Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL CRISPR-CAS12I SYSTEMS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefits of and priorities to CN Patent
Application No. 202111289092.6, filed on
November 2, 2021, entitled "NOVEL CRISPR-CAS12I SYSTEMS"; CN Patent
Application No. 202210081981.1,
filed on January 24, 2022, entitled "NOVEL CRISPR-CAS12I SYSTEMS"; and PCT
Patent Application No.
PCT/CN2022/089074, filed on April 25, 2022, entitled "NOVEL CRISPR-CAS12I
SYSTEMS", the entire
contents of which, including any sequence listing and drawings, are
incorporated herein by reference in its
entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic sequence listing ("xxx.xml"; Size is xxx bytes
and it was created on xxx) is
incorporated herein by reference in its entirety. Wherever a sequence is an
RNA sequence, the T in the sequence
shall be deemed as U.
TECHNICAL FIELD
The disclosure is generally directed to Cas12i polypeptides, fusion proteins
comprising such Cas12i polypeptides,
CRISPR-Cas12i systems comprising such Cas12i polypeptides or fusion proteins,
and methods of using the same.
BACKGROUND
The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas)
systems, including type II Cas9
and type V Cas12 systems, which serve in the adaptive immunity of prokaryotes
against viruses, have been
developed into genome editing toolsi 3. Compared with type II systems, the
type V systems including V-A to V-K
showed more functional diversity4' 5. Amongst them, Cas12i has a relatively
smaller size (1033-1093 aa),
compared to SpCas9 and Cas12a, and has a 5'-TTN protospacer adjacent motif
(PAM) preference4' 6' 7. Cas12i is
characterized by the capability of autonomously processing precursor crRNA
(pre-crRNA) to form short mature
crRNA. Cas12i mediates cleavage of dsDNA with a single RuvC domain, by
preferentially nicking the non-target
strand and then cutting the target strand810. These intrinsic features of
Cas12i enable multiplex high-fidelity
genome editing. However, the previous Cas12i (Cas12i1 and Cas12i2) showed low
editing efficiency which limits
their utility for therapeutic gene editing. It is thus needed to develop
CRISPR-Cas12i systems with higher
efficiency for practical use.
Citation or identification of any document in this application is not an
admission that such a document is available
as prior art to the disclosure.
SUMMARY
To address the limitations of previous Cas12i, the applicant screened ten
Cas12i and found one, xCas12i (also
referred to as "SiCas12i" herein), with robust high activity in HEK293T cells.
Engineering of xCas12i by arginine
substitutions at the PAM-interacting (PI), REC and RuvC domains led to the
production of a variant, high-fidelity
Cas12Max (hfCas12Max), with significantly elevated editing activity and
minimal off-target cleavage efficiency.
In addition, the applicant assessed the base editing efficiency of xCas12i-
based base editor, and thus expanded the
genome-editing toolbox. The applicant further demonstrated that hfCas12Max
could be an effective
genome-editing tool ex vivo and in vivo via ribonucleoprotein (RNP) and lipid
nanoliposomes (LNP) respectively,
suggesting the excellent potential for therapeutic genome editing
applications.
In some aspects, the disclosure provides a Cas12i polypeptide:
(1) as set forth in any one of SEQ ID NOs: 1-3, 6, and 10;
(2) comprising the amino acid sequence of any one of SEQ ID NOs: 1-3, 6, and
10; or
1
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(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any
one of SEQ ID NOs: 1-3, 6,
and 10.
In some aspects, the disclosure provides a Cas12i polypeptide comprising an
amino acid sequence having a
sequence identity of at least about 60% (e.g., at least about 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%, or 99.9%) and
less than 100% to the amino acid sequence of the reference Cas12i polypeptide
of any one of SEQ ID NOs: 1-3, 6,
and 10,
optionally wherein the Cas12i polypeptide has a function (e.g., a modified
function that is either increased or
decreased compared to that) of the reference Cas12i polypeptide
(e.g.,
(a) an ability to form a complex with a guide RNA capable of forming a complex
with the reference Cas12i
polypeptide; and/or,
(b) a spacer sequence-specific dsDNA cleavage activity).
In some embodiment, the Cas12i polypeptide has increased spacer sequence-
specific dsDNA and/or ssDNA
cleavage activity compared to that of the reference Cas12i polypeptide of any
one of SEQ ID NOs: 1-3, 6, and 10
when both used in combination with a same guide RNA, e.g., an increase by at
least about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
100%, 110%, 120%, 130%,
140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%,
270%, 280%, 290%,
300%, or more.
In some embodiment, the Cas12i polypeptide has decreased spacer sequence-
specific dsDNA and/or ssDNA
cleavage activity compared to that of the reference Cas12i polypeptide of any
one of SEQ ID NOs: 1-3, 6, and 10
when both used in combination with a same guide RNA, e.g., a decrease by at
least about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100%.
In some embodiment, the Cas12i polypeptide is a dead Cas12i polypeptide having
substantially no spacer
sequence-specific dsDNA and/or ssDNA cleavage activity, e.g., having at most
about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, or 50% of spacer sequence-specific dsDNA and/or ssDNA
cleavage activity of the
reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6, and 10.
In some embodiment, the Cas12i polypeptide comprise a substitution selected
from the group consisting of
D650A, D700A, E875A, and D1049A of SEQ ID NO: 1, or a combination thereof.
In some embodiment, the Cas12i polypeptide is a Cas12i nickase having spacer
sequence-specific ssDNA
cleavage activity.
In some embodiment, the Cas12i polypeptide is a Cas12i nickase having spacer
sequence-specific ssDNA
cleavage activity against the target strand of a target dsDNA.
In some embodiment, the Cas12i polypeptide is a Cas12i nickase having spacer
sequence-specific ssDNA
cleavage activity against the target strand of a target dsDNA, and having
substantially no spacer sequence-specific
dsDNA cleavage activity, e.g., having at most about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, or 50% of
spacer sequence-specific dsDNA cleavage activity of the reference Cas12i
polypeptide of any one of SEQ ID NOs:
1-3, 6, and 10.
In some embodiment, the Cas12i polypeptide comprise a substitution selected
from the group consisting of the
mutant in Tables 11-14 of SEQ ID NO: 1, or a combination thereof.
In some embodiment, the Cas12i polypeptide is not any one of SEQ ID NOs: 1-3,
6, and 10.
In some embodiment, the Cas12i polypeptide has decreased spacer sequence-
independent (off-target) dsDNA
and/or ssDNA cleavage activity compared to that of the reference Cas12i
polypeptide of any one of SEQ ID NOs:
1-3, 6, and 10 when both used in combination with a same guide RNA, e.g., a
decrease by at least about 5%, 10%,
2
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15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 100%.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids of the amino acid sequence of
the reference Cas12i polypeptide of any one of SEQ ID NOs: 1-3, 6, and 10.
In some embodiment, the one or more mutations are within a domain
corresponding to the PI domain, REC-I
domain, and/or RuvC-II domain of the reference Cas12i polypeptide of any one
of SEQ ID NOs: 1-3, 6, and 10.
In some embodiment, the one or more mutations are within the PI domain at
positions 173-291, the REC-I domain
at positions 427-473, and/or RuvC-II domain at positions 800-1082 of the
reference Cas12i polypeptide of SEQ
ID NO: 1.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids at one or more of the
following positions of the amino acid sequence of the reference Cas12i
polypeptide of any one of SEQ ID NOs:
1-3, 6, and 10:
any one of positions 1 to the end of the reference Cas12i polypeptide of any
one of SEQ ID NOs: 1-3, 6, and 10,
e.g., 1080, such as, position 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, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,
128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,
174, 175, 176, 177, 178, 179, 180, 181,
182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,
197, 198, 199, 200, 201, 202, 203, 204,
205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,
220, 221, 222, 223, 224, 225, 226, 227,
228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
243, 244, 245, 246, 247, 248, 249, 250,
251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,
266, 267, 268, 269, 270, 271, 272, 273,
274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288,
289, 290, 291, 292, 293, 294, 295, 296,
297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,
312, 313, 314, 315, 316, 317, 318, 319,
320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,
335, 336, 337, 338, 339, 340, 341, 342,
343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,
358, 359, 360, 361, 362, 363, 364, 365,
366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,
381, 382, 383, 384, 385, 386, 387, 388,
389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403,
404, 405, 406, 407, 408, 409, 410, 411,
412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,
427, 428, 429, 430, 431, 432, 433, 434,
435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449,
450, 451, 452, 453, 454, 455, 456, 457,
458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,
473, 474, 475, 476, 477, 478, 479, 480,
481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495,
496, 497, 498, 499, 500, 501, 502, 503,
504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,
519, 520, 521, 522, 523, 524, 525, 526,
527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,
542, 543, 544, 545, 546, 547, 548, 549,
550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564,
565, 566, 567, 568, 569, 570, 571, 572,
573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587,
588, 589, 590, 591, 592, 593, 594, 595,
596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610,
611, 612, 613, 614, 615, 616, 617, 618,
619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633,
634, 635, 636, 637, 638, 639, 640, 641,
642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656,
657, 658, 659, 660, 661, 662, 663, 664,
665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,
680, 681, 682, 683, 684, 685, 686, 687,
688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,
703, 704, 705, 706, 707, 708, 709, 710,
711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725,
726, 727, 728, 729, 730, 731, 732, 733,
734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748,
749, 750, 751, 752, 753, 754, 755, 756,
3
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WO 2023/078314 PCT/CN2022/129376
757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771,
772, 773, 774, 775, 776, 777, 778, 779,
780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,
795, 796, 797, 798, 799, 800, 801, 802,
803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817,
818, 819, 820, 821, 822, 823, 824, 825,
826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840,
841, 842, 843, 844, 845, 846, 847, 848,
849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863,
864, 865, 866, 867, 868, 869, 870, 871,
872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886,
887, 888, 889, 890, 891, 892, 893, 894,
895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909,
910, 911, 912, 913, 914, 915, 916, 917,
918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932,
933, 934, 935, 936, 937, 938, 939, 940,
941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955,
956, 957, 958, 959, 960, 961, 962, 963,
964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978,
979, 980, 981, 982, 983, 984, 985, 986,
987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001,
1002, 1003, 1004, 1005, 1006, 1007,
1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020,
1021, 1022, 1023, 1024, 1025,
1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038,
1039, 1040, 1041, 1042, 1043,
1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056,
1057, 1058, 1059, 1060, 1061,
1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074,
1075, 1076, 1077, 1078, 1079,
1080.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids at one or more of the
following positions of the amino acid sequence of the reference Cas12i
polypeptide of SEQ ID NO: 1:
any one of positions 1 to 1080, such as, position 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, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107,
108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,
123, 124, 125, 126, 127, 128, 129, 130,
131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 151, 152, 153,
154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,
169, 170, 171, 172, 173, 174, 175, 176,
177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,
192, 193, 194, 195, 196, 197, 198, 199,
200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,
215, 216, 217, 218, 219, 220, 221, 222,
223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 242, 243, 244, 245,
246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,
261, 262, 263, 264, 265, 266, 267, 268,
269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,
284, 285, 286, 287, 288, 289, 290, 291,
292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306,
307, 308, 309, 310, 311, 312, 313, 314,
315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,
330, 331, 332, 333, 334, 335, 336, 337,
338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,
353, 354, 355, 356, 357, 358, 359, 360,
361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375,
376, 377, 378, 379, 380, 381, 382, 383,
384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,
399, 400, 401, 402, 403, 404, 405, 406,
407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,
422, 423, 424, 425, 426, 427, 428, 429,
430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,
445, 446, 447, 448, 449, 450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,
468, 469, 470, 471, 472, 473, 474, 475,
476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,
491, 492, 493, 494, 495, 496, 497, 498,
499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513,
514, 515, 516, 517, 518, 519, 520, 521,
522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536,
537, 538, 539, 540, 541, 542, 543, 544,
545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559,
560, 561, 562, 563, 564, 565, 566, 567,
568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582,
583, 584, 585, 586, 587, 588, 589, 590,
591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605,
606, 607, 608, 609, 610, 611, 612, 613,
614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,
629, 630, 631, 632, 633, 634, 635, 636,
4
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637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651,
652, 653, 654, 655, 656, 657, 658, 659,
660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,
675, 676, 677, 678, 679, 680, 681, 682,
683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697,
698, 699, 700, 701, 702, 703, 704, 705,
706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720,
721, 722, 723, 724, 725, 726, 727, 728,
729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743,
744, 745, 746, 747, 748, 749, 750, 751,
752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,
767, 768, 769, 770, 771, 772, 773, 774,
775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789,
790, 791, 792, 793, 794, 795, 796, 797,
798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812,
813, 814, 815, 816, 817, 818, 819, 820,
821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,
836, 837, 838, 839, 840, 841, 842, 843,
844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858,
859, 860, 861, 862, 863, 864, 865, 866,
867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881,
882, 883, 884, 885, 886, 887, 888, 889,
890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904,
905, 906, 907, 908, 909, 910, 911, 912,
913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927,
928, 929, 930, 931, 932, 933, 934, 935,
936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950,
951, 952, 953, 954, 955, 956, 957, 958,
959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973,
974, 975, 976, 977, 978, 979, 980, 981,
982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996,
997, 998, 999, 1000, 1001, 1002, 1003,
1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016,
1017, 1018, 1019, 1020, 1021,
1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034,
1035, 1036, 1037, 1038, 1039,
1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052,
1053, 1054, 1055, 1056, 1057,
1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070,
1071, 1072, 1073, 1074, 1075,
1076, 1077, 1078, 1079, 1080.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids at one or more of the
following positions of the amino acid sequence of the reference Cas12i
polypeptide of SEQ ID NO: 1:
K109, N110, Y111, L112, M113, S114, N115, 1116, D117, S118, D119, F121, V122,
W123, V124, D125, C126,
127, K128, F129, A130, K131, D132, F133, A134, Y135, Q136, M137, E138, L139,
G140, F141, H142, E143,
F144, T145, V146, L147, A148, E149, T150, L151, L152, A153, N154, S155, 1156,
L157, V158, L159, N160,
E161, S162, T163, K164, A165, N166, W167, A168, W169, G170, T171, V172, S173,
A174, L175, Y176, G177,
G178, G179, D180, K181, E182, D183, S184, T185, L186, K187, S188, K189, 1190,
L191, L192, A193, F194,
V195, D196, A197, L198, N199, N200, H201, E202, L203, K204, T205, K206, E208,
1209, L210, N211, Q212,
V213, C214, E215, S216, L217, K218, Y219, Q220, S221, Y222, Q223, D224, M225,
Y226, V227, D228, F229,
S231, V232, V233, D234, E235, N236, G237, N238, K239, K240, S241, P242, N243,
G244, S245, M246, P247,
1248, V249, T250, K251, F252, E253, T254, D255, D256, L257, 1258, S259, D260,
N261, Q262, K264, A265,
M266, 1267, S268, N269, F270, T271, K272, N273, A274, A275, A276, K277, A278,
A279, K280, K281, P282,
1283, P284, Y285, L286, D287, 288, L289, K290, E291, M293, V294, S295, L296,
C297, D298, Y300, N301,
V302, Y303, A304, W305, A306, A307, A308, 1309, T310, N311, S312, N313, A314,
D315, V316, T317, A318,
N320, T321, L324, T325, F326, 1327, G328, E329, Q330, N331, S332, K335, E336,
L337, S338, V339, L340,
Q341, T342, T343, T344, N345, E346, K347, A348, K349, D350, 1351, L352, N353,
K354, N356, D357, N358,
L359, 1360, Q361, E362, V363, Y365, T366, P367, A368, K370, H371, L372, G373,
D375, L376, A377, N378,
L379, F380, D381, T382, L383, K384, E385, K386, D387, 1388, N389, N390, 1391,
E392, N393, E394, E395,
E396, K397, Q398, N399, V400, 1401, N402, D403, C404, 1405, E406, Q407, Y408,
V409, D410, D411, C412,
L415, N416, N418, P419, 1420, A421, A422, L423, L424, K425, H426, 1427, S428,
Y430, Y431, E432, D433,
F434, S435, A436, K437, N438, F439, L440, D441, G442, A443, K444, L445, N446,
V447, L448, T449, E450,
V451, V452, N453, Q455, K456, A457, H458, P459, T460, 1461, W462, S463, E464,
1800, S801, L802, K803,
M804, 1805, S806, D807, F808, K809, G810, V811, V812, Q813, S814, Y815, F816,
S817, V818, S819, G820,
C821, V822, D823, D824, A825, S826, K827, K828, A829, H830, D831, S832, M833,
L834, F835, T836, F837,
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M838, C839, A840, A841, E842, E843, K844, T846, N847, K848, E850, E851, K852,
T853, N854, A856, A857,
S858, F859, 1860, L861, Q862, K863, A864, Y865, L866, H867, G868, C869, K870,
M871, 1872, V873, C874,
E875, D876, D877, L878, P879, V880, A881, D882, G883, K884, T885, G886, K887,
A888, Q889, N890, A891,
D892, M894, D895, W896, C897, A898, A900, L901, A902, K903, K904, V905, N906,
D907, G908, C909, V910,
A911, M912, S913, 1914, C915, Y916, A918, P920, A921, Y922, M923, S924, S925,
H926, Q927, D928, P929,
F930, V931, H932, M933, Q934, D935, K936, K937, T938, S939, V940, L941, P943,
F945, M946, E947, V948,
N949, K950, D951, S952, 1953, D955, Y956, H957, V958, A959, G960, L961, L965,
N966, S967, K968, S969,
D970, A971, G972, T973, S974, V975, Y976, Y977, Q979, A980, A981, L982, H983,
F984, C985, E986, A987,
L988, G989, V990, S991, P992, E993, L994, V995, K996, N997, K998, K999, T1000,
H1001, A1002, A1003,
E1004, L1005, G1006, M1009, G1010, S1011, A1012, M1013, L1014, M1015, P1016,
W1017, G1019, G1020,
V1022, Y1023, 11024, A1025, S1026, K1027, K1028, L1029, T1030, S1031, D1032,
A1033, K1034, S1035,
V1036, K1037, Y1038, C1039, G1040, E1041, D1042, M1043, W1044, Q1045, Y1046,
H1047, A1048, D1049,
E1050, 11051, A1052, A1053, V1054, N1055, 11056, A1057, M1058, Y1059, E1060,
V1061, C1062, C1063,
Q1064, T1065, G1066, A1067, F1068, G1069, K1070, K1071, Q1072, K1073, K1074,
S1075, D1076, E1077,
L1078, P1079, and G1080.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids at one or more of the
following positions of the amino acid sequence of the reference Cas12i
polypeptide of SEQ ID NO: 1:
S118, D119, F121, W123, Q136, E138, E143, V146, S155, V158, E161, S162, T163,
A165, N166, G178, D180,
T185, K189, A193, D196, N199, N200, E202, L203, S221, V233, E235, N236, S241,
N243, S245, K251, D255,
L257, N273, D287, S295, V302, S332, E336, S338, V339, E362, D375, A377, N378,
D381, T382, E385, D387,
N390, E395, E396, Q398, N399, V400, D403, E406, Q407, V409, D411, C412, N416,
N418, L440, L448, V451,
Q455, E464, S806, S817, V818, S819, S832, M833, F835, T836, F837, C839, A840,
E842, E843, K844, T846,
N847, K848, N854, A856, S858, Q862, K863, Y865, L866, G868, K870, M871, D876,
D877, V880, G883, K884,
G886, K887, A888, A891, D892, M894, A900, K903, K904, N906, V910, M912, S913,
C915, Y916, A918, M923,
S925, H926, Q927, V931, M933, Q934, D935, K936, K937, T938, S939, V940, F945,
M946, V948, N949, K950,
D951, S952, D955, Y956, A959, G960, N966, S967, K968, S969, D970, A971, G972,
S974, V975, Y976, Q979,
A980, L982, H983, C985, E986, A987, G989, V990, S991, P992, E993, L994, V995,
K996, N997, K998, K999,
T1000, H1001, A1002, A1003, E1004, G1006, G1010, A1012, M1013, L1014, W1017,
V1022, K1028, D1032,
K1034, K1037, C1039, G1040, Q1045, H1047, C1063, and G1069.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids at one or more of the
following positions of the amino acid sequence of the reference Cas12i
polypeptide of SEQ ID NO: 1:
N243, E336, V880, G883, D892, and M923.
In some embodiment, the one or more mutation is a substitution with R.
In some embodiment, the Cas12i polypeptide further comprises one or more
mutations (such as, insertions,
deletions, or substitutions) at one or more amino acids corresponding to one
or more amino acids at one or more
of the following positions of the amino acid sequence of the reference Cas12i
polypeptide of SEQ ID NO: 1:
V880, G883, D892, and M923.
In some embodiment, the one or more mutation is a substitution with R.
In some embodiment, the Cas12i polypeptide comprises one or more mutations
(such as, insertions, deletions, or
substitutions) at one or more amino acids corresponding to one or more amino
acids at one or more of the
following positions of the amino acid sequence of the reference Cas12i
polypeptide of SEQ ID NO: 1:
K109, L112, D125, 127, F144, L147, A148, L151, L157, V195, Y226, F252, 1258,
M293, W305, A308, 1309,
S312, A314, D315, V316, A318, L324, 1327, A348, L352, Y365, L372, L376, L379,
L383, 1405, L424, 1427,
A436, F439, A443, V447, A457, H458, P459, T460, S463, S814, F859, A864, H867,
Y977, S1031, A1053, and
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F1068.
In some embodiment, the one or more mutation is a substitution with R:
In some embodiment, the substitution at N243 is a substitution with R, A, V.
L, I, M, F, W, S, T, C, Y, N, Q, E, K,
or H.
In some embodiment, the mutation is a substitution.
In some embodiment, the substitution is a substitution with a non-polar amino
acid residue (such as, Glycine
(Gly/G), Alanine (Ala/A), Valine (ValN), Cysteine (Cys/C), Proline (Pro/P),
Leucine (Leu/L), Isoleucine (Ile/I),
Methionine (Met/M), Tryptophan (Trp/W), Phenylalanine (Phe/F), a polar amino
acid residue (such as, Serine
(Ser/S), Threonine (Thr/T), Tyrosine (Tyr/Y), Asparagine (Asn/N), Glutamine
(Gln/Q)), a positively charged
amino acid residue (such as, Lysine (Lys/K), Arginine (Arg/R), Histidine
(His/H)), or a negatively charged amino
acid residue (such as, Aspartic Acid (Asp/D), Glutamic Acid (Glue/E)).
In some embodiment, the substitution is a substitution with a positively
charged amino acid residue, such as,
Arginine (R).
In some embodiment, the substitution is a substitution with a non-polar amino
acid residue, such as, Alanine (A).
In some embodiment, the Cas12i polypeptide comprises a substitution
corresponding to any one of the mutants in
Table 6, or a combination thereof, and wherein the amino acid location is
relative to SEQ ID NO: 1.
In some embodiment, the Cas12i polypeptide comprises a substitution
corresponding to any one of the mutants in
Table 6 with increased spacer sequence-specific dsDNA cleavage activity
compared to that of the reference
Cas12i polypeptide of SEQ ID NO: 1 when both used in combination with a same
guide RNA, e.g., an increase by
at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%,
90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%,
210%, 220%, 230%,
240%, 250%, 260%, 270%, 280%, 290%, 300%, or more, or a combination thereof,
and wherein the amino acid
location is relative to SEQ ID NO: 1.
In some embodiment, the Cas12i polypeptide is xCas12i-N243R mutant.
In some embodiment, the Cas12i polypeptide comprises a substitution
corresponding to any one of the mutants in
Table 8, or a combination thereof, and wherein the amino acid location is
relative to SEQ ID NO: 1.
In some embodiment, the Cas12i polypeptide is xCas12i- N243R+E336R+D892R
mutant.
In some embodiment, the Cas12i polypeptide is xCas12i- N243R+E336R+G883R
mutant.
In some aspect, the disclosure provides a Cas12i polypeptide:
(1) as set forth in the amino acid sequence of xCas12i-N243R mutant;
(2) comprising the amino acid sequence of xCas12i-N243R mutant; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of
xCas12i-N243R mutant.
In some aspect, the disclosure provides a Cas12i polypeptide:
(1) as set forth in the amino acid sequence of xCas12i-N243R+E336R+D892R
mutant;
(2) comprising the amino acid sequence of xCas12i-N243R+E336R+D892R mutant; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of
xCas12i-N243R+E336R+D892R
mutant.
