in the UNCCF13T line of human bronchial epithelial cells carrying the mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the cells were cultured and transiently transfected with a PEgRNA, ngRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by the PBS and the four terminal U nucleotides in the PEgRNAs lacking a 3’ linker and motif. These were designed to be used in combination to be able to correct the c.1624G->T (G542X) mutation.
  • human bronchial epithelial cells were seeded in collagen-coated 96-well plates at a density of 15,000 cells/well in PneumaCult Ex-Plus medium (STEMCELL Technologies) and then incubated at 37°C with 5% CO2.
  • Short term storage of gDNA was at 4°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 3 minutes, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 2 minutes for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • Table 47 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR genes of human bronchial epithelial cells with a PEgRNA and ngRNA (PE3 system) WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 1. Dosages represent total delivered RNA with a mass ratio of 3:1:4 of PEgRNA: ngRNA :mRNA respectively 2.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs and ngRNAs used a scaffold with sequence number 593 with the exception of PEgRNA sequence number 828 WSGR Docket No.59761-775.601 which uses scaffold sequence number 827 in addition to a linker with seq. number 634 and a 3’ motif with seq. number 407. 3. R1 indicates this is the first replicate out of 2 replicates. 4. R2 indicates this is the second replicate out of 2 replicates.
  • EXAMPLE 29 PE3 screening of PEgRNA and ngRNA combinations in human bronchial epithelial cells using a LNP delivery method for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis
  • a screen was performed in the UNCCF13T line of human bronchial epithelial cells carrying the mutation c.1624G->T (G542X) in both copies of the endogenous CFTR gene.
  • the cells were cultured and transiently transfected with a PEgRNA, ngRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs and ngRNAs used in this experiment were chemically synthesized by the PBS and the four terminal U nucleotides in the PEgRNAs lacking a 3’ linker and motif. These were designed to be used in combination to be able to correct the c.1624G->T (G542X) mutation.
  • human bronchial epithelial cells were seeded in collagen-coated 96-well plates at a density of 15,000 cells/well in PneumaCult Ex-Plus medium (STEMCELL Technologies) and then incubated at 37°C with 5% CO2.
  • gDNA QuickExtract Solution (Lucigen) was added into the 96-well plates after removal of culture medium and agitation using a pipette was applied to release and lyse cells. Individual suspensions were transferred into thermocycler compatible 96-well PCR plates. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 3 minutes, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 2 minutes for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • Table 48 Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR genes of human bronchial epithelial cells with a PEgRNA and ngRNA (PE3 system) WSGR Docket No.59761-775.601 1. Dosages represent total delivered RNA with a mass ratio of 3:1:4 of PEgRNA: ngRNA :mRNA respectively; in all dosage conditions, ngRNA seq. number 501 with spacer seq. number 475 is used. 2.
  • the PEgRNA and ngRNA sequences used experimentally contained 3’ mN*mN*mN*N and 5’mN*mN*mN*N modifications, where m indicates that the nucleotide contains a 2’-O-Me modification and a * indicates a phosphorothioate bond; all pegRNAs and ngRNAs used a scaffold with sequence number 593 with the exception of PEgRNA sequence number 828 which uses scaffold sequence number 827 in addition to a linker with sequence number 634 and a 3’ motif with sequence number 407. 3.
  • R1 indicates this is the first replicate out of 3 replicates.
  • R2 indicates this is the second replicate out of 3 replicates.
  • R3 indicates this is the third replicate out of 3 replicates.
  • EXAMPLE 30 Screening of PEgRNAs for editing of the c.1624G->T (G542X) mutation associated with Cystic Fibrosis WSGR Docket No.59761-775.601
  • a screen was performed in an HEK293T cell line edited in accordance with the methods in Example 1 to carry mutation c.1624G->T (G542X) in all three copies of the endogenous CFTR gene.
  • the mutation-carrying cell line was expanded and transiently transfected with a PEgRNA and an mRNA encoding a Prime Editor fusion protein in arrayed 96-well plates for assessment of editing by high-throughput sequencing.
  • the PEgRNAs used in this experiment were expressed from a plasmid containing an expression cassette with a U6 promoter and poly-T terminator.
  • An additional guanosine was present at the 5’ end of any spacer that started with a nucleotide other than guanosine to enable expression.
  • An additional adenine nucleotide was inserted between the 3’ structural motif and and the poly-T terminator.
  • the 3’ end of the expressed PEgRNA will comprise from 1-7 additional uridine nucleotides, e.g., 4 Us.
  • PEgRNAs were designed to restore the wild-type amino acid sequence, some PEgRNAs encode one or more synonymous edits including for example PAM-silencing mutations.
  • mutant HEK293T cells were plated in a 96-well plate at a density of about 10,000 cells per well. Before transfecting, cells were approximately 60-70% confluent. The cells were transfected using lipofectamine with a transfection cocktail containing a test PEgRNA plasmid and a plasmid encoding a Prime Editing fusion protein containing a Cas9 nickase compatible with the PAM associated with the tested PEgRNAs.
  • genomic DNA was harvested by removing the media from the cells and treating each well with 50 ⁇ L of QuickExtract Solution (Lucigen). The cells were resuspended in this solution and transferred to a new 96-well PCR plate compatible with the thermocycler. The plate was incubated at 65°C for 15 minutes, followed by an incubation at 98°C for 20 minutes. Short term storage of gDNA was at 4°C and long- term storage at -20°C.
  • the first PCR reaction on the gDNA used paired primers (Forward primer- (ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCTTTCAAATTCAGATTGAGC A (SEQ ID NO: 775)) and reverse primer- (TGGAGTTCAGACGTGTGCTCTTCCGATCTACCCACTAGCCATAAAACCCC (SEQ ID NO: 776)) in a 25 ⁇ L reaction with Q5 PCR Master Mix (NEB).
  • This reaction was run on a thermocycler at 98°C for 1 minute, then 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 30 seconds for 30 cycles total, followed by last step of 72°C for 2 minutes.
  • PCR1 reactions were stored at -20°C.
  • a second PCR reaction was set up with barcoded primers, 0.5 ⁇ L of the first PCR reaction, and Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo), which underwent 7 cycles of amplification.
  • the second PCR reactions were pooled and run on a 2% agarose E-gel for DNA purification.
  • the DNA samples were then run on the MiSeq for sequencing.
  • NGS results were analyzed using CRISPResso2 to measure percent correction and percent of indels for each mutation in the CFTR gene.
  • WSGR Docket No.59761-775.601 In total, 192 PEgRNAs were tested, each of which were designed to restore wild-type CFTR amino acid sequence. The results are shown in Table 49. [0732] Table 49: Prime Editing to correct c.1624G->T (G542X) mutations in the endogenous CFTR genes with a PEgRNA (PE2 system) WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 WSGR Docket No.59761-775.601 1.
  • the indicated PEgRNA sequence represents the expression cassette from the plasmids used experimentally and contains a poly-T terminator sequence; the PEgRNAs were expressed WSGR Docket No.59761-775.601 from a U6 promoter and will have contained anywhere from 1-7 uridine nucleotides (e.g., 4 uridine nucleotides) at the 3’ end due to read through of the poly-T terminator sequence.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des compositions et des méthodes d'utilisation de systèmes d'édition primaire comprenant des éditeurs primaires et des ARN guides d'édition primaire pour le traitement de troubles génétiques tels que la fibrose kystique.
PCT/US2024/013877 2023-02-01 2024-01-31 Compositions d'édition de génome et méthodes de traitement de la fibrose kystique Ceased WO2024163679A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
IL322419A IL322419A (en) 2023-02-01 2024-01-31 Genome editing agents and methods for treating cystic fibrosis
EP24750984.7A EP4658322A1 (fr) 2023-02-01 2024-01-31 Compositions d'édition de génome et méthodes de traitement de la fibrose kystique
AU2024215960A AU2024215960A1 (en) 2023-02-01 2024-01-31 Genome editing compositions and methods for treatment of cystic fibrosis