In some aspect, the disclosure provides a Cas12i polypeptide:
(1) as set forth in the amino acid sequence of xCas12i-N243R+E336R+G883R
mutant;
(2) comprising the amino acid sequence of xCas12i-N243R+E336R+G883R mutant; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
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99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of
xCas12i-N243R+E336R+G883R
mutant.
In some embodiment, the Cas12i polypeptide is capable of recognizing a target
adjacent motif (TAM)
immediately 5' to the protospacer sequence on the non-target strand of a
target dsDNA, and wherein the TAM is
5'-NTTN-3', wherein N is A, T, G, or C.
In some embodiment, the Cas12i polypeptide further comprises a functional
domain associated with the Cas12i
polypeptide.
In some embodiment, the functional domain has transposase activity, methylase
activity, demethylase activity,
translation activation activity, translation repression activity,
transcription activation activity, transcription
repression activity, transcription release factor activity, chromatin
modifying or remodeling activity, histone
modification activity, nuclease activity, single-strand RNA cleavage activity,
double-strand RNA cleavage activity,
single-strand DNA cleavage activity, double-strand DNA cleavage activity,
nucleic acid binding activity,
detectable activity, or any combination thereof.
In some aspect, the disclosure provides a fusion protein comprising the Cas12i
polypeptide of the disclosure and a
functional domain.
In some embodiments, the functional domain is fused N-terminally, C-
terminally, or internally with respect to the
C as12i polypeptide.
In some embodiments, the functional domain is fused to the Cas12i polypeptide
via a linker, e.g., a XTEN linker
(SEQ ID NO: 442), a GS linker containing multiple glycine and serine residues,
a GS linker containing multiple
glycine and serine residues and a XTEN linker (SEQ ID NO: 442), a GS linker
containing multiple glycine and
serine residues and a BP NLS (SEQ ID NO: 443).
In some embodiments, the functional domain is selected from the group
consisting of a nuclear localization signal
(NLS), a nuclear export signal (NES), a deaminase or a catalytic domain
thereof, an uracil glycosylase inhibitor
(UGI), an uracil glycosylase (UNG), a methylpurine glycosylase (MPG), a
methylase or a catalytic domain
thereof, a demethylase or a catalytic domain thereof, an transcription
activating domain (e.g., VP64 or VPR), an
transcription inhibiting domain (e.g., KRAB moiety or SID moiety), a reverse
transcriptase or a catalytic domain
thereof, an exonuclease or a catalytic domain thereof, a histone residue
modification domain, a nuclease catalytic
domain (e.g., FokI), a transcription modification factor, a light gating
factor, a chemical inducible factor, a
chromatin visualization factor, a targeting polypeptide for providing binding
to a cell surface portion on a target
cell or a target cell type, a reporter (e.g., fluorescent) polypeptide or a
detection label (e.g., GST, HRP, CAT, GFP,
HcRed, DsRed, CFP, YFP, BFP), a localization signal, a polypeptide targeting
moiety, a DNA binding domain
(e.g., MBP, Lex A DBD, Gal4 DBD), an epitope tag (e.g., His, myc, V5, FLAG,
HA, VSV-G, Trx, etc), a
transcription release factor, an HDAC, a moiety having ssRNA cleavage
activity, a moiety having dsRNA
cleavage activity, a moiety having ssDNA cleavage activity, a moiety having
dsDNA cleavage activity, a DNA or
RNA ligase, a functional domain exhibiting activity to modify a target DNA,
selected from the group consisting of:
methyltransferase activity, DNA repair activity, DNA damage activity,
dismutase activity, alkylation activity,
dealkylation activity, depurination activity, oxidation activity, deoxidation
activity, pyrimidine dimer forming
activity, integrase activity, transposase activity, recombinase activity,
polymerase activity, ligase activity, helicase
activity, photolyase activity, glycosylase activity, acetyl transferase
activity, deacetylase activity, kinase activity,
phosphatase activity, ubiquitin ligase activity, deubiquitination activity,
adenylation activity, deadenylation activity,
SUMOylation activity, deSUMOylation activity, ribosylation activity,
deribosylation activity, myristoylation
activity, demyristoylation activity, glycosylation activity (e.g., from 0-
G1cNAc transferase), deglycosylation
activity, and a catalytic domain thereof, and a functional fragment thereof,
and any combination thereof.
In some embodiments, the NLS comprises or is 5V40 NLS (SEQ ID NO: 444), bpSV40
NLS (BP NLS, bpNLS,
SEQ ID NO: 443), or NP NLS (Xenopus laevis Nucleoplasmin NLS, nucleoplasmin
NLS, SEQ ID NO: 445).
In some embodiments, the functional domain comprises a deaminase or a
catalytic domain thereof.
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In some embodiments, the deaminase or catalytic domain thereof is an adenine
deaminase (e.g., TadA, such as,
TadA8e, TadA8.17, TadA8.20, TadA9) or a catalytic domain thereof.
In some embodiments, the deaminase or catalytic domain thereof is a cytidine
deaminase (e.g., APOBEC, such as,
APOBEC3, for example, APOBEC3A, APOBEC3B, APOBEC3C; DddA) or a catalytic
domain thereof.
In some embodiments, the functional domain comprises an uracil glycosylase
inhibitor (UGI).
In some embodiments, the functional domain comprises an uracil glycosylase
(UNG).
In some embodiments, the functional domain comprises a methylpurine
glycosylase (MPG).
In some embodiments, the adenine deaminase domain is a wild type TadA or a
variant thereof
(1) as set forth in SEQ ID NO: 439;
(2) comprising the amino acid sequence of SEQ ID NO: 439; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 439.
In some embodiments, the adenine deaminase domain is TadA8e-V106W of SEQ ID
NO: 439 or TadA8e.
In some embodiments, the UGI domain
(1) is as set forth in SEQ ID NO: 441;
(2) comprises the amino acid sequence of SEQ ID NO: 441; or
(3) comprises an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 441.
In some embodiments, the cytidine deaminase domain is an APOBEC3 or a variant
thereof
(1) as set forth in SEQ ID NO: 440;
(2) comprising the amino acid sequence of SEQ ID NO: 440; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 440.
In some embodiments, the cytidine deaminase domain is human APOBEC3-W104A of
SEQ ID NO: 440.
In some embodiments, the functional domain comprises a reverse transcriptase
or a catalytic domain thereof.
In some embodiments, the functional domain comprises a methylase or a
catalytic domain thereof.
In some embodiments, the functional domain comprises a transcription
activating domain,
In some embodiments, the functional domain comprises an exonuclease or a
catalytic domain thereof, such as, T5
exonuclease (T5E) (SEQ ID NO: 449).
In some embodiments, the exonuclease is N-terminally or C-terminally fused to
the Cas12i polypeptide.
In some embodiments, the exonuclease is C-terminally fused to the Cas12i
polypeptide.
In some embodiments, the T5 exonuclease
(1) is as set forth in SEQ ID NO: 449;
(2) comprises the amino acid sequence of SEQ ID NO: 449; or
(3) comprises an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 449.
In some aspect, the disclosure provides a fusion protein comprising:
(1) an Cas12i polypeptide; and
(2) an adenine deaminase domain.
In some embodiments, the adenine deaminase domain is an adenine deaminase
(e.g., TadA, such as, TadA8e,
TadA8.17, TadA8.20, TadA9) or a catalytic domain thereof.
In some embodiments, the adenine deaminase domain is a wild type TadA or a
variant thereof
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(1) as set forth in SEQ ID NO: 439;
(2) comprising the amino acid sequence of SEQ ID NO: 439; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 439.
In some embodiments, the adenine deaminase domain is TadA8e-V106W of SEQ ID
NO: 439 or TadA8e.
In some aspect, the disclosure provides a fusion protein comprising:
(1) an Cas12i polypeptide; and
(2) a cytidine deaminase domain.
In some embodiments, the fusion protein further comprises an uracil
glycosylase inhibitor (UGI) domain.
In some embodiments, the UGI domain
(1) is as set forth in SEQ ID NO: 441;
(2) comprises the amino acid sequence of SEQ ID NO: 441; or
(3) comprises an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 441.
In some embodiments, the cytidine deaminase domain is a cytidine deaminase
(e.g., APOBEC (apolipoprotein B
mRNA-editing catalytic polypeptide-like), such as, APOBEC3, for example,
APOBEC3A, APOBEC3B,
APOBEC3C; DddA) or a catalytic domain thereof.
In some embodiments, the cytidine deaminase domain is an APOBEC3 or a variant
thereof
(1) as set forth in SEQ ID NO: 440;
(2) comprising the amino acid sequence of SEQ ID NO: 440; or
(3) comprising an amino acid sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of SEQ
ID NO: 440.
In some embodiments, the cytidine deaminase domain is human APOBEC3-W104A of
SEQ ID NO: 440.
In some embodiments, the fusion protein comprises the amino acid sequence of
SEQ ID NO: 85 or 184.
In some aspect, the disclosure provides a fusion protein comprising:
(1) an Cas12i polypeptide; and
(2) a non-LTR retrotransposon domain.
In some aspect, the disclosure provides a fusion protein comprising:
(1) an Cas12i polypeptide; and
(2) a transcription activating domain.
In some embodiments, the Cas12i polypeptide is the Cas12i polypeptide of the
disclosure.
In some embodiments, the adenine deaminase domain is N-terminally or C-
terminally fused to the Cas12i
polypeptide.
In some embodiments, the cytidine deaminase domain is N-terminally or C-
terminally fused to the Cas12i
polypeptide.
In some embodiments, the uracil glycosylase inhibitor domain is N-terminally
or C-terminally fused to the Cas12i
polypeptide.
In some embodiments, the uracil glycosylase inhibitor domain is N-terminally
or C-terminally fused to the
cytidine deaminase domain.
In some embodiments, the non-LTR retrotransposon domain is N-terminally or C-
terminally fused to the Cas12i
polypeptide.
In some embodiments, the fusion protein comprises one, two, three, or more UGI
domain.
In some embodiments, the fusion protein comprises one, two, three, or more UGI
domain in tandem via a linker or
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not.
In some embodiments, the fusion protein comprises one, two, three, four, or
more NLS and/or NES.
In some embodiments, the fusion protein comprises a NLS or a NES at the N-
terminus and/or C-terminus of the
C as12i polypeptide.
In some embodiments, the fusion protein comprises a NLS or a NES at the N-
terminus and/or C-terminus of the
adenine deaminase domain.
In some embodiments, the fusion protein comprises a NLS or a NES at the N-
terminus and/or C-terminus of the
cytidine deaminase domain.
In some embodiments, the fusion protein comprises a NLS or a NES at the N-
terminus and/or C-terminus of the
UGI domain.
In some embodiments, the fusion protein comprises a NLS or a NES at the N-
terminus and/or C-terminus of the
reverse transcriptase domain.
In some embodiments, the fusion protein comprises a NLS or a NES at the N-
terminus and/or C-terminus of the
non-LTR retrotransposon domain.
In some embodiments, the fusion is via a linker.
In some embodiments, the linker is a GS linker, a XTEN linker (SEQ ID NO:
442), a XTEN-containing linker, a
NLS or NES-containing linker, a XTEN-containing GS linker, a NLS or NES-
containing GS linker.
In some embodiments, the fusion protein comprises an inducible element, e.g.,
an inducible polypeptide.
In some embodiments, the NLS comprises or is 5V40 NLS (SEQ ID NO: 444), bpSV40
NLS (BP NLS, bpNLS,
SEQ ID NO: 443), or NP NLS (Xenopus laevis Nucleoplasmin NLS, nucleoplasmin
NLS, SEQ ID NO: 445).
In some aspect, the disclosure provides a vector, wherein the vector is an AAV
vector genome comprising:
(1) a polynucleotide encoding a fusion protein comprising of the disclosure
operably linked to a promoter; and
(2) a polynucleotide encoding a guide RNA operably linked to a promoter, the
guide RNA comprising:
(i) a direct repeat sequence capable of forming a complex with the Cas12i
polypeptide or the fusion protein; and
(ii) a spacer sequence capable of hybridizing to a target sequence on a target
strand of a target dsDNA, thereby
guiding the complex to the target dsDNA.
In some embodiments, the fusion protein has increased efficiency (e.g., base
editing efficiency, methylation
efficiency, transcription activating efficiency) compared to that of an
otherwise identical control fusion protein or
control conjugate or control fusion protein comprising the reference
polypeptide of any one of SEQ ID NOs: 1-3,
6, and 10, e.g., an increase in efficiency by at least about 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%,
150%, 160%, 170%,
180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%,
or more.
In some aspect, the disclosure provides a guide RNA comprising:
(1) a direct repeat sequence capable of forming a complex with an Cas12i
polypeptide or a fusion protein
comprising the Cas12i polypeptide and a functional domain; and
(2) a spacer sequence capable of hybridizing to a target sequence on a target
strand of a target dsDNA, thereby
guiding the complex to the target dsDNA.
In some embodiments, the direct repeat sequence is 5' to the spacer sequence.
In some embodiments, the guide RNA further comprises an aptamer.
In some embodiments, the guide RNA further comprises an extension to add an
RNA template.
In some embodiments, the guide RNA further comprises a donor sequence for
insertion into the target dsDNA.
In some embodiments, the direct repeat sequence:
(1) is as set forth in any one of SEQ ID NOs: 11-13, 16, 20, and 501-507;
(2) comprises the polynucleotide sequence of any one of SEQ ID NOs: 11-13, 16,
20, and 501-507; or
(3) comprises a polynucleotide sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
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99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the polynucleotide sequence of
any one of SEQ ID NOs: 11-13,
16, 20, and 501-507.
In some embodiments, the direct repeat sequence is a direct repeat sequence
comprising a polynucleotide
sequence having a sequence identity of at least about 60% (e.g., at least
about 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%,
or 99.9%) and less than 100% to the polynucleotide sequence of any one of SEQ
ID NOs: 11-13, 16, 20, and
501-507.
In some embodiments, the direct repeat sequence has substantially the same
secondary structure as the secondary
structure of any one of SEQ ID NOs: 11-13, 16, 20, and 501-507.
In some embodiments, the direct repeat sequence is not any one of SEQ ID NOs:
11-13, 16, and 20.
In some embodiments, when the guide RNA is used in combination with an Cas12i
polypeptide (e.g., the Cas12i
polypeptide of the disclosure), an increased spacer sequence-specific dsDNA
and/or ssDNA cleavage activity is
exhibited compared with that of an otherwise identical control guide RNA
comprising any one of SEQ ID NOs:
11-13, 16, 20, and 501-507 used in combination with the Cas12i polypeptide,
e.g., an increase by at least about
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%,
100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%,
230%, 240%, 250%,
260%, 270%, 280%, 290%, 300%, or more.
In some embodiments, when the guide RNA is used in combination with an Cas12i
polypeptide (e.g., the Cas12i
polypeptide of the disclosure), an decreased spacer sequence-specific dsDNA
and/or ssDNA cleavage activity is
exhibited compared with that of an otherwise identical control guide RNA
comprising any one of SEQ ID NOs:
11-13, 16, 20, and 501-507 used in combination with the Cas12i polypeptide,
e.g., an decrease by at least about
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or
100%.
In some embodiments, when the guide RNA is used in combination with a fusion
protein comprising an Cas12i
polypeptide (e.g., the Cas12i polypeptide of the disclosure) and a functional
domain (e.g., a functional domain of
the disclosure) (e.g., a fusion protein of the disclosure), an increased
efficiency (e.g., base editing efficiency,
methylation efficiency, transcription activating efficiency) is exhibited
compared to that of an otherwise identical
control guide RNA comprising any one of SEQ ID NOs: 11-13, 16, 20, and 501-507
used in combination with the
fusion protein, e.g., an increase by at least about 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%,
160%, 170%, 180%,
190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or
more.
In some embodiments, the direct repeat sequence comprises one or more
mutations (such as, insertions, deletions,
or substitutions) at one or more nucleotides corresponding to one or more
nucleotides of the polynucleotide
sequence of any one of SEQ ID NOs: 11-13, 16, 20, and 501-507.
In some embodiments, the one or more mutations are within a stem-loop region
corresponding to the stem-loop
region (e.g., R1 region, R2 region, R3 region, R4 region) of the
polynucleotide sequence of any one of SEQ ID
NOs: 11-13, 16, 20, and 501-507.
In some embodiments, the direct repeat sequence comprises one or more
mutations (such as, insertions, deletions,
or substitutions) at one or more nucleotides corresponding to one or more
nucleotides at one or more of the
following positions of the polynucleotide sequence of any one of SEQ ID NOs:
11-13, 16, 20, and 501-507:
any one of positions 1 to the end of any one of SEQ ID NOs: 11-13, 16, 20, and
501-507, e.g., 36, such as,
position 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.
In some embodiments, the direct repeat sequence comprises one or more
mutations (such as, insertions, deletions,
or substitutions) at one or more nucleotides corresponding to one or more
nucleotides at one or more of the
following positions of the polynucleotide sequence of SEQ ID NO: 11:
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any one of positions 1 to 36, such as, position 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.
In some embodiments, the mutation is a deletion.
In some embodiments, the mutation is a substitution.
In some embodiments, the mutation is a substitution with A, U, G, or C.
In some embodiments, the direct repeat sequence comprises a deletion.
In some embodiments, the deletion is within a stem-loop region (e.g., R1
region, R2 region, R3 region, R4 region,
R5 region) of the direct repeat sequence.
In some embodiments, the deletion 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,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100 nucleotides.
In some embodiments, the stem-loop region comprising the deletion retains 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, or 30
base pairs.
In some embodiments, the stem-loop region comprising the deletion retains at
most 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
base pairs.
In some embodiments, the stem-loop region comprising the deletion contains at
most 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
non-A-U or non-G-C mismatches.
In some embodiments, the direct repeat sequence comprises a substitution of
one or more thermodynamically
unstable base pairs with one or more G-C or C-G base pairs.
In some embodiments, the thermodynamically unstable base pair is a A-U or U-A
base pair, a A-G or G-A base
pair, or a U-G or G-U base pair.
In some embodiments, the thermodynamically unstable base pair is within the
stem of a stem-loop region of the
direct repeat sequence.
In some embodiments, the thermodynamically unstable base pair is the 1st, 2nd,
3rd, 4th, 5th, 6th, 7th, 8th, 9th,
10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21th, 22th,
23th, 24th, 25th, 26th, 27th, 28th, 29th,
or 30th base pair starting from and including the base pair shared by both the
stem and the loop of the stem-loop
region.
In some embodiments, the direct repeat sequence
(1) is as set forth in any one of SEQ ID NOs: 501-507;
(2) comprises the polynucleotide sequence of any one of SEQ ID NOs: 501-507;
or
(3) comprises a polynucleotide sequence having a sequence identity of at least
about 60% (e.g., at least about 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,
99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the polynucleotide sequence of
any one of SEQ ID NOs:
501-507.
In some embodiments, the target sequence comprises, consists essentially of,
or consists of at least about 16
contiguous nucleotides of a target gene, e.g., about 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, or 70 contiguous nucleotides of a target gene, or
in a numerical range between any of
two preceding values, e.g., from about 16 to about 50 contiguous nucleotides
of a target gene.
In some embodiments, the target sequence is at least about 16 nucleotides in
length, e.g., about 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, or
70 nucleotides in length, or in a length
of a numerical range between any of two preceding values, e.g., in a length of
from about 16 to about 50
nucleotides.
In some embodiments, the protospacer sequence comprises, consists essentially
of, or consists of at least about 16
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contiguous nucleotides of a target gene, e.g., about 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, or 70 contiguous nucleotides of a target gene, or
in a numerical range between any of
two preceding values, e.g., from about 16 to about 50 contiguous nucleotides
of a target gene.
In some embodiments, the protospacer sequence is at least about 16 nucleotides
in length, e.g., about 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, or 70 nucleotides in length, or in a
length of a numerical range between any of two preceding values, e.g., in a
length of from about 16 to about 50
nucleotides.
In some embodiments, the target sequence comprises a protospacer adjacent
motif (PAM) sequence 5' to the target
sequence.
In some embodiments, the target sequence comprises a protospacer adjacent
motif (PAM) sequence 5' to the
protospacer sequence reverse complementary to the target sequence.
In some embodiments, the spacer sequence is at least about 16 nucleotides in
length, e.g., about 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, or
70 nucleotides in length, or in a length
of a numerical range between any of two preceding values, e.g., in a length of
from about 16 to about 50
nucleotides.
In some embodiments, the spacer sequence is about 90 to 100% complementary to
the target sequence, and/or
contains no more than 1, 2, 3, 4, or 5 mismatches to the target sequence.
In some embodiments, the guide RNA comprises a plurality (e.g., 2, 3, 4, 5 or
more) of spacer sequences capable
of hybridizing to a plurality of target sequences, respectively.
In some embodiments, the plurality of target sequences are on a same
polynucleotide, or on separate
polynucleotides.
In some embodiments, the spacer sequence comprises at least 16 contiguous
nucleotides of any one of SEQ ID
NOs: 82-125, 130, 131-381, 382, 391, 398-438.
In some embodiments, the dsDNA is within a cell.
In some aspect, the disclosure provides a polynucleotide encoding the Cas12i
polypeptide or the fusion protein of
the disclosure.
In some aspect, the disclosure provides a polynucleotide encoding the guide
RNA of the disclosure.
In some embodiments, the polynucleotide is codon optimized for expression in
eukaryotic (e.g., mammalian, such
as, human) cells.
In some embodiments, the polynucleotide is a polydeoxyribonucleotide or a
polyribonucleotide.
In some embodiments, one or more of the nucleotides of the polynucleotide is
modified.
In some aspect, the disclosure provides a system or composition comprising:
(1) an Cas12i polypeptide or a fusion protein comprising the Cas12i
polypeptide and a functional domain, or a
polynucleotide encoding the Cas12i polypeptide or the fusion protein; and
(2) a guide RNA (also referred to as "CRISPR RNA" or "crRNA") or a
polynucleotide encoding the guide RNA,
the guide RNA comprising:
(i) a direct repeat sequence capable of forming a complex with the Cas12i
polypeptide or the fusion protein; and
(ii) a spacer sequence capable of hybridizing to a target sequence on a target
strand of a target dsDNA, thereby
guiding the complex to the target dsDNA.
In some embodiments, the system or composition is a non-naturally occurring,
engineered system or composition.
In some embodiments, the Cas12i polypeptide or the fusion protein is the
Cas12i polypeptide or the fusion protein
of the disclosure.
In some embodiments, the guide RNA is the guide RNA of the disclosure.
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In some embodiments, the direct repeat sequence is the direct repeat sequence
of the disclosure.
In some embodiments, the spacer sequence is the spacer sequence of the
disclosure.
In some embodiments, the system or composition further comprises an inducible
system, such as, TMP, DOX,
Degron.
In some embodiments, the inducible system comprises an inducing agent capable
of activating the fusion protein
comprising an inducible element.
In some embodiments, the inducible system comprises an inducing agent capable
of activating the expression of
the Cas12i polypeptide or the fusion protein comprising an inducible element.
In some embodiments, the system or composition comprises an activator capable
of activating the fusion protein
comprising a transcription activating domain.
In some embodiments, the coding sequence is a DNA coding sequence or an RNA
coding sequence.
In some embodiments, the system or composition further comprises a serine or
tyrosine recombinase.
In some embodiments, the system or composition further comprises a donor
construct comprising a donor
polynucleotide for insertion into the target dsDNA and located between two
binding elements capable of forming
a complex with the non-LTR retrotransposon protein.
In some embodiments, the Cas12i polypeptide is fused to the N-terminus of the
non-LTR retrotransposon protein.
In some embodiments, the Cas12i polypeptide is a nickase.
In some embodiments, the guide RNA guides the fusion protein to a target
sequence 5' of the targeted insertion
site, and wherein the Cas12i polypeptide generates a double-strand break at
the targeted insertion site.
In some embodiments, the guide RNA guides the fusion protein to a target
sequence 5' or 3' of the targeted
insertion site, and wherein the Cas12i polypeptide generates a double-strand
break at the targeted insertion site.
In some embodiments, the donor polynucleotide further comprises a polymerase
processing element to facilitate 5'
or 3' end processing of the donor polynucleotide sequence.
In some embodiments, the donor polynucleotide further comprises a homology
region to the target sequence on
the 5' end of the donor construct, the 3' end of the donor construct, or both.
In some embodiments, the homology region is from 8 to 25 base pairs.
In some aspect, the disclosure provides a vector comprising the polynucleotide
encoding the Cas12i polypeptide
or the fusion protein of the disclosure.
In some embodiments, the polynucleotide is operably linked to a promoter.
In some aspect, the disclosure provides a vector comprising the polynucleotide
encoding the guide RNA of the
disclosure.
In some embodiments, the polynucleotide is operably linked to a promoter.
In some aspect, the disclosure provides a vector comprising the polynucleotide
encoding the Cas12i polypeptide
or the fusion protein of the disclosure and the polynucleotide encoding the
guide RNA of the disclosure.
In some embodiments, the polynucleotide encoding the Cas12i polypeptide or the
fusion protein of the disclosure
and the polynucleotide encoding the guide RNA of the disclosure are operably
linked to a same promoter.
In some embodiments, the polynucleotide encoding the Cas12i polypeptide or the
fusion protein of the disclosure
and the polynucleotide encoding the guide RNA of the disclosure are each
operably linked to a promoter.
In some embodiments, the promoter is selected from the group consisting of a
ubiquitous promoter, a
tissue-specific promoter, a cell-type specific promoter, a constitutive
promoter, and an inducible promoter.
In some embodiments, the promoter comprises or is a promoter selected from the
group consisting of: a (human)
U6 promoter (such as SEQ ID NO: 446), an elongation factor la short (EFS)
promoter, a (human) Cbh promoter,
a MHCK7 promoter, a Cba promoter, a poll promoter, a pol II promoter, a pol
III promoter, a T7 promoter, a H1
promoter, a retroviral Rous sarcoma virus LTR promoter, a (human)
cytomegalovirus (CMV) promoter (such as
SEQ ID NO: 447), a 5V40 promoter, a dihydrofolate reductase promoter, a 13-
actin promoter, a 13 glucuronidase
(GUSB) promoter, a cytomegalovirus (CMV) immediate-early (le) enhancer and/or
promoter, a chicken 13-actin
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(CBA) promoter or derivative thereof such as a CAG promoter (such as SEQ ID
NO: 500), CB promoter, a
(human) elongation factor 1a-subunit (EF 1 a) promoter, a ubiquitin C (UBC)
promoter, a prion promoter, a
neuron-specific enolase (NSE) promoter, a neurofilament light (NFL) promoter,
a neurofilament heavy (NFH)
promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived
growth factor B -chain (PDGF-13)
promoter, a synapsin (Syn) promoter, a synapsin 1 (Synl) promoter, a methyl-
CpG binding polypeptide 2 (MeCP2)
promoter, a Ca2+/calmodulin-dependent polypeptide kinase II (CaMKII) promoter,
a metabotropic glutamate
receptor 2 (mGluR2) promoter, a 13-globin minigene n132 promoter, a
preproenkephalin (PPE) promoter, an
enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2)
promoter, a glial fibrillary acidic
polypeptide (GFAP) promoter, and a myelin basic polypeptide (MBP) promoter.
In some embodiments, the polynucleotide encoding the Cas12i polypeptide or the
fusion protein of the disclosure
is 5' or 3' to the polynucleotide encoding the guide RNA of the disclosure.
In some embodiments, the vector is a plasmid.
In some embodiments, the vector is a viral vector.
In some embodiments, the vector is a retroviral vector, a phage vector, an
adenoviral vector, a herpes simplex viral
(HSV) vector, an AAV vector, or a lentiviral vector.
In some embodiments, the AAV vector is a DNA-encapsidated AAV vector or a RNA-
encapsidated AAV vector.
In some embodiments, the AAV vector comprises a capsid with a serotype of
AAV1, AAV2, AAV3, AAV3A,
AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV-DJ,
AAV.PHP.eB, a member of the Clade to which any of the AAV1-AAV13 belong, a
functional truncated variant
thereof, or a functional mutant thereof.
In some aspect, the disclosure provides a recombinant AAV (rAAV) particle
comprising the vector of the
disclosure.
In some embodiments, the rAAV particle comprises a capsid with a serotype of
AAV1, AAV2, AAV3, AAV3A,
AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12,
AAV13, AAV-DJ,
AAV.PHP.eB, a member of the Clade to which any of the AAV1-AAV13 belong, a
functional truncated variant
thereof, or a functional mutant thereof, encapsidating the vector.
In some aspect, the disclosure provides a lipid nanoparticle (LNP) comprising
the polynucleotide encoding the
Cas12i polypeptide or the fusion protein of the disclosure and the guide RNA
of the disclosure.
In some embodiments, the polynucleotide encoding the Cas12i polypeptide or the
fusion protein of the disclosure
is in form of a mRNA.
In some embodiments, the polynucleotide encoding the Cas12i polypeptide or the
fusion protein comprise a 5'
UTR.
In some embodiments, the polynucleotide encoding the Cas12i polypeptide or the
fusion protein comprise a 3'
polyA tail.
In some aspect, the disclosure provides a method for modifying a target dsDNA,
comprising contacting the target
dsDNA with the system, vector, rAAV particle, or LNP of the disclosure,
wherein the spacer sequence is capable
of hybridizing to a target sequence of a target strand of the target dsDNA,
wherein the target sequence is modified
by the complex.
In some aspect, the disclosure provides use of the system, vector, rAAV
particle, or LNP of the disclosure in the
manufacture of an agent for modifying a target dsDNA, wherein the spacer
sequence is capable of hybridizing to a
target sequence of a target strand of the target dsDNA, wherein the target
sequence is modified by the complex.
In some aspect, the disclosure provides the system, vector, rAAV particle, or
LNP of the disclosure, for use in
modifying a target dsDNA, wherein the spacer sequence is capable of
hybridizing to a target sequence of a target
strand of the target dsDNA, wherein the target sequence is modified by the
complex.
In some embodiments, the target dsDNA is human TRAC gene.
In some embodiments, the spacer sequence comprises at least contiguous
nucleotides of any one of SEQ ID NOs:
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123-125.
In some aspect, the disclosure provides a cell or a progeny thereof comprising
the Cas12i polypeptide, the fusion
protein, the guide RNA, the system, the polynucleotide, the vector, the rAAV
particle, and/or the LNP of the
disclosure.
In some aspect, the disclosure provides a modified cell or a progeny thereof,
wherein the modified cell is modified
by the method of the disclosure.
In some embodiments, the cell is in vivo, ex vivo, or in vitro.
In some embodiments, the cell is a eukaryotic cell (e.g., an animal cell, a
vertebrate cell, a mammalian cell, a
non-human mammalian cell, a non-human primate cell, a rodent (e.g., mouse or
rat) cell, a human cell, a plant cell,
or a yeast cell) or a prokaryotic cell (e.g., a bacteria cell).
In some embodiments, the cell is a cultured cell, an isolated primary cell, or
a cell within a living organism.
In some embodiments, the cell is a T cell (such as, CAR-T cell), B cell, NK
cell (such as, CAR-NK cell), or stem
cell (such as, iPS cell, HSC cell).
In some embodiments, the cell is derived from or heterogenous to the subject.
In some aspect, the disclosure provides a host comprising the cell or progeny
thereof of the disclosure.
In some embodiments, the host is a non-human animal or a plant.
In some embodiments, the non-human animal is an animal (e.g., rodent or non-
human primate) model for a human
genetic disorder.
In some aspect, the disclosure provides a (e.g., pharmaceutical) composition
comprising the Cas12i polypeptide,
the fusion protein, the guide RNA, the polynucleotide, the system, the vector,
the rAAV particle, the LNP, and/or
the cell or progeny thereof of the disclosure.
In some embodiments, the composition comprises a pharmaceutically acceptable
excipient.
In some embodiments, the composition is formulated for delivery by
nanoparticles, e.g., lipid nanopaticles,
liposomes, exosomes, microvesicles, nucleic acid (e.g., DNA) nanoassemblies, a
gene gun, or an implantable
device.
In some aspect, the disclosure provides a delivery system comprising:
(1) a delivery vehicle, and
(2) the Cas12i polypeptide, the fusion protein, the guide RNA, the
polynucleotide, the system, the vector, the
rAAV particle, the LNP, the cell or progeny thereof, and/or the composition of
the disclosure.
In some embodiments, the delivery vehicle is a nanoparticle, e.g., a lipid
nanopaticle, a liposome, an exosome, a
microvesicle, a nucleic acid (e.g., DNA) nanoassembly, a gene-gun, or an
implantable device.
In some aspect, the disclosure provides a kit comprising the Cas12i
polypeptide, the fusion protein, the guide
RNA, the polynucleotide, the system, the vector, the rAAV particle, the LNP,
the cell or progeny thereof, the
composition, and/or the delivery system of the disclosure.
In some embodiments, the kit further comprising an instruction for modifying a
target dsDNA.
In some aspect, the disclosure provides a method for diagnosing, preventing,
or treating a disease or disorder in a
subject, comprising administering to the subject (e.g., an effective amount
of) the system, the vector, the rAAV
particle, the LNP, the cell or progeny thereof, the composition, the delivery
system, and/or the kit of the
disclosure.
In some aspect, the disclosure provides use of (e.g., an effective amount of)
the system, the vector, the rAAV
particle, the LNP, the cell or progeny thereof, the composition, the delivery
system, and/or the kit of the disclosure
in the manufacture of a medicament or kit for diagnosing, preventing, or
treating a disease or disorder in a subject.
In some aspect, the disclosure provides (e.g., an effective amount of) the
system, the vector, the rAAV particle, the
LNP, the cell or progeny thereof, the composition, the delivery system, and/or
the kit of the disclosure, for use in
diagnosing, preventing, or treating a disease or disorder in a subject.
In some embodiments, the disease or disorder is associated with an aberration
of a target dsDNA in the subject.
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In some embodiments, the spacer sequence is capable of hybridizing to a target
sequence of a target strand of the
target dsDNA, wherein the aberration of the target dsDNA is modified by the
complex.
In some embodiments, the method or use further comprises administering to the
subject an effective amount of a
homologous recombination donor template comprising a donor sequence for
insertion into a target dsDNA,
wherein the insertion of the donor sequence corrects the aberration of the
target dsDNA.
In some embodiments, the disease or disorder is prevented or treated by the
modified cell or progeny thereof.
In some embodiments, the disease or disorder is a TTR-associated disease or
disorder, e.g., ATTR.
In some embodiments, the spacer sequence comprises at least 16 contiguous
nucleotides of SEQ ID NO: 107.
In some embodiments, the disease or disorder is a PCSK9-associated disease or
disorder.
In some embodiments, the spacer sequence comprises at least 16 contiguous
nucleotides of SEQ ID NO: 122.
In some embodiments, the system further comprises a homologous recombination
donor template comprising a
donor sequence for insertion into a target dsDNA.
In some embodiments, said guiding the complex to the target dsDNA results in
binding of the complex to the
target dsDNA.
In some embodiments, said guiding the complex to the target dsDNA results in a
modification of the target
dsDNA.
In some embodiments, the modification of the target dsDNA comprises a double
strand break (DSB) of the target
dsDNA.
In some embodiments, the DSB results in generation of a deletion and/or
insertion mutation (Indel mutation).
In some embodiments, the Indel mutation modifies the transcription and/or
expression of the target dsDNA.
In some embodiments, a donor DNA template is inserted at the site of the DSB.
In some embodiments, the modification of the target dsDNA comprises a single
strand break (SSB) of the target
sequence of the target strand of the target dsDNA.
In some embodiments, the modification of the dsDNA comprises a substitution of
one or more nucleotides of the
protospacer sequence reverse complementary to the target sequence.
In some embodiments, the substitution is an A-to-T substitution, an A-to-G
substitution, an A-to-C substitution, a
C-to-A substitution, a C-to-T substitution, a C-to-G substitution, a T-to-A
substitution, a T-to-G substitution, a
T-to-C substitution, a G-to-A substitution, a G-to-T substitution, and/or a G-
to-C substitution.
In some embodiments, the modification of the dsDNA comprises a single strand
break (SSB) of the non-target
strand of the target dsDNA.
In some embodiments, the modification of the dsDNA comprises an insertion, a
deletion, and/or a substitution of
one or more nucleotides of the non-target strand.
In some embodiments, the modification: a. introduces one or more base edits;
b. corrects or introduces a
premature stop codon; c. disrupts a splice site; d. inserts or restores a
splice site; e. inserts a gene or gene fragment
at one or both alleles of the target polynucleotide; or f. a combination
thereof.
In some embodiments, the complex directs the reverse transcriptase domain to
the target sequence, and the reverse
transcriptase facilitates insertion of the donor sequence from the guide RNA
into the target dsDNA.
In some embodiments, the insertion of the donor sequence: a. introduces one or
more base edits; b. corrects or
introduces a premature stop codon; c. disrupts a splice site; d. inserts or
restores a splice site; e. inserts a gene or
gene fragment at one or both alleles of the target polynucleotide; or, f. a
combination thereof.
In some embodiments, the complex directs the non-LTR retrotransposon protein
to the target sequence, and the
non-LTR retrotransposon protein facilitates insertion of the donor
polynucleotide sequence from the donor
construct into the target dsDNA.
In some embodiments, the insertion of the donor sequence: a. introduces one or
more base edits; b. corrects or
introduces a premature stop codon; c. disrupts a splice site; d. inserts or
restores a splice site; e. inserts a gene or
gene fragment at one or both alleles of the target polynucleotide; or f. a
combination thereof.
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In some embodiments, said guiding the complex to the target dsDNA results in a
modification of the transcription
of the target dsDNA.
In some embodiments, the modification of the transcription is upregulated
transcription, downregulated
transcription, activated transcription, or inhibited transcription.
In some embodiments, the modification of the target dsDNA comprises
methylation or demethylation of one or
more nucleotides of the target dsDNA.
These and other aspects, objects, features, and advantages of the example
embodiments will become apparent to
those having ordinary skill in the art upon consideration of the following
detailed description of illustrated
example embodiments.
It should be understood that any one embodiment of the disclosure described
herein, including those described
only in the examples or claims, or only in one aspects / sections below, can
be combined with any other one or
more embodiments of the disclosure, unless explicitly disclaimed or improper.
BRIEF DESCRIPTION OF THE DRAWINGS
An understanding of the features and advantages of the disclosure will be
obtained by reference to the following
detailed description that sets forth illustrative embodiments, in which the
principles of the disclosure may be
utilized, and the accompanying drawings of which:
FIG. 1 shows that hfCas12Max, an engineered variant of xCas12i, mediated high-
efficient and -specificity
genome editing, and dCas12i base editor exhibited high base editing activity
in mammalian cells. A, xCas12i
mediated EGFP activation efficiency determined by flow cytometry. NC
represents non-specific (non-targeting)
control. B, Schematics of protein engineering strategy for mutants with high
efficiency and high fidelity using an
activatable EGFP reporter screening system with on-targeted and off-targeted
crRNA. C-D, Cas12Max exhibited
significantly increased cleavage activity than xCas12i at reporter plasmids
(C) or various genomic (D) target sites.
Each dot represents the mean indel frequency at one targeted site (n=3). E,
NGS analysis showed that
hfCas12Max retained comparable activity at TTR.2-ON targets and almost no at 6
OT sites, to Cas12Max. F, Both
Cas12Max and hfCas12Max exhibited a broader PAM recognition profile than other
Cas proteins, including
5'-TN and 5'-TNN PAM. G, Comparison of indel activity from Cas12Max,
hfCas12Max, LbCas12a, Ultra
AsCas12a, SpCas9 and KKH-saCas9 at TTR locus. hfCas12Max retained the
comparable activity of Cas12Max,
and higher gene-editing efficiency than other Cos proteins. Each dot
represents one of three repeats of single target
site. H, Schematics of different versions of dxCas12i adenine base editors. I,
Comparison of A-to-G editing
frequency and product purity at the KLF4 site from TadA8e.1-dxCas12i-v1.2,
v2.2 and v4.3, v4.3 showed a high
editing activity of 80%. TadA8e-dxCas12i-v4.3, named as ABE-dCas12Max.
TadA8e.1 represents TadA8e
V106W. J, Schematics of different versions of dxCas12i cytosine base editors.
K, Comparison of C-to-T editing
frequency and product purity at the DYRK1A site from hA3A.1-dxCas12i, -v1.2
v2.2 and v3.1, v3.1 showed a
high editing activity of 50%. hA3A.1-dxCas12i-v3.1, named as CBE-dCas12Max.
hA3A.1 represents human
APOBEC3A W104A.
FIG. 2 shows that hfCas12Max mediates high-efficiency gene editing ex vivo and
in vivo. A, Schematics of
hfCas12Max gene editing in primary human cells. B, Viability and indel
activity of human CD3+ T cells
following delivery of hfCas12Max RNPs with three different TRAC targeted
crRNAs at 1.6RM and 3.2RM
respectively (n=2 or 3). NC represents blank control, untreated with RNP. C,
Representative flow cytometric
analysis of edited CD3+ T cell 5 days after RNP delivery. NC represents blank
control, untreated with RNP. D,
Schematics of in vivo non-liposome delivery containing IVT-mRNA, LNP packaging
process. E, Editing
efficiency of LNP packaging with hfCas12Max mRNA and targeted Ttr crRNA at
increased concentrations in N2a
cells (n=8). F, Schematics of Ttr locus. G, Indel rates of LNP packaging with
hfCas12Max mRNA and targeted Ttr
crRNA at three dose (0.1, 0.3 and 0.5 mpk) in C57 mouse (n=6). H, The A to G
editing percentage of LNP
packaging with dCas12i-ABE mRNA and targeted Ttr crRNA at 3 mpk in C57 mouse
(n=2).
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FIG. 3 shows screen for functional Cas12i in HEK293T cells. A, Transfection of
plasmids coding Cas12i and
crRNA mediate EGFP activation. B, Five of ten Cas12i nuclease mediated EGFP-
activated efficiency in
HEK293T cells.
FIG. 4 shows identification and characterization of type V-I systems. A,
Nuclease domain organization of SpCas9,
LbCas12a, and xCas12i. B, Effective spacer sequence length for xCas12i. C, PAM
scope comparison of LbCas12a,
and xCas12i. xCas12i exhibited a higher dsDNA cleavage activity at 5'-TTN PAM
than Cas12a. D, Flow diagram
for detection of genome cleavage activity by transfection of an all-in-one
plasmid containing xCas12i and targeted
gRNA into HEK293T cells, followed by FACS and NGS analysis. E-F, xCas12i
mediated robust genome cleavage
(up to 90%) at the Ttr locus in N2a cells and TTR and PCSK9 in HEK293T cells.
FIG. 5 shows screen for engineered xCas12i mutants with increased dsDNA
cleavage activity. A, The relative
dsDNA cleavage activity of over 500 rationally engineered xCas12i mutants.
v1.1 represents xCas12i with N243R,
named as Cas12Max.
FIG. 6 shows other mutants mediated high-efficiency editing. A, Of the
saturated mutants of N243, N243R
increased the EGFP-activated fluorescent most. B-C, xCas12i mutant with N243R
increased 1.2, 5, 20-fold
activity at DMD.1, DMD.2 and DMD.3 locus. D, Both Cas12Max (xCas12i-N243R) and
Cas12Max-E336R
elevated EGFP-activated fluorescent at different PAM recognition sites.
FIG. 7 shows that Cas12Max induced off-target dsDNA cleavage activity at sites
with mismatches using the
reporter system (A) and targeted deep sequence (B).
FIG. 8 shows that hfCas12Max mediates high-efficiency and -specificity
editing. A, Rational protein engineering
screen of over 200 mutants for highly-fidelity Cas12Max.Four mutants show
significantly decreased activity at
both OT (off-target) sites and retains at ON.1 (on-target) site. B, Different
versions of xCas12i mutants. C, v6.3
reduced off-target at OT.1, OT.2 and OT.3 sites and retained indel activity at
TTR-ON targets, compared to
v1.1-Cas12Max. D, v6.3 exhibited comparable indel activity at DMD.1, DMD.2,
and higher at DMD.3 locus, than
v1.1-Cas12Max. v1.1, named as Cas12Max. v6.3, named as hfCas12Max.
FIG. 9 shows comparison of the gene-editing efficiency of hfCas12Max with
LbCas12a, Ultra AsCas12a, SpCas9
and KKH-saCas9 at TTR locus.
FIG. 10 shows that hfCas12Max mediated the high-efficient and -specific
editing. A-B, Off-target efficiency of
hfCas12Max, LbCas12a, and UltraAsCas12a at in-silico predicted off-target
sites, determined by targeted deep
sequencing. Sequences of on-target and predicted off-target sites are shown,
PAM sequences are in blue and
mismatched bases are in red.
FIG. 12 shows conserved cleavage sites of Cas12i. A, Sequence alignment of
xCas12i, Cas12i1 and Cas12i2
shows that D650, D700, E875 and D1049 are conserved cleavage sites at RuvC
domain. B, Introducing point
mutations of D650A, E875A, and D1049A result in abolished activity of xCas12i.
FIG. 13 shows engineering for high-efficiency dxCas12i-ABE. A, Engineering
schematic of TadA8e.1-dxCas12i.
Four parts for engineering are indicated. B, TadA8e.1-dxCas12i-v1.2 and v1.3
exhibits significantly increased
A-to-G editing activity among various variants at KLKF4 site of genome. C,
Increased A-to-G editing activity of
TadA8e-dxCas12i-v2.2 by combining v1.2 and v1.3. D, Unchanged or even
decreased editing activity from
various dCas12-ABEs carrying different NLS at N-terminal. E, Increased A-to-G
editing activity of
TadA8e-dxCas12i-v4.3 by combining v2.2, changed-NLS linker and high-activity
Tade8e.
FIG. 14 shows other strategies for high-efficiency dxCas12i-ABE. A, Schematics
of different versions of
dxCas12i adenine base editors. B, dxCas12i-ABE-N by TadA at the C-terminus of
dCas12 slightly increased
editing activity.
FIG. 15 shows comparison of editing frequencies induced by various dCas12-ABEs
at different genomic target
sites. A-B, Comparison of A-to-G editing frequencies induced by indicated
TadA8e.1-dxCas12i-v1.2, v2.2, and
TadA8e.1-dLbCas12a at PCSK9 and TTR genomic locus.
FIG. 16 shows characterization of dxCas12i-ABE in HEK293T cells. A-C,
dCas12Max-ABE base editing of each
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target sites with TTN (A), ATN (B), and CTN (C) PAM. D, dCas12Max-ABE base
editing product purity of each
target sites with TTN PAM of A. Target sites are indicated, with sequences of
each target protospacer and PAM
listed in Supplementary Table 4.
FIG. 17 shows comparison of editing frequencies induced by various dCas12-CBEs
at different genomic target
sites. A-B, Comparison of C-to-T editing frequencies and product purity
induced by indicated hA3A.1-dxCas12i,
v1.2, v2.2, and hA3A.1-dCas12a at DYRK1A and SITE4 genomic locus. hA3A.1
represents human
APOBEC3A-W104A.
FIG. 18 shows that hfCas12Max mediates high editing efficiency in HEK293
cells. A-C, Unchanged viability and
proliferation and increasing indel activity of HEK293 cells following delivery
of hfCas12Max RNPs with targeted
TTR or TRAC crRNA at increasing concentration (n=1).
FIG. 19 shows that hfCas12Max mediates high editing efficiency in mouse
blastocyst. A, Schematics of
hfCas12Max gene editing in mouse blastocyst. hfCas12Max mRNA and targeted Ttr
crRNA were injected into
mouse zygotes, and the injected zygotes were cultured into blastocyst stage
for genotyping analysis by targeted
deep sequencing. B, Indel rates of hfCas12Max targeted Ttr.3 and Ttr.12 in
mouse blastocyst (n=12).
FIG. 20 shows interaction of a guide RNA of CRISPR-Cas12i system and a target
dsDNA.
FIG. 21 shows the dsDNA cleavage activity of xCas12i when using various DR
sequence variant.
The figures herein are for illustrative purposes only and are not necessarily
drawn to scale.
DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS
Overview
In this study, the applicant demonstrate that the Type V-I Cas12i system
enables versatile and efficient genome
editing in mammalian cells. The applicant found a Cas12i, xCas12i (also
referred to as "SiCas12i" herein), that
shows high editing efficiency at TTN-PAM sites. By semi-rational design and
protein engineering of its PI, REC,
RuvC domains, the applicant obtained a high-efficiency, high-fidelity variant,
hfCas12Max, which contains
N243R, E336R, and D892R substitutions. In agreement with the hypothesis that
introducing arginine at key sites
could strengthen the binding between Cas and DNA, the introduction of N243R in
the PI domain and E336R at
REC domain significantly increased editing activity and expanded PAM
recognition. Interestingly, D892R or
G883R substitutions in the RuvC domain reduced off-target and retained on-
target cleavage activity, whereas
alanine substitutions28' 29, which has been used to reduce off-target
activity, did not (Fig. 56C). The D892R
substituted hfCas12Max was obviously more sensitive to mismatch, which
suggests that D892R or G883R
improved sgRNA binding specificity. According to sequence alignment and
predicted structure of xCas12i to
Cas12i2, asparagine 892 is located on NUC domain, together with RuvC domain to
forming a cleft, in which
crRNA:DNA heteroduplex was located. The variant with D892R did not alter the
on-target but eliminated
off-target activity, probably due to arginine substitution of asparagine
affecting the binding of non-target crRNA.
Our data suggests that a semi-rational engineering strategy with arginine
substitutions based on the
EGFP-activated reporter system could be used as a general approach to improve
the activity of CRISPR editing
tools.
Through engineering, the Cas12i system of the disclosure has achieved high
editing activity, high specificity and a
broad PAM range, comparable to SpCas9, and better than other Cas12 systems.
Given its smaller size, short
crRNA guide, and self-processing features4' 8' 10, the Type V-I Cas12i system
is suitable for in vivo multiplexed
gene editing applications, including AAV3 or LNP12' 13. Indeed, the data of
the disclosure indicates Type V-I
Cas12i system mediates the robust ex vivo or in vivo genome-editing
efficiencies via ribonucleoprotein (RNP)
delivery and lipid nanoliposomes (LNP) delivery respectively, demonstrating
the great potential for therapeutic
genome editing applications.
In addition, the applicant has confirmed that the Type V-I Cas12i system can
be used in base editing applications.
For base editor, the dCas12i system shows high A-to-G editing at A9-A11 sites
even A19 of KLF locus, and
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C-to-T editing at A7-A10 sites, which is similar to the dCas12a system but is
distinct from the dCas9/nCas9
system. Comparable to dCas12a, dCas12i-BE exhibited higher base editing
activity at KLF4, PCSK9 and
DYRK1A loci (Fig. 1K, Fig. S 13A, Fig. S 15A), suggesting it may have more
potential as a base editor. This
suggests that the dCas12i system is useful for broad genome engineering
applications, including epigenome
editing, genome activation, and chromatin imagingl' 31-34
In summary, the Cas12i system described here, which has robust editing
activity and high specificity, is a versatile
platform for genome editing or base editing in mammalian cells and could be
useful in the future for in vivo or ex
vivo therapeutic applications.
General Definitions
Cas12i is a programable RNA-guided dsDNA endonuclease that may generate a
double-strand break (DSB) on a
target dsDNA as guided by a programable RNA referred to as guide RNA (gRNA)
comprising a spacer sequence
and a direct repeat sequence. Without wishing to be bound by theory, it is
believed that the direct repeat sequence
is responsible for forming a complex with Cas12i and the spacer sequence is
responsible for hybridizing to a
target sequence of a target dsDNA, thereby guiding the complex of the gRNA and
the Cas12i to the target dsDNA.
Referring to FIG. 20, a target dsDNA is depicted to comprise a 5' to 3' upside
strand and a 3' to 5' downside
strand. A guide RNA is depicted to comprise a spacer sequence in green and a
direct repeat sequence in orange.
The spacer sequence is designed to hybridize to a part of the downside strand,
and so the spacer sequence "targets"
the part of the downside strand. And thus, the downside strand is referred to
as a "target DNA strand" or a "target
strand (TS)" of the target dsDNA, while the upside strand is referred to as a
"non-target DNA strand" or a
"non-target strand (NTS)" of the target dsDNA. The part of the target strand
based on which the spacer sequence
is designed and to which the spacer sequence may hybridize is referred to as a
"target sequence", while the
corresponding part of the part on the non-target strand is referred to as the
"reverse complementary sequence of
the target sequence" or "reverse complementary sequence" or "protospacer
sequence". In case of any conflict
with elsewhere of the disclosure, the definitions in this paragraph shall
prevail.
Unless otherwise specifically indicated, the invention will be practiced using
conventional methods of chemistry,
biochemistry, organic chemistry, molecular biology, microbiology, recombinant
DNA technology, genetics,
immunology, cell biology, stem cell protocols, cell culture, and transgenic
biology in the art, many of which are
described below for illustrative purposes. Such technologies are well
described in the literature.
All publications, patents and patent applications cited herein are
incorporated herein by reference in their entirety.
Unless otherwise specified, all technical and scientific terms used herein
have the meaning commonly understood
by one of ordinary skill in the art to which this invention belongs. For the
purposes of the invention, the following
terms are defined to conform to the meanings commonly understood in the art.
The articles "a/an" and the are used herein to refer to one or more than one
(i.e., at least one) grammatical object
of the article. For example, "element" means one element or more than one
element.
The use of alternatives (e.g. "or) is to be understood to mean either, both,
or any combination thereof.
The term "and/or" should be understood to mean either or both of the
alternatives.
As used herein, the term "about" or "approximately" refers to an amount,
level, value, quantity, frequency,
percentage, dimension, size, mass, weight, or length that is changed by up to
15%, 10%, 9%, 8%, 7%, 6%, 5%,
4%, 3%, 2%, or 1% as compared to the reference amount, level, value, quantity,
frequency, percentage, dimension,
size, mass, weight, or length. In one embodiment, the term "about" or
"approximately" refers to a range of amount,
level, value, quantity, frequency, percentage, dimension, size, mass, weight,
or length that is 15%, 10%, 9%,
8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% around the reference amount, level,
value, frequency, frequency,
percentage, scale, size, weight, quantity, weight, or length.
As used herein, the term "substantially/essentially" refers to a degree,
amount, level, value, quantity, frequency,
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percentage, dimension, size, mass, weight, or length that is about 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%,
97%, 98% or 99% or more of the reference degree, amount, level, value,
quantity, frequency, percentage,
dimension, size, mass, weight, or length.
A numerical range includes the end values of the range, and each specific
value within the range, for example, "16
to 100 nucleotides" includes 16 and 100, and each specific value between 16
and 100.
Throughout this specification, the terms "comprise", "include", "contain", and
have are to be understood as
implying that a stated step or element or a group of steps or elements is
included, but not excluding any other step
or element or group of steps or elements, unless the context requires
otherwise. In certain embodiments, the terms
"comprise", "include", "contain", and have are used synonymously.
"Consist or means including but limited to any element after the phrase
"consist or. Thus, the phrase "consist of
indicates that the listed elements are required or mandatory, and that no
other elements can be present.
"Consist essentially or is intended to include any element listed after the
phrase "consist essentially or and is
limited to other elements that do not interfere with or contribute to the
activities or actions specified in the
disclosure of the listed elements. Thus, the phrase "consist essentially or is
intended to indicate that the listed
elements are required or mandatory, but no other elements are optional, and
may or may not be present depending
on whether they affect the activities or actions of the listed elements.
Throughout the specification, reference to one embodiment", "embodiment", "a
specific embodiment", "a related
embodiment", an embodiment", "another embodiment" or "a further embodiment" or
a combination thereof
means that specific features, structures, or characteristics described in
connection with the embodiment are
included in at least one embodiment of the invention. Accordingly, the
appearances of the foregoing phrases in
various places throughout the specification are not necessarily all referring
to the same embodiments. Furthermore,
specific features, structures, or characteristics may be combined in any
suitable manner in one or more
embodiments.
"Sequence identity" between two polypeptides or nucleic acid sequences refers
to the percentage of the number of
identical residues between the sequences relative to the total number of the
residues, and the calculation of the
total number of residues is determined based on types of mutations. Types of
mutations include insertion
(extension) at either end or both ends of a sequence, deletions (truncations)
at either end or both ends of a
sequence, substitutions/replacements of one or more amino acids/nucleotides,
insertions within a sequence,
deletions within a sequence. Taking polypeptide as an example (the same for
nucleotide), if the mutation type is
one or more of the following: replacement/substitution of one or more amino
acids/nucleotides, insertion within a
sequence, and deletion within a sequence, then the number of residues of the
larger molecule in the compared
molecules is taken as the total number of residues. If the mutation type also
includes an insertion (extension) at
either end or both ends of the sequence or a deletion (truncation) at either
end or both ends of the sequence, the
number of amino acids inserted or deleted at either end or both ends (e.g.,
less than 20 inserted or deleted at both
ends) is not counted in the total number of residues. In calculating the
percentage of identity, the sequences being
compared are aligned in a manner that produces the largest match between the
sequences, and the gaps (if present)
in the alignment are resolved by a particular algorithm.
Conservative substitutions of non-critical amino acids may be made without
affecting the normal functions of the
protein. Conservative substitutions refer to the substitution of amino acids
with chemically or functionally similar
amino acids. Conservative substitution tables that provide similar amino acids
are well known in the art. For
example, in some embodiments, the amino acid groups provided below are
considered to be mutual conservative
substitutions.
In certain embodiments, selected groups of amino acids considered as mutual
conservative substitutions are as
follows:
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Acidic residues ID and E
Basic residues K, R and H
Hydrophilic uncharged residues S, T, N, and Q
Aliphatic uncharged residues 3, A, V, L and I
Nonpolar uncharged residues 2. M and P
Aromatic residues Y and W
In certain embodiments, other selected groups of amino acids considered as
mutual conservative substitutions are
as follows:
Group 1 LA, S and T
Croup 2 0 and E
!Group 3 N and Q
Croup 4 0 and K
!Group 5 L and M
1Group 6 F,YandW
In certain embodiments, other selected groups of amino acids considered as
mutual conservative substitutions are
as follows:
Group A A and G
Group B P and E
Group C N and Q
Group D R, K and H
Group E E, L, M, V
Group F , Y and W
Group G LS' and T
Group H IC and M
The term "amino acid" means twenty common naturally occurring amino acids.
Naturally occurring amino acids
include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic
acid (Asp; D), cysteine (Cys; C);
glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His;
H), isoleucine (Ile; I), leucine (Leu;
L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline
(Pro; P), serine (Ser; S), threonine (Thr;
T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
As used herein, the term "Cas12i protein" is used in its broadest sense and
includes parental or reference Cas12i
proteins (e.g., Cas12i protein comprising any of SEQ ID NOs: 1-10),
derivatives or variants thereof, and
functional fragments such as oligonucleotide-binding fragments thereof.
As used herein, the term "crRNA" is used interchangeably with guide molecule,
gRNA, and guide RNA, and
refers to nucleic acid-based molecules, which include but are not limited to
RNA-based molecules capable of
forming complexes with CRISPR-Cas proteins (e.g., any of Cas12i proteins
described herein) (e.g., via direct
repeat, DR), and comprises sequences (e.g., spacers) that are sufficiently
complementary to a target nucleic acid
sequence to hybridize to the target nucleic acid sequence and guide sequence-
specific binding of the complex to
the target nucleic acid sequence.
As used herein, the term "CRISPR array" refers to a nucleic acid (e.g., DNA)
fragment comprising CRISPR
repeats and spacers, which begins from the first nucleotide of the first
CRISPR repeat and ends at the last
nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in the
CRISPR array is located between
two repeats. As used herein, the term "CRISPR repeat" or "CRISPR direct
repeat" or "direct repeat" refers to a
plurality of short direct repeat sequences that exhibit very little or no
sequence variation in a CRISPR array.
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Appropriately, V-I direct repeats may form a stem-loop structure.
"Stem-loop structure" refers to a nucleic acid having a secondary structure
including a nucleotide region known or
predicted to form a double strand (stem) connected on one side by a region
(loop) which is mainly a
single-stranded nucleotide. The terms "hairpin" and "fold-back" structures are
also used herein to refer to
stem-loop structures. Such structures are well known in the art and these
terms are used in accordance with their
well-known meanings in the art. As known in the art, the stem-loop structure
does not require accurate base
pairing. Thus, the stem may include one or more base mismatches.
Alternatively, the base pairing may be accurate,
i.e., no mismatch is included.
As use herein, target nucleic acid is used interchangeably with target
sequence or target nucleic acid sequence to
refer to a specific nucleic acid comprising a nucleic acid sequence
complementary to all or part of a spacer in a
crRNA. In some examples, the target nucleic acid comprises a gene or a
sequence within the gene. In some
examples, the target nucleic acid comprises a non-coding region (e.g., a
promoter). In some examples, the target
nucleic acid is single-stranded. In some examples, the target nucleic acid is
double-stranded.
As used herein, "donor template nucleic acid" or "donor template" is used
interchangeably to refer to a nucleic
acid molecule that can be used by one or more cell proteins to alter the
structure of a target nucleic acid after the
CRISPR enzyme described herein alters the target nucleic acid. In some
examples, the donor template nucleic acid
is a double-stranded nucleic acid. In some examples, the donor template
nucleic acid is a single-stranded nucleic
acid. In some examples, the donor template nucleic acid is linear. In some
examples, the donor template nucleic
acid is circular (e.g., plasmid). In some examples, the donor template nucleic
acid is an exogenous nucleic acid
molecule. In some examples, the donor template nucleic acid is an endogenous
nucleic acid molecule (e.g.,
chromosome).
The target nucleic acid should be associated with PAM (protospacer adjacent
motif), that is, short sequences
recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas
protein, the target sequence
should be selected such that its complementary sequence (the complementary
sequence of the target sequence) in
the DNA duplex is upstream or downstream of PAM. In an embodiment of the
invention, the complementary
sequence of the target sequence is downstream or 3' of PAM. The requirements
for exact sequence and length of
PAM vary depending on the Cas12i protein used.
It will be understood by one of ordinary skill in the art that uracil and
thymine can both be represented by 't',
instead of `u' for uracil and 't' for thymine; in the context of a ribonucleic
acid, it will be understood that 't' is
used to represent uracil unless otherwise indicated.
As use herein, the term "cleavage" refers to DNA breakage in a target nucleic
acid produced by a nuclease of the
CRISPR system described herein. In some examples, the cleavage is double-
stranded DNA breakage. In some
examples, the cleavage is single-stranded DNA breakage.
As used herein, the meanings of "cleaving target nucleic acid" or "modifying
target nucleic acid" may overlap.
Modifying a target nucleic acid includes not only modification of a
mononucleotide but also insertion or deletion
of a nucleic acid fragment.
Cas12i proteins
The present application provides Cas12i proteins, such as those of SEQ ID NOs:
1-10, which have single-stranded
or double-stranded DNA cleavage activity. The Cas12i proteins described herein
have less than about 50%
sequence identity to other known Cas12i, are smaller and have better delivery
efficiency than other C as such as
Cas9 or Cas12. In some embodiments, the Cas12i protein comprises a sequence of
any of SEQ ID NOs: 1-10,
such as any of SEQ ID NOs: 1-3, 6, and 10, or SEQ ID NO: 1. In some
embodiments, the Cas12i protein is
isolated. In some embodiments, the Cas12i protein is engineered. In some
embodiments, the Cas12i protein is
man-made.
Cas12i proteins described herein, such as SiCas12i, Si2Cas12i, WiCas12i, and
SaCas12i, have excellent cleavage
activity for exogenous or endogenous genes in vitro or at the cellular level,
comparable to or even better than the
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cleavage activity of SpCas9, LbCas12a, and Cas12i.3. The cleavage activity of
Cas12i proteins described herein,
such as SiCas12i, Si2Cas12i, WiCas12i, and SaCas12i, for specific target
sequences of exogenous or endogenous
genes can be greater than about any of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95% or even greater
than 99% at the cellular level. Generally speaking, the cleavage activity of
Cas12i proteins described herein for
specific target sequences of exogenous or endogenous genes at the cellular
level is superior to that of Cas12i.3.
The cleavage activity of SiCas12i for exogenous or endogenous genes in vitro
or at the cellular level is
comparable to, or even better than that of SpCas9 or LbCas12a, and
significantly better than that of Cas12i.3. Its
cleavage activity for specific target sequences of exogenous or endogenous
genes at the cellular level may be
greater than about any of 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or
even greater than 99%. In
general, the cleavage activity of SiCas12i for specific target sequences of
exogenous or endogenous genes at the
cellular level is significantly superior to that of Cas12i.3.
The above Cas12i proteins may also comprise amino acid mutations that do not
substantially affect (e.g., affect no
more than about any of 5%, 4%, 3%, 2%, 1%, or smaller) the catalytic activity
(endonuclease cleavage activity) or
nucleic acid binding function of the Cas12i.
In some embodiments, the Cas12i proteins of the present invention (including
variants, dCas, nickases, etc.), such
as SiCas12i, comprise one or more nuclear localization sequences (NLSs) at its
N-terminus and/or C-terminus,
preferably one NLS at its N-terminus and one NLS at C-terminus. In some
embodiments, the NLS is an SV40
NLS (e.g., as set forth in SEQ ID NO: 444), preferably when the Cas12i protein
is used for cleavage. In some
embodiments, the NLS is a BP NLS, such as shown in SEQ ID NO: 443, preferably
when the Cas12i protein is
used for base editing, more preferably the Cas12i protein is fused at its N-
terminus a BP NLS of SEQ ID NO: 443,
and fused at its C-terminus a BP NLS of SEQ ID NO: 443.
Cas12i protein variants
The present invention also provides variants of any of the Cas12i proteins
described herein, such as Cas12i
variants with at least about 80% (e.g., at least about any of 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher) but less
than 100% identical sequence
to any of SEQ ID NOs: 1-10 (preferably, SEQ ID NOs: 1-3, 6, and 10, more
preferably, SEQ ID NO: 1). In some
embodiments, the Cas12i variant comprises one or more substitutions,
insertions, deletions, or truncations relative
to the amino acid sequence of a reference Cas12i protein (e.g., a Cas12i
protein comprising the amino acid
sequence of any one of SEQ ID NOs: 1-10).
As used herein, "variant" refers to a polynucleotide or a polypeptide that
differs from a reference (e.g., parental)
polynucleotide or polypeptide, respectively, but retains the necessary
properties. A typical variant of a
polynucleotide differs in nucleic acid sequence from a reference
polynucleotide. Nucleotide changes may or may
not alter the amino acid sequence of the polypeptide encoded by the reference
polynucleotide. Nucleotide changes
can result in amino acid substitutions, additions, deletions, or truncations
in the polypeptide encoded by the
reference polynucleotide. A typical variant of a polypeptide differs in amino
acid sequence from a reference
polypeptide. Typically, this difference is limited such that the sequences of
the reference and variant polypeptides
are generally very similar and identical in many regions. The amino acid
sequences of the variant polypeptide and
the reference polypeptide may differ by any combination of one or more of
substitutions, additions, deletions, or
truncations. A substituted or inserted amino acid residue may or may not be an
amino acid residue encoded by the
genetic code. Variants of a polynucleotide or polypeptide may be naturally
occurring (such as allelic variants), or
may be non-naturally occurring. Non-naturally occurring variants of
polynucleotides and polypeptides can be
prepared by mutagenesis techniques, by direct synthesis, or by other
recombinant methods known to those of skill
in the art.
As used herein, the term "wild-type" has the meaning commonly understood by
those skilled in the art and means
the typical form of an organism, strain, gene or trait. It can be isolated
from resources in nature and has not been
deliberately decorated.
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As used herein, the terms "non-naturally occurring" and "engineered" are used
interchangeably and refer to
artificial involvement. When these terms are used to describe a nucleic acid
molecule or polypeptide, it is meant
that the nucleic acid molecule or polypeptide is at least substantially free
of at least one other component with
which it is naturally associated or occurs in nature.
In some embodiments, the Cas12i variant is isolated. In some embodiments, the
Cas12i variant is engineered or
non-naturally occurring. In some embodiments, the Cas12i variant is
artificially synthesized. In some
embodiments, the Cas12i variant has one or more amino acid mutations (e.g.,
insertions, deletions, or substitutions)
in one or more domains relative to a reference Cas12i protein (e.g., the
parental Cas12i protein), such as PI
domain, Helical domain, RuvC domain, WED domain, Nuc domain, etc.
In some embodiments, the Cas12i variant is a variant relative to SiCas12i (SEQ
ID NO: 1). This means that the
Cas12i variant (e.g., a variant of Si2Cas12i) in its original sequence (e.g.,
Si2Cas12i, SEQ ID NO: 2) and the
original SiCas12i (SEQ ID NO: 1) can be aligned, and the one or more positions
with amino acid mutations (such
as insertions, deletions or substitutions) can be identified. In some
embodiments, the Cas12i variant is an
engineered SiCas12i.
In some embodiments, the Cas12i variant (e.g., a SiCas12i variant) has a
higher spacer-specific endonuclease
cleavage activity against a target sequence of a target DNA that is
complementary to the guide sequence,
compared to the corresponding reference Cas12i protein (e.g., Cas12i protein
comprising any of SEQ ID NOs:
1-10), such as at least about 1.2-fold (e.g., at least about any of 1.3, 1.4,
1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5 , 3, 3.5, 4, 5,
10, 20, 50-fold, or higher) higher than the corresponding reference Cas12i
protein.
In some embodiments, the original reference Cas12i protein (e.g., Cas12i
protein comprising any of SEQ ID NOs:
1-10) has a higher spacer-specific endonuclease cleavage activity against a
target sequence of a target DNA that is
complementary to the guide sequence, compared to the corresponding Cas12i
variant (e.g., SiCas12i variant),
such as at least about 1.2-fold (e.g., at least about any of 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2, 2.5 , 3, 3.5, 4, 5, 10, 20,
50-fold, or higher) higher than the Cas12i variant.
In some embodiments, the spacer-specific endonuclease cleavage activity of the
Cas12i variant (e.g., a SiCas12i
variant) against a target sequence of a target DNA that is complementary to a
guide sequence is the same as or not
significantly different from (e.g., within about 1.2-fold) that of the
corresponding original Cas12i protein (e.g.,
Cas12i protein comprising any of SEQ ID NOs: 1-10). For example, in some
embodiments, the Cas12i variant has
the same spacer-specific endonuclease cleavage activity against the target
sequence of the target DNA that is
complementary to the guide sequence as the corresponding original Cas12i
protein. In some embodiments, the
Cas12i variant has a spacer-specific endonuclease cleavage activity against a
target sequence of a target DNA that
is complementary to a guide sequence of no more than about 1.2-fold higher
than the corresponding original
Cas12i protein (e.g., less than or equal to about any of 1.2, 1.19, 1.15, 1.1,
1.01, 1.001-fold, etc.). In some
embodiments, the spacer-specific endonuclease cleavage activity of the
original Cas12i protein against a target
sequence of a target DNA that is complementary to the guide sequence is no
more than about 1.2-fold higher than
that of the corresponding Cas12i variant (e.g., less than or equal to about
any of 1.2, 1.19, 1.15, 1.1, 1.01,
1.001-fold, etc.).
Cas12i proteins substantially lacking catalytic activity (dCas12i)
The present invention also provides dead Cas12i (dCas12i) proteins lacking or
substantially lacking catalytic
activity. For example, in some embodiments, the dCas12i protein retains less
than about 50% (e.g., less than about
any of 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%,
5%, 4%, 3%, 2.5%, 2%, 1%
or less) spacer-specific endonuclease cleavage activity of the corresponding
parental Cas12i protein (e.g., Cas12i
protein comprising any of SEQ ID NOs: 1-10) for a target sequence of a target
DNA that is complementary to a
guide sequence. In some embodiments, the dCas12i protein comprises one or more
amino acid substitutions in the
RuvC domain (e.g., RuvC domain of a Cas12i protein comprising any of SEQ ID
NOs: 1-10), resulting in
substantial lack of catalytic activity. In some embodiments, the DNA cleavage
activity of dCas12i is zero or
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negligible compared to the non-mutated Cas12i form. In some embodiments, the
dCas12i is a Cas12i protein
without catalytic activity, which contains mutation(s) in the RuvC domain that
allow for formation of a CRISPR
complex and successful binding to a target nucleic acid while not allowing for
successful nuclease activity
(catalytic/cleavage activity).