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363482785P 2023-02-01 2023-02-01
US63/482,785 2023-02-01
US202363596171P 2023-11-03 2023-11-03
US63/596,171 2023-11-03

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AU (1) AU2024215960A1 (fr)
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200102561A1 (en) * 2017-02-06 2020-04-02 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2021138469A1 (fr) * 2019-12-30 2021-07-08 The Broad Institute, Inc. Édition de génome à l'aide de complexes crispr activés et entièrement actifs de la transcriptase inverse
WO2022204270A1 (fr) * 2021-03-23 2022-09-29 Recode Therapeutics, Inc. Compositions de polynucléotide, formulations associées et leurs méthodes d'utilisation
WO2023070110A2 (fr) * 2021-10-21 2023-04-27 Prime Medicine, Inc. Compositions d'édition génomique et méthodes de traitement de la rétinite pigmentaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200102561A1 (en) * 2017-02-06 2020-04-02 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
WO2021138469A1 (fr) * 2019-12-30 2021-07-08 The Broad Institute, Inc. Édition de génome à l'aide de complexes crispr activés et entièrement actifs de la transcriptase inverse
WO2022204270A1 (fr) * 2021-03-23 2022-09-29 Recode Therapeutics, Inc. Compositions de polynucléotide, formulations associées et leurs méthodes d'utilisation
WO2023070110A2 (fr) * 2021-10-21 2023-04-27 Prime Medicine, Inc. Compositions d'édition génomique et méthodes de traitement de la rétinite pigmentaire

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AU2024215960A1 (en) 2025-08-21
IL322419A (en) 2025-09-01
EP4658322A1 (fr) 2025-12-10

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