In some embodiments, the dCas12i is a dSiCas12i substantial lacking catalytic
activity. In some embodiments, the
dSiCas12i comprises one or more substitutions at amino acid residues 650, 700,
875, and/or 1049 relative to SEQ
ID NO: 1. In some embodiments, the dSiCas12i comprises one or more
substitutions selected from the group
consisting of D700A, D700V, D650A, D650V, E875A, E875V, D1049A, and D1049V
relative to SEQ ID NO: 1.
In one embodiment, the dSiCas12i comprises the amino acid sequence of any of
dSiCas12i-D700A,
dSiCas12i-D650A, dSiCas12i-E857A, and dSiCas12i-D1049A, respectively. In some
embodiments, the
dSiCas12i comprises one or more substitutions selected from the group
consisting of D650A, D700A, E875A,
D1049A, D650A+D700A, D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A,
E875A+D1049A, D650A+D700A+E875A, D650A+D700A+D1049A,
D650A+E875A+D1049A,
D700A+E875A+D1049A, and D650A+D700A+E875A+D1049A, relative to SEQ ID NO: 1.
In addition, the dCas12i may contain mutations other than those previously
described that do not substantially
affect (e.g., affect no more than about any of 5%, 4%, 3%, 2%, 1%, or smaller)
the catalytic activity or nucleic
acid binding function of the dCas12i protein. The dCas12i protein, which
substantially lacks catalytic activity, can
be used as a DNA-binding protein.
In some embodiments, the dCas12i described herein can be fused with an
adenosine deaminase (ADA) or a
cytidine deaminase (CDA), or a catalytic domain thereof, to achieve single-
base editing. In some embodiments,
the single-base editing efficiency of a fusion protein comprising any of the
dCas12i proteins described herein and
an ADA or a CDA (or catalytic domain thereof) is at least about 10% higher
(e.g., at least about any of 20%, 30%,
40%, 50%, 60%, 70%, 80% 90%, 100%, 120%, 150%, 200%, 500%, 1000%, or higher)
than that of a fusion
protein comprising a dCas12i not from present invention and a sane ADA or CDA
(or catalytic domain thereof).
The number of amino acids in a full-length sequence of any of the Cas12i or
dCas12i proteins described above is
remarkably less than that of Cas12 proteins of other types, and their smaller
molecular size facilitates the
subsequent assembly and delivery of the Cas system in vivo.
In some embodiments, the adenosine deaminase is TadA8e, such as TadA8e
comprising the sequence of SEQ ID
NO: 439.
In some embodiments, the C' terminus of a deaminase, such as adenosine
deaminase, is fused to the N' terminus
of a dCas12i via an optional peptide linker, such as a peptide linker
comprising SEQ ID NO: 442. In some
embodiments, the N' terminus of a deaminase, such as adenosine deaminase, is
fused to the C' terminus of a
dCas12i via an optional peptide linker, such as a peptide linker comprising
SEQ ID NO: 442. In some
embodiments, there is provided a fusion protein comprising dSiCas12i and an
adenosine deaminase (e.g., TadA8e),
such as fusion protein TadA8e-dSiCas12i-D1049A, or fusion protein TadA8e-
dSiCas12i-E875A.
Unless otherwise specified, "Cas12i," or "Cas12i protein" described herein
include any Cas12i protein described
in the present invention and its variants (such as mutants), derivatives (such
as Cas12i fusion proteins), as well as
dCas12i proteins substantially lacking catalytic activity and derivatives
thereof (such as dCas12i fusion proteins,
such as dCas12i-TadA). The present invention also provides nucleotide
sequences encoding any of the Cas12i
proteins and variants and derivatives thereof, such as the polynucleotide
sequences of any of SEQ ID NOs: 21-40.
CRISPR (crRNA) or guide RNA (gRNA)
Typically, crRNAs (exchangeable with guide RNA / gRNA) described herein
comprise, consist essentially of, or
consist of a direct repeat (DR) and a spacer. In some embodiments, the crRNA
comprises, consists essentially of,
or consists of a DR linked to a spacer. In some embodiments, the crRNA
comprises a DR, a spacer, and a DR
(DR-spacer-DR). This is a typical configuration of a pre-crRNA. In some
embodiments, the crRNA comprises a
DR, a spacer, a DR, and a spacer (DR-spacer-DR-spacer). In some embodiments,
the crRNA comprises two or
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more DRs and two or more spacers. In some embodiments, the crRNA comprises a
truncated DR, and a spacer.
This is typical for processed or mature crRNAs. In some embodiments, the
CRISPR-Cas12i effector protein forms
a complex with the crRNA, and the spacer directs the complex to a target
nucleic acid that is complementary to
the spacer for sequence-specific binding.
In some embodiments, the CRISPR-Cas12i system described herein comprises one
or more crRNAs (e.g., 1, 2, 3,
4, 5, 10, 15, or more), or nucleic acids encoding thereof. In some
embodiments, the two or more crRNAs target
different target sites, e.g., 2 target sites of the same target DNA or gene,
or 2 target sites of 2 different target DNA
or genes.
The sequences and lengths of the crRNAs described herein can be optimized. In
some embodiments, the optimal
length of the crRNA can be determined by identifying the processed form of the
crRNA or by empirical length
studies of the crRNA. In some embodiments, the crRNA comprises base
modifications.
Direct Repeat (DR)
Table A exemplifies DR sequences of corresponding Cas12i protein of the
present invention. For example, the DR
sequence corresponding to SiCas12i (or a variant or derivative thereof, or
dSiCas12i or a fusion protein thereof)
may comprise the nucleotide sequence set forth in SEQ ID NO: 11 or a
functional variant thereof. Any DR
sequence that can mediate the binding of the Cas12i protein described herein
to the corresponding crRNA can be
used in the present invention. In some embodiments, the DR comprises the RNA
sequence of any one of SEQ ID
NOs: 11-20 and 501-507. In some embodiments, the DR is a "functional variant"
of any of the RNA sequences
of SEQ ID NOs: 11-20, such as a "functionally truncated version,"
"functionally extended version," or
"functionally replacement version." For example, DR sequence of SEQ ID NO: 501
or 502 is a part of SEQ ID
NO: 11 (truncated version), it still has DR function, as demonstrated in
Example, and is therefore a functional
variant, or a functionally truncated DR variant. A "functional variant" of a
DR is a 5' and/or 3' extended
(functionally extended version) or truncated (functionally truncated version)
variant of a reference DR (e.g., a
parental DR), or comprises one or more insertions, deletions, and/or
substitutions (functional replacement version)
of one or more nucleotides relative to the reference DR (e.g., a parental DR),
while still retaining at least about 20%
(such as at least about any of 30%, 40%, 50%, 60%, 60%, 70%, 80%, 90%, 95%, or
higher) functionality of the
reference DR, i.e., the function to mediate the binding of a Cas12i protein to
the corresponding crRNA. DR
functional variants typically retain stem-loop-like secondary structure or
portions thereof available for Cas12i
protein binding. As shown in FIG. 21, DR-T2 (SEQ ID NO: 502) is one of the
functionally truncated versions of
the DR shown in SEQ ID NO: 11. In some embodiments, the DR or functional
variant thereof comprises a
stem-loop-like secondary structure or portion thereof available for binding by
the Cas12i protein. In some
embodiments, the DR or functional variant thereof comprises at least two
(e.g., 2, 3, 4, 5 or more) stem-loop-like
secondary structures or portions thereof available for binding by the Cas12i
protein.
In some embodiments, the DR or functional variant thereof comprises at least
about 16 nucleotides (nt), such as
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 or more nucleotides.
In some embodiments, the DR comprises about 20nt to about 40nt, such as about
20nt to about 30nt, about 22nt to
about 40nt, about 23nt to about 38nt, about 23nt to about 36nt, or about 30nt
to about 40nt. In some embodiments,
the DR comprises 22nt, 23nt, or 24nt. In some embodiments, the DR comprises
35nt, 36nt, or 37nt.
In some embodiments, the DR sequence comprises a stem-loop structure near the
3' end (immediately adjacent to
the spacer sequence). "Stem-loop structure" refers to a nucleic acid having a
secondary structure that includes
regions of nucleotides known or predicted to form a double-strand (stem)
portion and connected at one end by a
linking region (loop) of substantially single-stranded nucleotides. The term
"hairpin" structure is also used herein
to refer to stem-loop structures. Such structures are well known in the art,
and these terms are used in accordance
with their commonly known meanings in the art. Stem-loop structures do not
require precise base pairing. Thus,
the stem may comprise one or more base mismatches. Alternatively, base pairing
may be exact, i.e., not including
any mismatches.
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The crRNA of the present invention comprises a DR comprising a stem-loop
structure near the 3' end of the DR
sequence. The DR stem-loop structure of SiCas12i is exemplified in FIG 11. In
some embodiments, the stem
contained in the DR consists of 5 pairs of complementary bases that hybridize
to each other, and the loop length is
6, 7, 8, or 9 nucleotides. In some embodiments, the loop length is 7
nucleotides. In some embodiments, the stem
can comprise at least 2, at least 3, at least 4, or at least 5 base pairs. In
some embodiments, the DR comprises two
complementary stretches of nucleotides about 5 nucleotides in length separated
by about 7 nucleotides. In some
embodiments, the stem-loop structure comprises a first stem nucleotide chain
of 5 nucleotides in length; a second
stem nucleotide chain of 5 nucleotides in length, wherein the first and the
second stem nucleotide chains can
hybridize to each other; and a cyclic nucleotide chain arranged between the
first and second stem nucleotide
chains, wherein the cyclic nucleotide chain comprises 6, 7 or 8 nucleotides.
As used herein, the secondary structure of two or more crRNAs are
substantially identical or not substantially
different means that these crRNAs contain stems and/or loops differing by no
more than 1, 2, or 3 nucleotides in
length; in terms of nucleotide type (A, U, G, or C), the nucleotide sequences
of these crRNAs when compared by
sequence alignment differ by no more than 1, 2, 3, 4, 5, 6, 7 or 8
nucleotides. In some embodiments, the secondary
structure of two or more crRNAs are substantially identical or not
substantially different means that the crRNAs
contain stems that differ by at most one pair of complementary bases, and/or
loops that differ by at most one
nucleotide in length, and/or contain stems with same length but with
mismatched bases. In some embodiments,
the stem-loop structure comprises 5'-X1X2X3X4X5NNNnNNNX6X7X8X9X10-3', wherein
Xi, X2, X3, X4, X5, X6,
X7, X8, X9, and Xio can be any base, n can be any base or deletion, and N can
be any base; wherein X iX2X3X4X5
and X6X7X8X9X10 can hybridize to each other to form a stem and make NNNnNNN
form a loop. In some
embodiments, the stem-loop structure comprises the sequence of any one of SEQ
ID NOs: 503-507.
In some embodiments, the DR sequence that can direct any of the Cas12i of the
invention to the target site
comprises one or more nucleotide changes selected from the group consisting of
nucleotide additions, insertions,
deletions, and substitutions that do not result in substantial differences in
secondary structure compared to DR
sequence set forth in any of SEQ ID NOs: 11-20 and 501-507 or functionally
truncated version thereof.
Spacer
In some embodiments, the length of the spacer sequence is at least about 16
nucleotides, preferably about 16 to
about 100 nucleotides, more preferably about 16 to about 50 nucleotides (e.g.,
about any of 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
nucleotides). In some embodiments, the spacer is about 16 to about 27
nucleotides, such as any of about 17 to
about 24 nucleotides, about 18 to about 24 nucleotides, or about 18 to about
22 nucleotides.
In some embodiments, the spacer is at least about 70% (e.g., at least about
any of 75%, 80%, 85%, 90%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) complementary to the
target sequence. In some
embodiments, there are at least about 15 (e.g., at least about any of 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35, 40, 45, 50 or more) between the spacer sequence and the target
sequence of the target nucleic acid
(e.g., DNA).
Complete complementarity is not required for spacers, provided that there is
sufficient complementarity for the
crRNA to function (i.e., directing Cas12i protein to the target site). The
cleavage efficiency by Cas12i mediated by
the crRNA can be adjusted by introducing one or more mismatches (e.g., 1 or 2
mismatches between the spacer
sequence and the target sequence, including the positions along the mismatches
of the spacer/target sequence).
Mismatches, such as double mismatches, have greater impact on cleavage
efficiency when they are located more
central to the spacer (i.e., not at the 3' or 5' end of the spacer). Thus, by
choosing the position of mismatches along
the spacer sequence, the cleavage efficiency of Cas12i can be tuned. For
example, if less than 100% cleavage of
the target sequence is desired (e.g., in a population of cells), 1 or 2
mismatches between the spacer sequence and
the target sequence can be introduced into the spacer sequence.
PAM
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In some embodiments, the Cas12i protein of the present invention can recognize
PAM (protospacer adjacent motif,
protospacer adjacent motif) to act on the target sequence. In some
embodiments, the PAM comprises or consists of
5'-NTTN-3' (wherein N is A, T, G, or C). In some embodiments, the PAM
comprises or consists of 5'-TTC-3',
5'-TTA-3', 5'-TTT-3', 5'-TTG-3', 5'-ATA-3', or 5'-ATG-3'. In some embodiments,
the PAM comprises or
consists of 5'-TTC-3'.
The invention provides the following embodiments:
1. A Cas12i protein comprising an amino acid sequence having at least about
80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or
100% identity to the
amino acid sequence as set forth in any one of SEQ ID NOs: 1-10 (preferably,
SEQ ID NOs: 1-3, 6, and 10, and
more preferably, SEQ ID NO: 1).
The Cas12i protein may also contain amino acid mutations that do not
substantially affect the catalytic activity
(endonuclease cleavage activity) or nucleic acid binding function of Cas12i.
2. The Cas12i protein according to any one of the preceding embodiments,
wherein the Cas12i protein
substantially lacks (e.g., retains less than 50%, 40%, 35%, 30%, 27.5%, 25%,
22.5%, 20%, 17.5%, 15%, 12.5%,
10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1% or less) spacer-specific endonuclease
cleavage activity of the
corresponding parental Cas12i protein (e.g., Cas12i protein comprising any of
SEQ ID NOs: 1-10) for a target
sequence of a target DNA complementary to a guide sequence.
In one embodiment, the Cas12i substantially lacks (e.g., retains less than
50%, 40%, 35%, 30%, 27.5%, 25%,
22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1%, or less)
spacer-specific endonuclease
cleavage activity or spacer non-specific collateral activity of the
corresponding parental Cas12i protein (e.g.,
Cas12i protein comprising any of SEQ ID NOs: 1-10).
3. The Cas12i protein according to any one of the preceding embodiments,
wherein the Cas12i protein comprises
one or more amino acid variations in its RuvC domain such that the Cas12i
protein substantially lacks (e.g.,
retains less than 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%,
12.5%, 10%, 7.5%, 5%, 4%, 3%,
2.5%, 2%, 1% or less) spacer-specific endonuclease cleavage activity of the
corresponding parental Cas12i protein
(e.g., Cas12i protein comprising any of SEQ ID NOs: 1-10) for a target
sequence of a target DNA complementary
to a guide sequence.
4. The Cas12i protein according to any one of the preceding embodiments,
wherein the amino acid variation is
selected from the group consisting of amino acid additions, insertions,
deletions, and substitutions.
5. The Cas12i protein according to any one of the preceding embodiments,
wherein the Cas12i protein comprises
an amino acid substitution at one or more positions corresponding to positions
700 (D700), 650 (D650), 875
(E875) or 1049 (D1049) of the sequence as set forth in SEQ ID NO: 1.
The amino acid at the above amino acid site (D700, D650, E875 or D1049) may be
mutated to another amino acid
different from the corresponding amino acid on the parental sequence (e.g.,
parental Cas12i protein comprising
any of SEQ ID NOs: 1-10) to substantially lose endonuclease cleavage activity.
The Cas12i protein may also contain other mutations that have no substantial
effect on the catalytic activity or
nucleic acid binding function of the Cas12i.
6. The Cas12i protein according to any one of the preceding embodiments,
wherein the amino acid substitution is
selected from the group consisting of D700A/V, D650AN E875A/V, and D1049AN.
7. The Cas12i protein according to any one of the preceding embodiments,
wherein the amino acid substitution is
selected from the group consisting of D700A, D650A, E875A, and D1049A.
8. The Cas12i protein according to any one of the preceding embodiments,
wherein the amino acid substitution is
selected from the group consisting of D700A, D650A, E875A, D1049A,
D700A+D650A, D700A+E875A,
D700A+D1049A, D650A+E875A, D650A+D1049A, E875A+D1049A, D700A+D650A+E875A,
D700A+D650A+D1049A, D650A+E875A+D1049A, and D700A+D650A+E875A+D1049A.
10. The Cas12i protein according to any one of the preceding embodiments,
wherein the Cas12i protein is linked
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to one or more functional domains.
11. The Cas12i protein according to any one of the preceding embodiments,
wherein the functional domain is
linked to the N-terminus and/or C-terminus of the Cas12i protein.
The linking may be a direct linking or an indirect linking through a linker.
12. The Cas12i protein according to any one of the preceding embodiments,
wherein the functional domain is
selected from the group consisting of a nuclear localization signal (NLS),
nuclear export signal (NES), deaminase
(e.g., adenosine deaminase or cytidine deaminase) catalytic domain, a DNA
methylation catalytic domain, a DNA
demethylation catalytic domain, a histone residue modification domain, a
nuclease catalytic domain, a fluorescent
protein, a transcription modification factor (e.g., a transcription activation
catalytic domain, a transcription
inhibition catalytic domain), a light gating factor, a chemical inducible
factor, a chromatin visualization factor, a
targeting polypeptide for providing binding to a cell surface portion on a
target cell or a target cell type.
13. The Cas12i protein according to any one of the preceding embodiments,
wherein the functional domain
exhibits activity to modify a target DNA, selected from the group consisting
of nuclease activity, methylation
activity, demethylation activity, DNA repair activity, DNA damage activity,
deamination activity, dismutase
activity, alkylation activity, depurination activity, oxidation activity,
pyrimidine dimer formation activity, integrase
activity, transposase activity, recombinase activity, polymerase activity,
ligase activity, helicase activity,
photolyase activity, glycosylase activity, acetyl transferase activity,
deacetylase activity, kinase activity,
phosphatase activity, ubiquitin ligase activity, deubiquitination activity,
adenylation activity, deadenylation activity,
SUMOylation activity, deSUMOylation activity, ribosylation activity,
deribosylation activity, myristoylation
activity, demyristoylation activity, glycosylation activity (e.g., from 0-
G1cNAc transferase), deglycosylation
activity, transcription inhibition activity, transcription activation
activity.
14. The Cas12i protein according to any one of the preceding embodiments,
wherein the functional domain is
selected from an adenosine deaminase catalytic domain or a cytidine deaminase
catalytic domain.
15. The Cas12i protein according to any one of the preceding embodiments,
wherein the functional domain is a
full length or functional fragment of TadA8e.
17. The Cas12i protein according to any one of the preceding embodiments,
wherein the Cas12i protein is
modified to reduce or eliminate spacer non-specific endonuclease collateral
activity.
18. A polynucleotide encoding the Cas12i protein according to any one of the
preceding embodiments.
19. The polynucleotide according to any one of the preceding embodiments,
wherein the polynucleotide is codon
optimized for expression in eukaryotic cells.
20. The polynucleotide according to any one of the preceding embodiments,
wherein the polynucleotide
comprises a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the
nucleotide sequence as set
forth in any one of SEQ ID NOs: 21-40.
21. A vector comprising the polynucleotide according to any one of the
preceding embodiments.
22. The vector according to any one of the preceding embodiments, wherein the
polynucleotide is operably linked
to a promoter.
23. The vector according to any one of the preceding embodiments, wherein the
promoter is a constitutive
promoter, an inducible promoter, a ubiquitous promoter, a cell type specific
promoter, or a tissue specific
promoter.
24. The vector according to any one of the preceding embodiments, wherein the
vector is a plasmid.
25. The vector according to any one of the preceding embodiments, wherein the
vector is a retroviral vector, a
phage vector, an adenovirus vector, a herpes simplex virus (HSV) vector, an
adeno-associated virus (AAV) vector,
or a lentiviral vector.
26. The vector according to any one of the preceding embodiments, wherein the
AAV vector is selected from the
group consisting of recombinant AAV vectors of serotypes AAV1, AAV2, AAV4,
AAV5, AAV6, AAV7, AAVrh74,
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AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.
27. A delivery system comprising (1) a delivery medium; and (2) the Cas12i
protein, polynucleotide or vector
according to any one of the preceding embodiments.
28. The delivery system according to any one of the preceding embodiments,
wherein the delivery medium is
nanoparticle, liposome, exosome, microvesicle, or gene gun.
29. An engineered, non-naturally occurring CRISPR-Cas system comprising:
the Cas12i protein or a polynucleotide encoding the Cas12i protein according
to any one of the preceding
embodiments; and
a CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, the crRNA
comprising:
a spacer capable of hybridizing to a target sequence of a target DNA, and
a Direct Repeat (DR) linked to the spacer and capable of guiding the Cas12i
protein to bind to the crRNA to form
a CRISPR-Cas complex targeting the target sequence.
The Cas12i protein is capable of binding to the crRNA and targeting the target
sequence, wherein the target
sequence is a single-stranded or double-stranded DNA or RNA.
30. A CRISPR-Cas system comprising one or more vectors, wherein the one or
more vectors comprise:
a first regulatory element operably linked to a nucleotide sequence encoding
the Cas12i protein according to any
one of the preceding embodiments; and
a second regulatory element operably linked to a polynucleotide encoding a
CRISPR RNA (crRNA), the crRNA
comprising:
a spacer capable of hybridizing to a target sequence of a target DNA, and
a Direct Repeat (DR) linked to the spacer that is capable of guiding the
Cas12i protein to bind to the crRNA to
form a CRISPR-Cas complex targeting the target sequence;
wherein the first regulatory element and the second regulatory element are
located on the same or different vectors
of the CRISPR-Cas vector system.
31. An engineered, non-naturally occurring CRISPR-Cas complex comprising:
the Cas12i protein according to any one of the above embodiments; and
a CRISPR RNA (crRNA), the crRNA comprising:
a spacer capable of hybridizing to a target sequence of a target DNA, and
a Direct Repeat (DR) linked to the spacer; the DR guides the Cas12i protein to
bind to the crRNA.
32. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the spacer
is greater than 16 nucleotides in length, preferably 16 to 100 nucleotides,
more preferably 16 to 50 nucleotides
(e.g., 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 nucleotides), more preferably 16 to 27 nucleotides,
more preferably 17 to 24 nucleotides,
more preferably 18 to 24 nucleotides, and most preferably 18 to 22
nucleotides.
33. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the DR has
a secondary structure substantially identical to the secondary structure of
the DR as set forth in any one of SEQ ID
NOs: 11-20.
34. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the DR has
nucleotide additions, insertions, deletions or substitutions without causing
substantial differences in the secondary
structure as compared to the DR as set forth in any one of SEQ ID NOs: 11-20.
35. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the DR
comprises a stem-loop structure near the 3' end of the DR,
wherein the stem-loop structure comprises 5'-X1X2X3X4X5NNNnNNNX6X7X8X9X10-3'
(X1, X2, X3, X4, X5, X6,
X7, X8, X9, X10 are any base, n is any nucleobase or deletion, N is any
nucleobase); wherein X1X2X3X4X5 and
X6X7X8X9X10 can hybridize to each other.
36. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the DR
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comprises a stem-loop structure selected from any one of the following:
5'-CUCCCNNNNNNUGGGAG-3' (SEQ ID NO:) near the 3' end of the DR, wherein N is
any nucleobase;
5'-CUCCUNNNNNNUGGGAG-3' (SEQ ID NO:) near the 3' end of the DR, wherein N is
any nucleobase;
5'-GUCCCNNNNNNUGGGAC-3' (SEQ ID NO:) near the 3' end of the DR, wherein N is
any nucleobase;
5'-GUGUCNNNNNNUGACAC-3' (SEQ ID NO:) near the 3' end of the DR, wherein N is
any nucleobase;
5'-GUGCCNNNNNNUGGCAC-3' (SEQ ID NO:) near the 3' end of the DR, wherein N is
any nucleobase;
5'-UGUGUNNNNNNUCACAC-3' (SEQ ID NO:) near the 3' end of the DR, wherein N is
any nucleobase;
5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO:) near the 3' end of the DR, where N is any
nucleobase;
5'-GUUUCNNNNNNUGAAAC-3' (SEQ ID NO:) near the 3' end of the DR, where N is any
nucleobase;
5'-GUGUUNNNNNNUAACAC-3' (SEQ ID NO:) near the 3' end of the DR, where N is any
nucleobase; and
5'-UUGUCNNNNNNUGACAA-3' (SEQ ID NO:) near the 3' end of the DR, where N is any
nucleobase.
37. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, further comprising
a target DNA capable of hybridizing to the spacer.
38. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the target
DNA is a eukaryotic DNA.
39. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the target
DNA is in cells; preferably the cells are selected from the group consisting
of prokaryotic cells, eukaryotic cells,
animal cells, plant cells, fungal cells, vertebrate cells, invertebrate cells,
rodent cells, mammalian cells, primate
cells, non-human primate cells, and human cells.
40. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the crRNA
hybridizes to and forms a complex with the target sequence of the target DNA,
causing the Cas12i protein to
cleave the target sequence.
41. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the target
sequence is at the 3' end of a protospacer adjacent motif (PAM).
42. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the PAM
comprises a 5'-T-rich motif.
43. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the PAM is
5'-TTA, 5'-TTT, 5'-TTG, 5'-TTC, 5'-ATA or 5'-ATG.
44. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the one or
more vectors comprise one or more retroviral vectors, phage vectors,
adenoviral vectors, herpes simplex virus
(HSV) vectors, adeno-associated virus (AAV) vectors, or lentiviral vectors.
45. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the AAV
vector is selected from the group consisting of recombinant AAV vectors of
serotypes AAV1, AAV2, AAV4,
AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.
46. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the
regulatory element comprises a promoter.
47. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the
promoter is selected from the group consisting of a constitutive promoter, an
inducible promoter, a ubiquitous
promoter, a cell type specific promoter, or a tissue specific promoter.
48. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the
promoter is functional in eukaryotic cells.
49. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein the
eukaryotic cells include animal cells, plant cells, fungal cells, vertebrate
cells, invertebrate cells, rodent cells,
mammalian cells, primate cells, non-human primate cells, and human cells.
50. The CRISPR-Cas system or complex according to any one of the preceding
embodiments, further comprising
a DNA donor template optionally inserted at a locus of interest by homology-
directed repair (HDR).
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51. A cell or descendant thereof comprising the Cas12i protein,
polynucleotide, vector, delivery system,
CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein preferably, the cell
is selected from the group consisting of prokaryotic cells, eukaryotic cells,
animal cells, plant cells, fungal cells,
vertebrate cells, invertebrate cells, rodent cells, mammalian cells, primate
cells, non-human primate cells, and
human cells.
52. A non-human multicellular organism, comprising the cell or descendant
thereof according to any one of the
preceding embodiments; preferably, the non-human multicellular organism is an
animal (e.g., rodent or
non-human primate) model for human gene related diseases.
53. A method of modifying a target DNA, comprising contacting a target DNA
with the CRISPR-Cas system or
complex according to any one of the preceding embodiments, the contacting
resulting in modification of the target
DNA by the Cas12i protein.
54. The method according to any one of the preceding embodiments, wherein the
modification occurs outside
cells in vitro.
55. The method according to any one of the preceding embodiments, wherein the
modification occurs inside cells
in vitro.
56. The method according to any one of the preceding embodiments, wherein the
modification occurs inside cells
in vivo.
57. The method according to any one of the preceding embodiments, wherein the
cell is a eukaryotic cell.
58. The method according to any one of the preceding embodiments, wherein the
eukaryotic cell is selected from
the group consisting of animal cells, plant cells, fungal cells, vertebrate
cells, invertebrate cells, rodent cells,
mammalian cells, primate cells, non-human primate cells, and human cells.
59. The method according to any one of the preceding embodiments, wherein the
modification is cleavage of the
target DNA.
Optionally, the cleavage is performed in a manner of cleaving a single-
stranded DNA, or optionally, in a manner
of sequentially cleaving the same site or different sites of a double-stranded
DNA.
60. The method according to any one of the preceding embodiments, wherein the
cleavage results in deletion of a
nucleotide sequence and/or insertion of a nucleotide sequence.
61. The method according to any one of the preceding embodiments, wherein the
cleavage comprises cleaving the
target nucleic acid at two sites resulting in deletion or inversion of a
sequence between the two sites.
62. The method according to any one of the preceding embodiments, wherein the
modification is a base variation,
preferably A¨>G or C¨>T base variation.
63. A cell or descendant thereof from the method according to any one of the
preceding embodiments, comprising
the modification absent in a cell not subjected to the method.
64. The cell or descendant thereof according to any one of the preceding
embodiments, wherein a cell not
subjected to the method comprises abnormalities and the abnormalities in the
cell from the method have been
resolved or corrected.
65. A cell product from the cell or descendant thereof according to any one of
the preceding embodiments,
wherein the product is modified relative to the nature or quantity of a cell
product from a cell not subjected to the
method.
66. The cell product according to any one of the preceding embodiments,
wherein cells not subjected to the
method comprise abnormalities and the cell product reflects that the
abnormalities have been resolved or corrected
by the method.
67. A method of non-specifically cleaving a non-target DNA, comprising
contacting the target DNA with the
CRISPR-Cas system or complex according to any one of the preceding
embodiments, whereby hybridization of
the spacer to the target sequence of the target DNA and cleavage of the target
sequence by the Cas12i protein
make the Cas12i protein cleave the non-target DNA by spacer non-specific
endonuclease collateral activity.
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68. A method of detecting a target DNA in a sample, comprising:
contacting the sample with the CRISPR-Cas system or complex according to any
one of the preceding
embodiments and a reporter nucleic acid capable of releasing a detectable
signal after being cleaved, whereby
hybridization of the spacer to the target sequence of the target DNA and
cleavage of the target sequence by the
Cas12i protein make the Cas12i protein cleave the reporter nucleic acid by
spacer non-specific endonuclease
collateral activity; and
measuring a detectable signal generated by cleavage of the reporter nucleic
acid, thereby detecting the presence of
the target DNA in the sample.
69. The method according to any one of the preceding embodiments, further
comprising comparing the level of
the detectable signal to the level of a reference signal and determining the
level of the target DNA in the sample
based on the level of the detectable signal.
70. The method according to any one of the preceding embodiments, wherein the
measurement is performed using
gold nanoparticle detection, fluorescence polarization, colloidal phase
change/dispersion, electrochemical
detection, or semiconductor-based sensing.
71. The method according to any one of the preceding embodiments, wherein the
reporter nucleic acid comprises
a fluorescence emission dye pair, a fluorescence resonance energy transfer
(FRET) pair, or a quencher/fluorophore
pair, and cleavage of the reporter nucleic acid by the Cas12i protein results
in an increase or decrease in the level
of the detectable signal produced by cleavage of the reporter nucleic acid.
72. A method of treating a condition or disease in a subject in need thereof,
comprising administering to the
subject the CRISPR-Cas system according to any one of the preceding
embodiments.
73. The method according to any one of the preceding embodiments, wherein the
condition or disease is a cancer
or infectious disease or neurological disease,
optionally, the cancer is selected from the group consisting of:
Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma,
neuroblastoma, melanoma, skin cancer,
breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer,
kidney cancer, pancreatic cancer, lung
cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal
cancer, gastric cancer, head and neck cancer,
thyroid myeloid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma,
acute lymphocytic leukemia,
acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic
leukemia, Hodgkin's lymphoma,
non-Hodgkin's lymphoma and urinary bladder cancer;
optionally, the infectious disease is caused by:
human immunodeficiency virus (HIV), herpes simplex virus-1 (HSV1) and herpes
simplex virus-2 (HSV2);
optionally, the neurological disorder is selected from the group consisting
of:
glaucoma, age-related loss of RGC, optic nerve injury, retinal ischemia,
Leber's hereditary optic neuropathy,
neurological diseases associated with RGC neuronal degeneration, neurological
diseases associated with
functional neuronal degeneration in the striatum of subjects in need,
Parkinson's disease, Alzheimer's disease,
Huntington's disease, schizophrenia, depression, drug addiction, dyskinesia
such as chorea, choreoathetosis and
dyskinesia, bipolar affective disorder, autism spectrum disorder (ASD) or
dysfunction.
74. The method according to any one of the preceding embodiments, wherein the
condition or disease is selected
from the group consisting of cystic fibrosis, progressive pseudohypertrophic
muscular dystrophy, Becker muscular
dystrophy, alpha-1 -antitrypsin deficiency, Pompe disease, myotonic dystrophy,
Huntington's disease, fragile X
syndrome, Friedreich ataxia, amyotrophic lateral sclerosis, frontotemporal
dementia, hereditary chronic kidney
disease, hyperlipidemia, hypercholesterolemia, Leber congenital amaurosis,
sickle cell disease, and beta
thalassemia.
75. The method according to any one of the preceding embodiments, wherein the
condition or disease is caused by
the presence of a pathogenic point mutation.
76. A kit comprising the CRISPR-Cas system according to any one of the
preceding embodiments; preferably the
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components of the system are in the same container or in separate containers.
77. A sterile container comprising the CRISPR-Cas system according to any one
of the preceding embodiments;
preferably the sterile container is a syringe.
78. An implantable device comprising the CRISPR-Cas system according to any
one of the preceding
embodiments; preferably the CRISPR-Cas system is stored in a reservoir.
Collateral activity
The Cas12i protein may have collateral activity, that is, under certain
conditions, the activated Cas12i protein
remains active after binding to the target sequence and continues to non-
specifically cleave non-target
oligonucleotides. This collateral activity enables detection of the presence
of specific target oligonucleotides using
the Cas12i system. In one embodiment, the Cas12i system is engineered to non-
specifically cleave ssDNA or
transcript. In certain embodiments, Cas12i is transiently or stably provided
or expressed in an in vitro system or
cell and is targeted or triggered to non-specifically cleave cellular nucleic
acids, such as ssDNA, such as viral
ssDNA. In some embodiments, the Cas12i protein described herein is modified to
reduce (e.g., reduce at least
about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher) or
eliminate spacer non-specific
endonuclease cleavage activity. In some embodiments, the Cas12i protein
described herein substantially lacks
(e.g., lacks at lease about any of 50%, 60%, 70%, 80%, 90%, 95%, or 100%)
spacer non-specific endonuclease
collateral activity of the parental/reference Cas12i protein (e.g., Cas12i
protein of any of SEQ ID NOs: 1-10)
against a non-target DNA.
The collateral activity has recently been used in a highly sensitive and
specific nucleic acid detection platform
known as SHERLOCK which can be used in many clinical diagnostics (Gootenberg,
J.S. et al., Nucleic acid
detection with CRISPR-Cas13a/C2c2. Science 356, 438-442 (2017)).
Reporter nucleic acid
A "reporter nucleic acid" refers to a molecule that can be cleaved or
otherwise deactivated by the activated
CRISPR system protein as described herein. The reporter nucleic acid comprises
a nucleic acid element cleavable
by the CRISPR protein. Cleavage of the nucleic acid element releases an agent
or produces a conformational
change allowing for the generation of a detectable signal. The reporter
nucleic acid prevents the generation or
detection of a positive detectable signal prior to cleavage or when the
reporter nucleic acid is in an "active" state.
It will be appreciated that in certain exemplary embodiments, minimal
background signals may be generated in
the presence of the active reporter nucleic acid. The positive detectable
signal may be any signal that may be
detected using optical, fluorescent, chemiluminescent, electrochemical or
other detection methods known in the
art. For example, in certain embodiments, a first signal (i.e., a negative
detectable signal) may be detected when a
reporter nucleic acid is present, and then it is converted to a second signal
(e.g., a positive detectable signal) when
the target molecule is detected and the reporter nucleic acid is cleaved or
deactivated by the activated CRISPR
protein.
Functional domains
Functional domains are used in their broadest sense and include proteins such
as enzymes or factors themselves or
specific functional fragments (domains) thereof.
A Cas12i protein (e.g., dCas12i) is associated with one or more functional
domains selected from the group
consisting of a deaminase (e.g., adenosine deaminase or cytidine deaminase)
catalytic domain, a DNA methylation
catalytic domain, a DNA demethylation catalytic domain, a histone residue
modification domain, a nuclease
catalytic domain, a fluorescent protein, a transcription modification factor
(e.g., a transcription activation catalytic
domain, a transcription inhibition catalytic domain), a nuclear localization
signal (NLS), nuclear export signal
(NES), a light gating factor, a chemical inducible factor, or a chromatin
visualization factor; preferably, the
functional domain is selected from the group consisting of an adenosine
deaminase catalytic domain or cytidine
deaminase catalytic domain.
In some embodiments, the functional domain may be a transcription activation
domain. In some embodiments, the
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functional domain is a transcription repression domain. In some embodiments,
the functional domain is an
epigenetic modification domain such that an epigenetic modification enzyme is
provided. In some embodiments,
the functional domain is an activation domain. In some embodiments, the Cas12i
protein is associated with one or
more functional domains; and the Cas12i protein contains one or more mutations
within the RuvC domain, and
the resulting CRISPR complex can deliver epigenetic modifiers, or transcript
or translate activation or repression
signals.
In some embodiments, the functional domain exhibits activity to modify a
target DNA or proteins associated with
the target DNA, wherein the activity is one or more selected from the group
consisting of nuclease activity (e.g.,
HNH nuclease, RuvC nuclease, Trex 1 nuclease, Trex2 nuclease), methylation
activity, demethylation activity,
DNA repair activity, DNA damage activity, deamination activity, dismutase
activity, alkylation activity,
depurination activity, oxidation activity, pyrimidine dimer formation
activity, integrase activity, transposase
activity, recombinase activity, polymerase activity, ligase activity, helicase
activity, photolyase activity,
glycosylase activity, acetyl transferase activity, deacetylase activity,
kinase activity, phosphatase activity, ubiquitin
ligase activity, deubiquitination activity, adenylation activity,
deadenylation activity, SUMOylation activity,
deSUMOylation activity, ribosylation activity, deribosylation activity,
myristoylation activity, demyristoylation
activity, glycosylation activity (e.g., from 0-G1cNAc transferase),
deglycosylation activity, transcription inhibition
activity, and transcription activation activity. Target DNA associated
proteins include, but not limited to, proteins
that can bind to target DNA, or proteins that can bind to proteins bound to
target DNA, such as histones,
transcription factors, Mediator, etc.
The functional domain may be, for example, one or more domains from the group
consisting of methylase activity,
demethylase activity, transcription activation activity, transcription
repression activity, transcription release factor
activity, histone modification activity, RNA cleavage activity, DNA cleavage
activity, nucleic acid binding activity,
and molecular switches (e.g., photo-inducible). When more than one functional
domain is included, the functional
domains may be the same or different.
Base editing
In certain exemplary embodiments, Cas12i (e.g., dCas12i) may be fused to
adenosine deaminase or cytidine
deaminase for base editing purposes.
Adenosine deaminase
As used herein, the term "adenosine deaminase" or "adenosine deaminase
protein" refers to a protein, polypeptide,
or one or more functional domains of a protein or polypeptide that can
catalyze hydrolytic deamination reaction to
convert adenine (or the adenine portion of a molecule) to hypoxanthine (or the
hypoxanthine portion of a
molecule), as shown below. In some embodiments, the adenine-containing
molecule is adenosine (A) and the
hypoxanthine-containing molecule is inosine (I). The adenine-containing
molecule may be deoxyribonucleic acid
(DNA) or ribonucleic acid (RNA).
According to the present disclosure, adenosine deaminases that can be used in
combination with the present
disclosure include, but are not limited to, enzyme family members referred to
as adenosine deaminase acting on
RNA (ADAR), enzyme family members referred to as adenosine deaminase acting on
tRNA (ADAT), and other
family members comprising adenosine deaminase domain (ADAD). According to the
present disclosure, the
adenosine deaminase is capable of targeting adenine in RNA/DNA and RNA
duplexes. In fact, Zheng et al.
(Nucleic Acids Res. 2017, 45 (6): 3369-3377) demonstrated that ADAR can edit
adenosine to inosine in
RNA/DNA and RNA/RNA duplexes. In specific embodiments, adenosine deaminase has
been modified to
increase its ability to edit DNA in the RNA/DNA heteroduplex of the RNA
duplex, as described in detail below.
In some embodiments, the adenosine deaminase is derived from one or more
metazoan species, including but not
limited to mammals, birds, frogs, squid, fish, flies, and worms. In some
embodiments, the adenosine deaminase is
human, squid, or drosophila adenosine deaminase.
In some embodiments, the adenosine deaminase is human ADAR, including hADAR1,
hADAR2, and hADAR3.
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In some embodiments, the adenosine deaminase is Caenorhabditis elegans ADAR
protein, including ADR-1 and
ADR-2. In some embodiments, the adenosine deaminase is drosophila ADAR
protein, including dAdar. In some
embodiments, the adenosine deaminase is squid (Loligo pealeii) ADAR protein,
including sqADAR2a and
sqADAR2b. In some embodiments, adenosine deaminase is human ADAT protein. In
some embodiments, the
adenosine deaminase is drosophila ADAT protein. In some embodiments, the
adenosine deaminase is human
ADAD protein, including TENR (hADAD1) and TENRL (hADAD2).
In some embodiments, the adenosine deaminase is TadA protein, such as E. coli
TadA. See Kim et al.,
Biochemistry 45: 6407-6416 (2006); Wolf et al., EMBO J. 21: 3841-3851 (2002).
In some embodiments, the
adenosine deaminase is mouse ADA. See Grunebaum et al., Curr. Opin. Allergy
Clin. Immunol. 13: 630-638
(2013). In some embodiments, the adenosine deaminase is human ADAT2. See Fukui
et al., J. Nucleic Acids 2010:
260512 (2010). In some embodiments, the deaminase (e.g., adenosine or cytidine
deaminase) is one or more of
those described in: Cox et al., Science. Nov. 24, 2017; 358(6366): 1019-1027;
Komore et al., Nature. May 19,
2016; 533 (7603): 420-4; and Gaudelli et al., Nature. Nov. 23, 2017; 551
(7681): 464-471.
In some embodiments, the adenosine deaminase protein recognizes one or more
target adenosine residues in a
double-stranded nucleic acid substrate and converts them to inosine residues.
In some embodiments, the
double-stranded nucleic acid substrate is an RNA-DNA heteroduplex. In some
embodiments, the adenosine
deaminase protein recognizes a binding window on a double-stranded substrate.
In some embodiments, the
binding window comprises at least one target adenosine residue. In some
embodiments, the binding window is in
the range of about 3 bp to about 100 bp. In some embodiments, the binding
window is in the range of about 5 bp
to about 50 bp. In some embodiments, the binding window is in the range of
about 10 bp to about 30 bp. In some
embodiments, the binding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp,
15 bp, 20 bp, 25 bp, 30 bp, 40 bp,
45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp or
100 bp.
In some embodiments, the adenosine deaminase protein comprises one or more
deaminase domains. Without
wishing to be bound by a particular theory, it is contemplated that the
deaminase domain is used to recognize one
or more target adenosine (A) residues contained in a double-stranded nucleic
acid substrate and convert them to
inosine (I) residues. In some embodiments, the deaminase domain comprises an
active center. In some
embodiments, the active center comprises zinc ions. In some embodiments,
during A-I editing, the base pair at the
target adenosine residue is destroyed and the target adenosine residue is
"flipped" out of the double helix to
become accessible by the adenosine deaminase. In some embodiments, amino acid
residues in or near the active
center interact with one or more nucleotides 5' of the target adenosine
residue. In some embodiments, amino acid
residues in or near the active center interact with one or more nucleotides 3'
of the target adenosine residue. In
some embodiments, amino acid residues in or near the active center further
interact with nucleotides
complementary to the target adenosine residues on the opposite chain. In some
embodiments, the amino acid
residue forms a hydrogen bond with the 2' hydroxyl group of the nucleotide.
In some embodiments, the adenosine deaminase comprises human ADAR2 whole
protein (hADAR2) or
deaminase domain (hADAR2-D) thereof. In some embodiments, the adenosine
deaminase is a member of the
ADAR family homologous to hADAR2 or hADAR2-D.
In particular, in some embodiments, the homologous ADAR protein is human ADAR1
(hADAR1) or deaminase
domain (hADAR1-D) thereof. In some embodiments, glycine 1007 of hADAR1-D
corresponds to glycine
487hADAR2-D, and glutamic acid 1008 of hADAR1-D corresponds to glutamic acid
488 of hADAR2-D.
In some embodiments, the adenosine deaminase comprises the wild-type amino
acid sequence of hADAR2-D. In
some embodiments, the adenosine deaminase comprises one or more mutations in
the hADAR2-D sequence such
that the editing efficiency and/or substrate editing preference of hADAR2-D
are changed as desired.
In some embodiments, the adenosine deaminase is TadA8e, such as TadA8e
comprising the sequence of SEQ ID
NO: 182. In some embodiments, the Cas12i protein described herein (e.g.,
dCas12i) is fused to TadA8e or
functional fragment thereof (i.e., capable of A-to-I single base editing).
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Cytidine deaminase
In some embodiments, the deaminase is cytidine deaminase. As used herein, the
term "cytidine deaminase" or
"cytidine deaminase protein" refers to a protein, polypeptide, or one or more
functional domains of a protein or
polypeptide that can catalyze hydrolytic deamination reaction to convert
cytosine (or the cytosine portion of a
molecule) to uracil (or the uracil portion of a molecule), as shown below. In
some embodiments, the
cytosine-containing molecule is cytidine (C) and the uracil-containing
molecule is uridine (U). The
cytosine-containing molecule may be deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA).
According to the present disclosure, cytidine deaminases that can be used in
combination with the present
disclosure include, but are not limited to, members of an enzyme family known
as apolipoprotein B mRNA
editing complex (APOBEC) family deaminases, activation-induced deaminase
(AID), or cytidine deaminase 1
(CDA1), and in specific embodiments, the deaminase in APOBEC1 deaminases,
APOBEC2 deaminases,
APOBEC3A deaminases, APOBEC3B deaminases, APOBEC3C deaminases and APOBEC3D
deaminases,
APOBEC3E deaminases, APOBEC3F deaminases, APOBEC3G deaminases, APOBEC3H
deaminases or
APOBEC4 deaminases.
In the methods and systems of the invention, the cytidine deaminase is capable
of targeting cytosines in a DNA
single strand. In certain exemplary embodiments, the cytidine deaminase can
edit on a single strand present
outside of the binding component, e.g., bind to Cas13. In other exemplary
embodiments, the cytidine deaminase
may edit at localized bubbles, such as those formed at target editing sites
but with guide sequence mismatching. In
certain exemplary embodiments, the cytidine deaminase may comprise mutations
that contribute to focus activity,
such as those described in Kim et al., Nature Biotechnology (2017) 35 (4): 371-
377 (doi: 10.1038/nbt.3803).
In some embodiments, the cytidine deaminase is derived from one or more
metazoan species, including but not
limited to mammals, birds, frogs, squid, fish, flies, and worms. In some
embodiments, the cytidine deaminase is
human, primate, bovine, canine, rat, or mouse cytidine deaminase.
In some embodiments, the cytidine deaminase is human APOBEC, including
hAPOBEC1 or hAPOBEC3. In
some embodiments, the cytidine deaminase is human AID.
In some embodiments, the cytidine deaminase protein recognizes one or more
target cytosine residues in a
single-stranded bubble of a RNA duplex and converts them to uracil residues.
In some embodiments, the cytidine
deaminase protein recognizes a binding window on a single-stranded bubble of
an RNA duplex. In some
embodiments, the binding window comprises at least one target cytosine
residue. In some embodiments, the
binding window is in the range of about 3 bp to about 100 bp. In some
embodiments, the binding window is in the
range of about 5 bp to about 50 bp. In some embodiments, the binding window is
in the range of about 10 bp to
about 30 bp. In some embodiments, the binding window is about 1 bp, 2 bp, 3
bp, 5 bp, 7 bp, 10 bp, 15 bp, 20 bp,
25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp,
85 bp, 90 bp, 95 bp or 100 bp.
In some embodiments, the cytidine deaminase protein comprises one or more
deaminase domains. Without
wishing to be bound by theory, it is contemplated that deaminase domains are
used to recognize one or more
target cytosine (C) residues contained in a single-stranded bubble of a RNA
duplex and convert them to uracil (U)
residues. In some embodiments, the deaminase domain comprises an active
center. In some embodiments, the
active center comprises zinc ions. In some embodiments, amino acid residues in
or near the active center interact
with one or more nucleotides at 5' of the target cytosine residue. In some
embodiments, amino acid residues in or
near the active center interact with one or more nucleotides at 3' of the
target cytosine residue.
In some embodiments, the cytidine deaminase comprises human APOBEC1 whole
protein (hAPOBEC1) or its
deaminase domain (hAPOBEC1-D) or its C-terminal truncated form (hAPOBEC-T). In
some embodiments, the
cytidine deaminase is a member of the APOBEC family homologous to hAPOBEC1,
hAPOBEC-D, or
hAPOBEC-T. In some embodiments, the cytidine deaminase comprises human AID1
whole protein (hAID) or its
deaminase domain (hAID-D) or its C-terminal truncated form (hAID-T). In some
embodiments, the cytidine
deaminase is a member of the AID family homologous to hAID, hAID-D, or hAID-T.
In some embodiments,
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hAID-T is hAID with the C-terminus truncated by about 20 amino acids.
In some embodiments, the cytidine deaminase comprises the wild-type amino acid
sequence of cytosine
deaminase. In some embodiments, the cytidine deaminase comprises one or more
mutations in the cytosine
deaminase sequence such that the editing efficiency and/or substrate editing
preference of the cytosine deaminase
are changed as desired.
As used herein, "associated is used in its broadest sense and encompasses both
the case where two functional
modules form a fusion protein directly or indirectly (via a linker) and the
case where two functional modules are
each independently bonded together by covalent bonds (e.g., disulfide bond) or
non-covalent bonds.
The term "vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid attached thereto.
It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA
segment can be inserted to effect
replication of the inserted segment. Typically, the vector is capable of
replication when combined with suitable
control elements.
In some cases, the vector system comprises a single vector. Alternatively, the
vector system comprises a plurality
of vectors. The vector may be a viral vector.
The vector includes, but are not limited to, a single-stranded, double-
stranded or partially double-stranded nucleic
acid molecule; a nucleic acid molecule comprising one or more free ends, or
without a free end (e. g., circular); a
nucleic acid molecule comprising DNA, RNA or both; and other polynucleotide
variants known in the art. One
type of vector is "plasmid", which refers to a circular double-stranded DNA
ring into which other DNA segments
can be inserted, for example by standard molecular cloning techniques. Another
type of vector is viral vector in
which a viral-derived DNA or RNA sequence is present for packaging into a
virus (e.g., retrovirus,
replication-defective retrovirus, adenovirus, replication-defective
adenovirus, and adeno-associated virus). The
viral vector also comprises a polynucleotide carried by the virus for
transfection into a host cell. Certain vectors
are capable of autonomous replication in the host cells into which they are
introduced (e.g., bacterial vectors
having origins of bacterial replication and episomal mammalian vectors). After
these vectors are introduced into
the host cells, other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genomes of the host
cells for replication with the host genomes. In addition, certain vectors are
capable of guiding expression of genes
operably linked thereto. Such vectors are referred to herein as "expression
vectors". Vectors expressed in
eukaryotic cells and vectors resulting in expression in eukaryotic cells may
be referred to herein as "eukaryotic
expression vectors". Common expression vectors useful in recombinant DNA
techniques are usually in the forms
of plasmids.
The recombinant expression vector may comprise the nucleic acid of the
invention in a form suitable for
expression in a host cell, which means that the recombinant expression vector
comprises one or more regulatory
elements that can be selected according to the host cell to be used for
expression, and the nucleic acid is operably
linked to a nucleic acid sequence to be expressed. Within recombinant
expression vectors, "operably linked" is
intended to mean that the nucleotide sequence of interest is linked to a
regulatory element in a manner that allows
expression of the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a host cell when
the vector is introduced into the host cell). Advantageous vectors include
lentiviruses and adeno-associated viruses,
and the type of these vectors may also be selected to target specific types of
cells.
The term "regulatory element" is intended to include promoters, enhancers,
internal ribosome entry sites (IRES),
and other expression control elements (e.g., transcription termination signals
such as polyadenylation signals and
poly-U sequences). Such regulatory elements are described, for example, in
Goeddel, GENE EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990)
(1990).
Regulatory elements include those that guide constitutive expression of
nucleotide sequences in many types of
host cells and those that guide expression of nucleotide sequences only in
certain host cells (e.g., tissue-specific
regulatory sequences). Tissue-specific promoters may guide expression
primarily in desired target tissues such as
muscle, neuron, bone, skin, blood, particular organs (e. g., liver, pancreas)
or particular cell types (e.g.,
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lymphocytes). Regulatory elements may also guide expression in a time-
dependent manner, e.g., in a cell cycle
dependent or developmental stage dependent manner, which may or may not be
tissue or cell type specific.
In some embodiments, the vector encodes a Cas12i protein comprising one or
more nuclear localization sequences
(NLSs), e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more NLSs. More specifically, the vector
comprises one or more NLSs that are not naturally occurring in the Cas12i
protein. Most particularly, the NLS is
present in 5' and/or 3' of the vector for the Cas12i protein sequence. In some
embodiments, the protein targeting
RNA comprises about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more NLSs at or near the amino terminus
and about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at
or near the carboxyl terminus, or a
combination of these (e.g., 0 or at least one or more NLSs at the amino
terminus and 0 or one or more NLSs at the
carboxyl terminus). When more than one NLSs are present, each of them may be
selected independently of the
others such that a single NLS may be present in more than one copies and/or in
combination with one or more
other NLSs in one or more copies. In some embodiments, NLS is considered to be
near the N-terminus or
C-terminus when its nearest amino acid is within about 1, 2, 3, 4, 5, 10, 15,
20, 25, 30, 40, 50, or more amino
acids along the polypeptide chain from the N-terminus or C-terminus.
"Codon optimization" refers to a method of modifying a nucleic acid sequence
in a target host cell to enhance
expression by replacing at least one codon (e.g., about or greater than about
1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or
more codons) of a natural sequence with a codon that is more frequently or
most frequently used in the gene of the
host cell while maintaining the natural amino acid sequence. A variety of
species show particular bias towards
certain codons for particular amino acids. Codon bias (the difference in codon
usage among organisms) is
generally related to the translation efficiency of messenger RNA (mRNA), which
in turn is thought to depend,
inter alia, on the characteristics of the translated codons and the
availability of specific transfer RNA (tRNA)
molecules. The dominance of the selected tRNA in the cell generally reflects
the codons most commonly used in
peptide synthesis. Thus, genes can be tailored to optimize gene expression in
a given organism based on codon
optimization. Codon usage tables are readily available, for example, in the
"codon usage database" in
www.kazusa.orjp/codon/, and may be modified in a number of ways. See Nakamura,
Y., et al. "Codon usage
tabulated from the international DNA Sequence databases: status for the year
2000" Nucl. Acids Res. 28: 292
(2000). Computerized algorithms for codon optimization of specific sequences
for expression in specific host cells
are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some
embodiments, one or more codons (e.g., 1,
2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all codons) in a sequence encoding
the Cas protein targeting DNA/RNA
correspond to the codons most commonly used for particular amino acids. For
codon usage in yeast, reference can
be made to the online saccharomyces genome
database available from
www.yeastgenome.org/community/codon_usage.shtml, or Codon selection in yeast,
Bennetzen and Hall, J Biol
Chem. March 25 1982; 257(6): 3026-31. For codon usage in plants including
algae, see Codon usage in higher
plants, green algae, and cyanobacteria, Campbell and Gown, Plant Physiol.,
January 1990; 92(1): 1-11.; and
Codon usage in plant genes, Murray et al., Nucleic Acids Res. January 25,
1989; 17(2): 477-98; or Selection on
the codon bias of chloroplast and cyanelle genes in different plant and algal
lineages, Morton BR, J Mol Evol.
April 1998; 46(4): 449-59.
Delivery system
In some embodiments, the components of the CRISPR-Cas system may be delivered
in various forms, such as a
combination of DNA/RNA or RNA/RNA or protein RNA. For example, the Cas12i
protein may be delivered as a
polynucleotide encoding DNA or a polynucleotide encoding RNA or as a protein.
The guide may be delivered as a
polynucleotide encoding DNA or RNA. All possible combinations are
contemplated, including mixed delivery
forms.
In some aspects, the invention provides a method for delivering one or more
polynucleotides, such as one or more
vectors, one or more transcripts thereof, and/or one or more proteins
transcribed therefrom as described herein, to
host cells.
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In some embodiments, one or more vectors that drive expression of one or more
elements of the nucleic acid
targeting system are introduced into host cells such that expression of
elements of the nucleic acid targeting
system guides formation of the nucleic acid targeting complex at one or more
target sites. For example, the
nucleic acid encoding effector enzymes and the nucleic acid encoding guide
RNAs may each be operably linked
to separate regulatory elements on separate vectors. The RNA of the nucleic
acid targeting system can be
delivered to a transgenic nucleic acid targeting effector protein animal or
mammal, e.g., an animal or mammal that
constitutively or inductively or conditionally expresses the nucleic acid
targeting effector protein; or an animal or
mammal that otherwise expresses the nucleic acid targeting effector protein or
has cells containing the nucleic
acid targeting effector protein, for example, by administering thereto one or
more vectors encoding and expressing
the in vivo nucleic acid targeting effector protein in advance. Alternatively,
two or more elements regulated by the
same or different regulatory elements may be combined in a single vector,
while one or more additional vectors
provide any components of the nucleic acid targeting system not contained in
the first vector. The elements of the
nucleic acid targeting system combined in the single vector may be arranged in
any suitable orientation, for
example, one element is positioned 5' ("upstream") relative to the second
element or 3' ("downstream") relative to
the second element. The coding sequence of one element may be on the same or
opposite chain of the coding
sequence of the second element and oriented in the same or opposite direction.
In some embodiments, a single
promoter drives the expression of transcripts encoding the nucleic acid
targeting effector protein and the nucleic
acid targeting guide RNA, and the transcripts are embedded into one or more
intron sequences (e.g., each in a
separate intron, two or more in at least one intron, or all in a single
intron). In some embodiments, the nucleic acid
targeting effector protein and the nucleic acid targeting guide RNA may be
operably linked to the same promoter
and expressed from the same promoter. Delivery vehicles, vectors, particles,
nanoparticles, formulations and
components thereof for expressing one or more elements of the nucleic acid
targeting system are as used in the
previous documents such as WO 2014/093622 (PCT/US2013/074667; the content of
which is incorporated herein
by reference in its entirety). In some embodiments, the vector comprises one
or more insertion sites, such as a
restriction endonuclease recognition sequence (also referred to as a "cloning
site"). In some embodiments, one or
more insertion sites (e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or more insertion sites) are located
upstream and/or downstream of one or more sequence elements of one or more
vectors. When a plurality of
different guide sequences are used, a single expression construct may be used
to target nucleic acids to various
corresponding target sequences within active target cells. For example, a
single vector may comprise about or
greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide
sequences. In some embodiments, about or
greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such vectors
containing guide sequences may be provided
and optionally delivered to the cells. In some embodiments, the vector
comprises a regulatory element operably
linked to an enzyme coding sequence encoding the nucleic acid targeting
effector protein. The nucleic acid
targeting effector protein or one or more nucleic acid targeting guide RNAs
may be delivered separately; and
advantageously at least one of these is delivered via a particle complex. The
nucleic acid targeting effector protein
mRNA may be delivered prior to the nucleic acid targeting guide RNA to allow
time for expression of the nucleic
acid targeting effector protein. The nucleic acid targeting effector protein
mRNA may be administered 1-12 h
(preferably about 2-6 h) prior to administration of the nucleic acid targeting
guide RNA. Alternatively, the nucleic
acid targeting effector protein mRNA and the nucleic acid targeting guide RNA
may be administered together.
Advantageously, the second boosted dose of guide RNA may be administered 1-12
h (preferably about 2-6 h) after
the initial administration of the nucleic acid targeting effector protein mRNA
+ guide RNA. The additional
administration of the nucleic acid targeting effector protein mRNA and/or
guide RNA may be useful to achieve
the most effective level of genomic modification.
Conventional viral and non-viral based gene transfer methods can be used to
introduce nucleic acids into
mammalian cells or target tissues. Such methods can be used to administer
nucleic acids encoding the components
of a nucleic acid targeting system to cells in culture or in a host organism.
A non-viral vector delivery system
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comprises DNA plasmids, RNA (e.g., transcripts of vectors as described
herein), naked nucleic acids, and nucleic
acids complexed with a delivery vehicle such as liposome. Viral vector
delivery systems comprise DNA and RNA
viruses that have episomal or integrated genomes upon delivery to cells. For a
review of gene therapy procedures,
see Anderson, Science 256: 808-813 (1992); Nabel and Felgner, TIBTECH 11: 211-
217 (1993); Mitani and
Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,
Nature 357: 455-460 (1992);
Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative
Neurology and Neuroscience 8: 35-36
(1995); Kremer and Perricaudet, British Medical Bulletin 51(1): 31-44 (1995);
Haddada et al., Current Topics in
Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al.,
Gene Therapy 1:13-26 (1994).
Non-viral delivery methods for nucleic acids include lipid transfection,
nuclear transfection, microinjection,
biolistics, virosomes, liposomes, immunoliposomes, polycations or lipids:
nucleic acid conjugates, naked DNA,
artificial virosomes, and reagent-enhanced DNA uptake. Lipid transfection is
described, for example, in U.S. Pat.
Nos. 5,049,386, 4,946,787; and 4,897,355, and lipid transfection reagents are
commercially available (e.g.,
TransfectamTm and LipofectinTm). Cationic and neutral lipids suitable for
effective receptor recognition lipid
transfection for polynucleotides include those in Felgner, WO 91/17424; WO
91/16024, which can be delivered to
cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in
vivo administration).
Plasmid delivery involves cloning the guide RNA into a plasmid expressing the
CRISPR-Cas protein and
transfecting DNA in cell culture. The plasmid backbone is commercially
available and does not require specific
equipment. Advantageously, they are modularized, and can carry CRISPR-Cas
coding sequences of different sizes,
including sequences encoding larger-sized protein, as well as selection
markers. Also, plasmids are advantageous
in that they ensure transient but continuous expression. However, the delivery
of plasmids is not direct, usually
leading to low in vivo efficiency. Continuous expression may also be
disadvantageous in that it can increase
off-target editing. In addition, excessive accumulation of CRISPR-Cas proteins
may be toxic to cells. Finally,
plasmids always have the risk of random integration of dsDNA into the host
genome, more particularly
considering the risk of double-stranded breakage (on-target and off-target).
The preparation of lipid: nucleic acid complexes (including targeting
liposomes, such as immunolipid complexes)
are well known to those skilled in the art (see, for example, Crystal, Science
270: 404-410 (1995); Blaese et al.,
Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-
389 (1994); Remy et al.,
Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722
(1995); Ahmad et al., Cancer Res.
52: 4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,
4,261,975, 4,485,054, 4,501,728,
4,774,085, 4,837,028 and 4,946,787), as will be discussed in more detail
below.
The use of RNA or DNA virus-based systems to deliver nucleic acids takes
advantage of a highly evolved process
of targeting viruses to specific cells in vivo and transporting viral payloads
to the nuclei. The viral vectors may be
administered directly to a patient (in vivo) or they may be used to treat
cells in vitro, and the modified cells may
optionally be administered to a patient (ex vivo). Conventional virus-based
systems may include retrovirus,
lentivirus, adenovirus, adeno-associated virus and herpes simplex virus
vectors for gene transfer. Integration into
the host genome by retroviral, lentiviral and adeno-associated virus gene
transfer methods often results in
long-term expression of the inserted transgene. In addition, high transduction
efficiency has been observed in
many different cell types and target tissues.
The tropism of a retrovirus can be altered by incorporation of a foreign
envelope protein to expand the potential
target population of target cells. Lentiviral vectors are retroviral vectors
that can transduce or infect non-dividing
cells and generally produce high viral titers. Therefore, the choice of a
retroviral gene transfer system will depend
on the target tissue. Retroviral vectors consist of cis-acting long terminal
repeats with a packaging capacity up to
6-10 kb of foreign sequences. The minimal cis-acting LTR is sufficient to
replicate and package the vector, which
is then used to integrate therapeutic genes into target cells to provide
permanent transgene expression. Widely
used retroviral vectors include vectors based on murine leukemia virus (MuLV),
gibbon ape leukemia virus
(GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus
(HIV), and combinations thereof
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(see, e.g., Buchscher et al., J. Virol. 66: 2731-2739 (1992); Johann et al.,
J. Virol. 66: 1635-1640 (1992);
Sommnerfelt et al., Virol. 176: 58-59 (1990); Wilson et al., J. Virol. 63:
2374-2378 (1989); Miller et al., J. Virol.
65: 2220-2224 (1991); PCT/US94/05700).
In applications where transient expression is preferred, adenovirus-based
systems may be used. Adenovirus-based
vectors provide high transduction efficiency in many cell types and do not
require cell division. With such vectors,
high titers and expression levels have been achieved. The vector can be mass
produced in a relatively simple
system. Adeno-associated virus ("AAV") vectors can also be used to transduce
cells with target nucleic acids, e.g.,
in the in vitro production of nucleic acids and peptides, as well as in in
vivo and ex vivo gene therapy procedures
(see, e.g., West et al., Virology 160: 38-47 (1987); U.S. Patent No.
4,797,368; WO 93/24641; Kotin, Human Gene
Therapy 5: 793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994)).
Construction of recombinant AAV
vectors is described in numerous publications, including U.S. Pat. No.
5,173,414; Tratschin et al., Mol. Cell. Biol.
5: 3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4: 2072-2081 (1984);
Hermonat and Muzyczka, PNAS 81:
6466-6470 (1984); and Samulski et al., J. Virol. 63: 03822-3828 (1989).
The invention provides AAV comprising or consisting essentially of an
exogenous nucleic acid molecule encoding
a CRISPR system, e.g., a plurality of cassettes comprising or consisting of a
first cassette comprising or consisting
essentially of a promoter, a nucleic acid molecule encoding a CRISPR
associated (Cas) protein (putative nuclease
or helicase protein ), e.g., Cas12i and a terminator, and one or more,
advantageously up to the packaging size limit
of the vector, for example five cassettes in total (including the first
cassette) comprising or consisting essentially
of a promoter, a nucleic acid molecule encoding guide RNA (gRNA) and a
terminator (for example, each cassette
is schematically represented as promoter - gRNA1 - terminator, promoter -
gRNA2 - terminator ... promoter -
gRNA(N)- terminator, where N is the upper limit of the package size limits of
the insertable vectors), or two or
more individual rAAVs, wherein each rAAV contains one or more cassettes of the
CRISPR system, for example, a
first rAAV contains a first cassette comprising or consisting essentially of a
promoter, a Cas-encoding nucleic acid
molecule such as Cas (Cas12i) and a terminator, and a second rAAV contains one
or more cassettes, each cassette
comprising or consisting essentially of a promoter, a nucleic acid molecule
encoding guide RNA (gRNA) and a
terminator (e.g., each cassette is schematically represented as promoter -
gRNA1 - terminator, promoter - gRNA2
- terminator ... promoter - gRNA(N) - terminator, where N is the upper limit
of the package size limits of the
insertable vectors). Alternatively, a single crRNA/gRNA array can be used for
multiplex gene editing, since
Cas12i can process its own crRNA/gRNA. Thus, rather than comprising a
plurality of cassettes to deliver gRNA,
rAAV can contain a single cassette comprising or consisting essentially of a
promoter, a plurality of crRNA/gRNA,
and a terminator (e.g., schematically represented as promoter - gRNA1 - gRNA2
... gRNA(N) - terminator, where
N is the upper limit of the package size limits of the insertable vector). See
Zetsche et al., Nature Biotechnology
35, 31-34 (2017), which is incorporated herein by reference in its entirety.
Since rAAV is a DNA virus, the nucleic
acid molecule in the discussion herein with respect to AAV or rAAV is
advantageously DNA. In some
embodiments, the promoter is advantageously human synaptophysin I promoter
(hSyn). Other methods for
delivering nucleic acids to cells are known to those skilled in the art. See,
for example, U520030087817, which is
incorporate herein by reference.
In another embodiment, cocal vesiculovirus enveloped pseudoretrovirus vector
particles are considered (see, for
example, U.S. Patent Publication No. 20120164118 assigned to Fred Hutchinson
Cancer Research Center). Cocal
virus belongs to the genus vesiculovirus and is the pathogen of vesicular
stomatitis in mammals. The cocal virus
was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet.
Res. 25: 236-242 (1964)), and cocal
virus infections have been identified in insects, cattle, and horses in
Trinidad, Brazil, and Argentina. Many
vesicular viruses that infect mammals have been isolated from naturally
infected arthropods, suggesting that they
are vector-borne. Antibodies to vesicular viruses are widely available in
rural areas where the viruses are obtained
locally and in laboratories; their infections in humans usually cause flu-like
symptoms. The envelope glycoprotein
of cocal virus shares 71.5% identity to VSV-G Indiana at the amino acid level,
and phylogenetic comparison of
CA 03237337 2024-05-02
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the vesicular virus envelope gene shows that cocal virus is serologically
distinct from, but most closely related to,
the VSV-G Indiana strain of vesicular virus. Jonkers et al., Am. J. Vet. Res.
25: 236-242 (1964) and Travassos da
Rosa et al., AM. J. Tropical Med. & Hygiene 33: 999-1006 (1984). Cocal
vesicular virus envelope
pseudoretrovirus vector particles may include, for example, lentivirus, alpha
retrovirus, beta retrovirus, gamma
retrovirus, delta retrovirus and epsilon retrovirus vector particles, which
may comprise retrovirus Gag, Pol and/or
one or more helper proteins and cocal vesicular virus envelope proteins. In
certain aspects of these embodiments,
the Gag, Pol and helper proteins are lentiviruses and/or gamma retroviruses.
In some embodiments, host cells are transiently or non-transiently transfected
with one or more vectors described
herein. In some embodiments, when the cells are naturally present in the
subject, the cells are transfected, and
optionally reintroduced therein. In some embodiments, the transfected cells
are taken from a subject. In some
embodiments, the cells are derived from cells from a subject, such as cell
lines. A wide variety of cell lines for
tissue culture are known in the art. Examples of cell lines include, but are
not limited to, C8161, CCRF-CEM,
MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa,
MiaPaCell, Pancl,
PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul,
SW480, SW620, SKOV3,
SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bc1-
1, BC-3, IC21, DLD2,
Raw264.7, NRK, NRK-52E, MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6,
COS-M6A, BS-C-1
monkey kidney epithelium, BALB/3T3 mouse embryonic fibroblasts, 3T3 Swiss, 3T3-
L1, 132-d5 human fetal
fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR,
A2780cis, A172, A20, A253, A431,
A-549, ALC, B16, B35, BCP-1 cell, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-
10T1/2, C6/36, Cal-27,
CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr-/-, COR-L23, COR-L23/CPR,
COR-L23/5010,
COR-L23/R23, COS-7, COV-434, CML Ti, CMT, CT26, D17, DH82, DU145, DuCaP, EL4,
EM2, EM3,
EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,
Hepalc1c7, HL-60,
HMEC, HT-29, Jurkat, JY cell, K562 cell, Ku812, KCL22, KG1, KY01, LNCap, Ma-
Mel 1-48, MC-38, MCF-7,
MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-
MAC 6,
MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,
NALM-1,
NW-145, OPCN/OPCT cell line, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-
2 cell, Sf-9, SkBr3,
T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cell, WM39, WT-49,
X63, YAC-1, YAR and
transgenic varieties thereof. Cell lines may be obtained from a variety of
sources known to those skilled in the art
(see, for example, the American Type Culture Collection (ATCC) (Manassus,
Va.)).
In particular embodiments, the transient expression and/or presence of one or
more components of an
AD-functionalized CRISPR system may be of interest, for example, to reduce off-
target effects. In some
embodiments, cells transfected with one or more vectors described herein are
used to establish novel cell lines
comprising one or more vector derived sequences. In some embodiments, cells
transiently transfected (e.g.,
transiently transfected with one or more vectors, or transfected with RNA)
with components of the
AD-functionalized CRISPR system as described herein and modified by the
activity of the CRISPR complex are
used to establish new cell lines comprising cells containing the modifications
but lacking any other exogenous
sequence. In some embodiments, cells transiently or non-transiently
transfected with one or more vectors
described herein, or cell lines derived from such cells, are used to evaluate
one or more test compounds.
In some embodiments, direct introduction of RNA and/or protein into host cells
is contemplated. For example, the
CRISPR-Cas protein may be delivered as encoded mRNA along with guide RNA from
in vitro transcription. Such
methods may reduce and ensure the action time of the CRISPR-Cas protein and
further prevent long-term
expression of the components of the CRISPR system.
In some embodiments, the RNA molecules of the invention are delivered as
liposomes or lipofectin formulations
and the like, and may be prepared by methods well known to those skilled in
the art. Such methods are described,
for example, in U.S. Pat. Nos. 5,593,972, 5,589,466 and 5,580,859, which are
incorporated herein by reference in
their entirety. Delivery systems specifically designed to enhance and improve
the delivery of siRNA into
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mammalian cells have been developed (see, e.g., Shen et al., FEBS Let. 2003,
539: 111-114; Xia et al., Nat.
Biotech. 2002, 20: 1006-1010; Reich et al., Mol. Vision. 2003, 9: 210-216;
Sorensen et al., J. Mol. Biol. 2003, 327:
761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108; and Simeoni et al., NAR
2003, 31, 11: 2717-2724) and may
be applied to the invention. siRNA have recently been successfully used to
inhibit gene expression in primates
(see, for example, Tolentino et al., Retina 24 (4): 660), which can also be
applied to the invention.
In fact, RNA delivery is a useful method of delivery in vivo. Cas12i,
adenosine deaminase, and guide RNA may
be delivered to cells using liposomes or particles. Thus, the delivery of
CRISPR-Cas proteins (e.g., Cas12i), the
delivery of adenosine deaminase (which may be fused to CRISPR-Cas proteins or
adaptor proteins) and/or the
delivery of RNA of the invention may be in the form of RNA and via
microvesicles, liposomes or particles or
nanoparticles. For example, Cas12i mRNA, adenosine deaminase mRNA, and guide
RNA may be packaged into
liposome particles for delivery in vivo. Liposome transfection reagents, such
as lipofectamine from Life
Technologies and other reagents on the market, can efficiently deliver RNA
molecules into the liver. In some
embodiments, the lipid nanoparticle (LNP) comprises ALC-0315: Cholesterol: PEG-
DMG: DOPE at a molar ratio
of 50mM: 50mM: 10mM: 20mM. In some embodiments, the LNP encapsulates both
Cas12i and its corresponding
crRNA (e.g., SiCas12i:crRNA with a weight ratio of 1:1), or nucleic acid(s)
encoding thereof. In some
embodiments, the LNP comprising Cas12i and/or crRNA (or nucleic acid(s)
encoding thereof) is administered to
an individual (e.g., human) by intravenous infusion.
Delivery of RNA also preferably includes RNA delivery via particles (Cho, S.,
Goldberg, M., Son, S., Xu, Q.,
Yang, F., Mei, Y., Bogatyrev, S., Langer, R., and Anderson, D., Lipid-like
nanoparticles for small interfering RNA
delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118,
2010) or via exosomes (Schroeder,
A., Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-based
nanotherapeutics for siRNA delivery,
Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641). In fact,
exosomes have been shown to be
particularly useful in delivering siRNA, and this system is somewhat similar
to the CRISPR system. For example,
El-Andaloussi S et al. ("Exosome-mediated delivery of siRNA in vitro and in
vivo." Nat Protoc. December 2012;
7 (12): 2112-26. doi: 10.1038/nprot.2012.131. Electronically published on
November 15, 2012) describes how
exosomes can become promising tools for drug delivery across different
biological barriers and for in vitro and in
vivo delivery of siRNA. Their method involves generating targeting exosomes by
transfecting an expression
vector comprising an exosome protein fused to a peptide ligand. The exosome is
then purified and characterized
from the transfected cell supernatant, and the RNA is loaded into the exosome.
Delivery or administration
according to the invention may be performed using exosomes, particularly (but
not limited to) the brain. Vitamin
E (a-tocopherol) can be conjugated with CRISPR Cas and delivered to the brain
along with high-density
lipoprotein (HDL), for example, in a manner similar to that of Uno et al.
(HUMAN GENE THERAPY 22:
711-719 (June 2011)) for delivery of short interfering RNA (siRNA) to the
brain. Infusion to mice is performed
via an Osmotic micro-pump (Model 1007D; Alzet, Cupertino, CA) filled with
phosphate buffered saline (PBS) or
free TocsiBACE or Toc-siBACE/HDL and connected to brain infusion kit 3
(Alzet). A brain infusion cannula is
placed approximately 0.5 mm posterior to the anterior fontanel at the midline
for infusion into the dorsal side of
the third ventricle. Uno et al. found that Toc-siRNA containing HDL as low as
3 nmol could induce the target
reduction considerably by the same ICV infusion method. In the invention, for
humans, similar doses of CRISPR
Cos conjugated to a-tocopherol and co-administered with brain-targeted HDL may
be considered, for example,
about 3 nmol to about 3 amol of brain-targeted CRISPR Cas may be considered.
Zou et al. (HUMAN GENE
THERAPY 22: 465-475 (April 2011)) describes a lentivirus-mediated delivery
method of short hairpin RNA
targeting PKCy for in vivo gene silencing in the spinal cords of rats. Zou et
al. administered approximately 10 pl
of recombinant lentivirus through an intrathecal catheter with a titer of 1
x109 transducing units (TU)/ml. In the
invention, for humans, a similar dose of CRISPR Cas expressed in a brain-
targeted lentivirus vector may be
considered, for example, about 10-50 ml of brain-targeted CRISPR Cas in a
lentivirus with a titer of 1x109
transduced units (TU)/m1 may be considered.
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Other suitable modifications and variations of the methods of the invention
described herein will be apparent to
those skilled in the art and may be made using suitable equivalents without
departing from the scope of the
invention or the embodiments disclosed herein.
EXEMPLARY EMBODIMENTS
Embodiment 1. A Cas12i protein comprising an amino acid sequence having at
least 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or 100% identity to
an amino acid sequence as set forth in any one of SEQ ID NOs: 1-10
(preferably, SEQ ID NOs: 1-3, 6, and 10,
and more preferably, SEQ ID NO: 1).
Embodiment 2. The Cas12i protein according to any one of the preceding
embodiments, wherein the Cas12i
protein substantially lacks (e.g., retains less than 50%, 40%, 35%, 30%,
27.5%, 25%, 22.5%, 20%, 17.5%, 15%,
12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1% or less) spacer-specific
endonuclease cleavage activity of the
corresponding parental Cas12i protein (e.g., Cas12i protein comprising any of
SEQ ID NOs: 1-10) for a target
sequence of a target DNA complementary to a guide sequence.
Embodiment 3. The Cas12i protein according to any one of the preceding
embodiments, wherein the Cas12i
protein comprises one or more amino acid variations in its RuvC domain such
that the Cas12i protein substantially
lacks (e.g., retains less than 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%,
17.5%, 15%, 12.5%, 10%, 7.5%,
5%, 4%, 3%, 2.5%, 2%, 1% or less) spacer-specific endonuclease cleavage
activity of the corresponding parental
Cas12i protein (e.g., Cas12i protein comprising any of SEQ ID NOs: 1-10) for a
target sequence of a target DNA
complementary to a guide sequence.
Embodiment 4. The Cas12i protein according to any one of the preceding
embodiments, wherein the amino acid
variation is selected from the group consisting of amino acid additions,
insertions, deletions, and substitutions.
Embodiment 5. The Cas12i protein according to any one of the preceding
embodiments, wherein the Cas12i
protein comprises an amino acid substitution at one or more positions
corresponding to positions 700 (D700), 650
(D650), 875 (E875) or 1049 (D1049) of the sequence as set forth in SEQ ID NO:
1.
Embodiment 6. The Cas12i protein according to any one of the preceding
embodiments, wherein the amino acid
substitution is selected from the group consisting of D700A/V, D650AN,
E875A/V, and D1049A/V.
Embodiment 7. The Cas12i protein according to any one of the preceding
embodiments, wherein the amino acid
substitution is selected from the group consisting of D700A, D650A, E875A, and
D1049A.
Embodiment 8. The Cas12i protein according to any one of the preceding
embodiments, wherein the amino acid
substitution is selected from the group consisting of D700A, D650A, E875A,
D1049A, D700A+D650A,
D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A, E875A+D1049A,
D700A+D650A+E875A,
D700A+D650A+D1049A, D650A+E875A+D1049A, and D700A+D650A+E875A+D1049A.
Embodiment 10. The Cas12i protein according to any one of the preceding
embodiments, wherein the Cas12i
protein is linked to one or more functional domains.
Embodiment 11. The Cas12i protein according to any one of the preceding
embodiments, wherein the functional
domain is linked to the N-terminus and/or C-terminus of the Cas12i protein.
Embodiment 12. The Cas12i protein according to any one of the preceding
embodiments, wherein the functional
domain is selected from the group consisting of a nuclear localization signal
(NLS), a nuclear export signal (NES),
a deaminase (e.g., adenosine deaminase or cytidine deaminase) catalytic
domain, a DNA methylation catalytic
domain, a histone residue modification domain, a nuclease catalytic domain, a
fluorescent protein, a transcription
modification factor, a light gating factor, a chemical inducible factor, a
chromatin visualization factor, a targeting
polypeptide for providing binding to a cell surface portion on a target cell
or a target cell type.
Embodiment 13. The Cas12i protein according to any one of the preceding
embodiments, wherein the functional
domain exhibits activity to modify a target DNA, selected from the group
consisting of nuclease activity,
methylation activity, demethylation activity, DNA repair activity, DNA damage
activity, deamination activity,
dismutase activity, alkylation activity, depurination activity, oxidation
activity, pyrimidine dimer formation
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activity, integrase activity, transposase activity, recombinase activity,
polymerase activity, ligase activity, helicase
activity, photolyase activity, glycosylase activity, acetyl transferase
activity, deacetylase activity, kinase activity,
phosphatase activity, ubiquitin ligase activity, deubiquitination activity,
adenylation activity, deadenylation activity,
SUMOylation activity, deSUMOylation activity, ribosylation activity,
deribosylation activity, myristoylation
activity, demyristoylation activity, glycosylation activity (e.g., from 0-
G1cNAc transferase), deglycosylation
activity, transcription inhibition activity, transcription activation
activity.
Embodiment 14. The Cas12i protein according to any one of the preceding
embodiments, wherein the functional
domain is selected from an adenosine deaminase catalytic domain or a cytidine
deaminase catalytic domain.
Embodiment 15. The Cas12i protein according to any one of the preceding
embodiments, wherein the functional
domain is a full length or functional fragment of TadA8e.
Embodiment 17. The Cas12i protein according to any one of the preceding
embodiments, wherein the Cas12i
protein is modified to reduce or eliminate spacer non-specific endonuclease
collateral activity.
Embodiment 18. A polynucleotide encoding the Cas12i protein according to any
one of the preceding
embodiments.
Embodiment 19. The polynucleotide according to any one of the preceding
embodiments, wherein the
polynucleotide is codon optimized for expression in eukaryotic cells.
Embodiment 20. The polynucleotide according to any one of the preceding
embodiments, comprising a nucleotide
sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99%, 99.5% or 100% identity to any one of the
nucleotide sequences as set forth in
SEQ ID NOs: 21-40.
Embodiment 21. A vector comprising the polynucleotide according to any one of
the preceding embodiments.
Embodiment 22. The vector according to any one of the preceding embodiments,
wherein the polynucleotide is
operably linked to a promoter.
Embodiment 23. The vector according to any one of the preceding embodiments,
wherein the promoter is a
constitutive promoter, an inducible promoter, a ubiquitous promoter, a cell
type specific promoter, or a tissue
specific promoter.
Embodiment 24. The vector according to any one of the preceding embodiments,
wherein the vector is a plasmid.
Embodiment 25. The vector according to any one of the preceding embodiments,
wherein the vector is a retroviral
vector, a phage vector, an adenovirus vector, a herpes simplex virus (HSV)
vector, an adeno-associated virus
(AAV) vector, or a lentiviral vector.
Embodiment 26. The vector according to any one of the preceding embodiments,
wherein the AAV vector is
selected from the group consisting of recombinant AAV vectors of serotypes
AAV1, AAV2, AAV4, AAV5, AAV6,
AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.
Embodiment 27. A delivery system comprising (1) a delivery medium; and (2) the
Cas12i protein, polynucleotide
or vector according to any one of the preceding embodiments.
Embodiment 28. The delivery system according to any one of the preceding
embodiments, wherein the delivery
medium is nanoparticle, liposome, exosome, microvesicle, or gene gun.
Embodiment 29. An engineered, non-naturally occurring CRISPR-Cas system
comprising:
the Cas12i protein or a polynucleotide encoding the Cas12i protein according
to any one of the preceding
embodiments; and
a CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, the crRNA
comprising:
a spacer capable of hybridizing to a target sequence of a target DNA, and
a Direct Repeat (DR) linked to the spacer and capable of guiding the Cas12i
protein to bind to the crRNA to form
a CRISPR-Cas complex targeting the target sequence.
Embodiment 30. A CRISPR-Cas system comprising one or more vectors, wherein the
one or more vectors
comprise:
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a first regulatory element operably linked to a nucleotide sequence encoding
the Cas12i protein according to any
one of the preceding embodiments; and
a second regulatory element operably linked to a polynucleotide encoding a
CRISPR RNA (crRNA), the crRNA
comprising:
a spacer capable of hybridizing to a target sequence of a target DNA, and
a Direct Repeat (DR) linked to the spacer and capable of guiding the Cas12i
protein to bind to the crRNA to form
a CRISPR-Cas complex targeting the target sequence;
wherein the first regulatory element and the second regulatory element are
located on the same or different vectors
of the CRISPR-Cas vector system.
Embodiment 31. An engineered, non-naturally occurring CRISPR-Cas complex
comprising:
the Cas12i protein according to any one of the preceding embodiments; and
a CRISPR RNA (crRNA), the crRNA comprising:
a spacer capable of hybridizing to a target sequence of a target DNA, and
a Direct Repeat (DR) linked to the spacer; the DR guides the Cas12i protein to
bind to the crRNA.
Embodiment 32. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the spacer is greater than 16 nucleotides in length, preferably 16 to
100 nucleotides, more preferably 16 to
50 nucleotides, more preferably 16 to 27 nucleotides, more preferably 17 to 24
nucleotides, more preferably 18 to
24 nucleotides, and most preferably 18 to 22 nucleotides.
Embodiment 33. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the DR has a secondary structure substantially identical to the
secondary structure of the DR as set forth
in any one of SEQ ID NOs: 11-20.
Embodiment 34. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the DR has nucleotide additions, insertions, deletions or
substitutions without causing substantial
differences in the secondary structure as compared to the DR as set forth in
any one of SEQ ID NOs: 11-20.
Embodiment 35. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the DR comprises a stem-loop structure near the 3' end of the DR,
wherein the stem-loop structure comprises 5'-X1X2X3X4X5NNNnNNNX6X7X8X9X10-3'
(X1, X2, X3, X4, X5, X6,
X7, X8, X9, X10 are any base, n is any nucleobase or deletion, N is any
nucleobase); wherein X1X2X3X4X5 and
X6X7X8X9X10 can hybridize to each other.
Embodiment 36. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the DR comprises a stem-loop structure selected from any one of the
following:
5' CUCCC GGGAG 3' near the 3' end of the DR, wherein N is any
nucleobase;
5' CUCC UGGGAG 3' near the 3' end of the DR, wherein N is any
nucleobase;
5' GUCCC UGGGAC 3' near the 3' end of the DR, wherein N is any
nucleobase;
5' GUGUC UGACAC 3' near the 3' end of the DR, wherein N is any
nucleobase;
5' GUGCC GGCAC 3' near the 3' end of the DR, wherein N is any
nucleobase;
5' UGUG UCACAC 3' near the 3' end of the DR, wherein N is any
nucleobase; and
5' CCGUC UGACGG 3' near the 3 end of the DR, where N is any nucleobase;
5' GTTTC UGAAAC 3' near the 3' end of the DR, where N is any nucleobase;
5' GTGTT AACAC 3' near the 3' end of the DR, where N is any nucleobase;
5' TTGTC GACAA 3' near the 3' end of the DR, where N is any nucleobase.
Embodiment 37. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
further comprising a target DNA capable of hybridizing to the spacer.
Embodiment 38. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the target DNA is a eukaryotic DNA.
Embodiment 39. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
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wherein the target DNA is in cells; preferably the cells are selected from the
group consisting of prokaryotic cells,
eukaryotic cells, animal cells, plant cells, fungal cells, vertebrate cells,
invertebrate cells, rodent cells, mammalian
cells, primate cells, non-human primate cells, and human cells.
Embodiment 40. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the crRNA hybridizes to and forms a complex with the target sequence
of the target DNA, causing the
Cas12i protein to cleave the target sequence.
Embodiment 41. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the target sequence is at the 3' end of a protospacer adjacent motif
(PAM).
Embodiment 42. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the PAM comprises a 5'-T-rich motif.
Embodiment 43. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the PAM is 5'-TTA, 5'-TTT, 5'-TTG, 5'-TTC, 5'-ATA or 5'-ATG.
Embodiment 44. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the one or more vectors comprise one or more retroviral vectors, phage
vectors, adenovirus vectors,
herpes simplex virus (HSV) vectors, adeno-associated virus (AAV) vectors, or
lentiviral vectors.
Embodiment 45. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the AAV vector is selected from the group consisting of recombinant
AAV vectors of serotypes AAV1,
AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, and
AAV13.
Embodiment 46. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the regulatory element comprises a promoter.
Embodiment 47. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the promoter is selected from the group consisting of a constitutive
promoter, an inducible promoter, a
ubiquitous promoter, a cell type specific promoter, or a tissue specific
promoter.
Embodiment 48. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the promoter is functional in eukaryotic cells.
Embodiment 49. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
wherein the eukaryotic cells include animal cells, plant cells, fungal cells,
vertebrate cells, invertebrate cells,
rodent cells, mammalian cells, primate cells, non-human primate cells, and
human cells.
Embodiment 50. The CRISPR-Cas system or complex according to any one of the
preceding embodiments,
further comprising a DNA donor template optionally inserted at a locus of
interest by homology-directed repair
(HDR).
Embodiment 51. A cell or descendant thereof, comprising the Cas12i protein,
polynucleotide, vector, delivery
system, CRISPR-Cas system or complex according to any one of the preceding
embodiments, wherein preferably,
the cell is selected from the group consisting of prokaryotic cells,
eukaryotic cells, animal cells, plant cells, fungal
cells, vertebrate cells, invertebrate cells, rodent cells, mammalian cells,
primate cells, non-human primate cells,
and human cells.
Embodiment 52. A non-human multicellular organism, comprising the cell or
descendant thereof according to any
one of the preceding embodiments; preferably, the non-human multicellular
organism is an animal (e.g., rodent or
non-human primate) model for human gene related diseases.
Embodiment 53. A method of modifying a target DNA, comprising contacting a
target DNA with the
CRISPR-Cas system or complex according to any one of the preceding
embodiments, the contacting resulting in
modification of the target DNA by the Cas12i protein.
Embodiment 54. The method according to any one of the preceding embodiments,
wherein the modification
occurs outside cells in vitro.
Embodiment 55. The method according to any one of the preceding embodiments,
wherein the modification
occurs inside cells in vitro.
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Embodiment 56. The method according to any one of the preceding embodiments,
wherein the modification
occurs inside cells in vivo.
Embodiment 57. The method according to any one of the preceding embodiments,
wherein the cell is a eukaryotic
cell.
Embodiment 58. The method according to any one of the preceding embodiments,
wherein the eukaryotic cell is
selected from the group consisting of animal cells, plant cells, fungal cells,
vertebrate cells, invertebrate cells,
rodent cells, mammalian cells, primate cells, non-human primate cells, and
human cells.
Embodiment 59. The method according to any one of the preceding embodiments,
wherein the modification is
cleavage of the target DNA.
Embodiment 60. The method according to any one of the preceding embodiments,
wherein the cleavage results in
deletion of a nucleotide sequence and/or insertion of a nucleotide sequence.
Embodiment 61. The method according to any one of the preceding embodiments,
wherein the cleavage
comprises cleaving the target nucleic acid at two sites resulting in deletion
or inversion of a sequence between the
two sites.
Embodiment 62. The method according to any one of the preceding embodiments,
wherein the modification is a
base variation, preferably A¨>G or C¨>T base variation.
Embodiment 63. A cell or descendant thereof from the method according to any
one of the preceding
embodiments, comprising the modification absent in a cell not subjected to the
method.
Embodiment 64. The cell or descendant thereof according to any one of the
preceding embodiments, wherein a
cell not subjected to the method comprises abnormalities and the abnormalities
in the cell from the method have
been resolved or corrected.
Embodiment 65. A cell product from the cell or descendant thereof according to
any one of the preceding
embodiments, wherein the product is modified relative to the nature or
quantity of a cell product from a cell not
subjected to the method.
Embodiment 66. The cell product according to any one of the preceding
embodiments, wherein cells not subjected
to the method comprise abnormalities and the cell product reflects that the
abnormalities have been resolved or
corrected by the method.
Embodiment 67. A method of non-specifically cleaving a non-target DNA,
comprising contacting the target DNA
with the CRISPR-Cas system or complex according to any one of the preceding
embodiments, whereby
hybridization of the spacer to the target sequence of the target DNA and
cleavage of the target sequence by the
Cas12i protein make the Cas12i protein cleave the non-target DNA by spacer non-
specific endonuclease collateral
activity.
Embodiment 68. A method of detecting a target DNA in a sample, comprising:
(1) contacting the sample with the CRISPR-Cas system or complex according to
any one of the preceding
embodiments and a reporter nucleic acid capable of releasing a detectable
signal after being cleaved, whereby
hybridization of the spacer to the target sequence of the target DNA and
cleavage of the target sequence by the
Cas12i protein make the Cas12i protein cleave the reporter nucleic acid by
spacer non-specific endonuclease
collateral activity; and
(2) measuring a detectable signal generated by cleavage of the reporter
nucleic acid, thereby detecting the
presence of the target DNA in the sample.
Embodiment 69. The method according to any one of the preceding embodiments,
further comprising comparing
the level of the detectable signal to the level of a reference signal and
determining the content of the target DNA in
the sample based on the level of the detectable signal.
Embodiment 70. The method according to any one of the preceding embodiments,
wherein the measurement is
performed using gold nanoparticle detection, fluorescence polarization,
colloidal phase change/dispersion,
electrochemical detection, or semiconductor-based sensing.
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Embodiment 71. The method according to any one of the preceding embodiments,
wherein the reporter nucleic
acid comprises a fluorescence emission dye pair, a fluorescence resonance
energy transfer (FRET) pair, or a
quencher/fluorophore pair, and cleavage of the reporter nucleic acid by the
Cas12i protein results in an increase or
decrease in the level of the detectable signal produced by cleavage of the
reporter nucleic acid.
Embodiment 72. A method of treating a condition or disease in a subject in
need thereof, comprising
administering to the subject the CRISPR-Cas system according to any one of the
preceding embodiments.
Embodiment 73. The method according to any one of the preceding embodiments,
wherein the condition or
disease is a cancer or infectious disease or neurological disease,
optionally, the cancer is selected from the group consisting of:
Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma,
neuroblastoma, melanoma, skin cancer,
breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer,
kidney cancer, pancreatic cancer, lung
cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal
cancer, gastric cancer, head and neck cancer,
thyroid myeloid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma,
acute lymphocytic leukemia,
acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic
leukemia, Hodgkin's lymphoma,
non-Hodgkin's lymphoma and urinary bladder cancer;
optionally, the infectious disease is caused by:
human immunodeficiency virus (HIV), herpes simplex virus-1 (HSV1) and herpes
simplex virus-2 (HSV2);
optionally, the neurological disease is selected from the group consisting of:
glaucoma, age-related loss of RGC, optic nerve injury, retinal ischemia,
Leber's hereditary optic neuropathy,
neurological diseases associated with RGC neuronal degeneration, neurological
diseases associated with
functional neuronal degeneration in the striatum of subjects in need,
Parkinson's disease, Alzheimer's disease,
Huntington's disease, schizophrenia, depression, drug addiction, dyskinesia
such as chorea, choreoathetosis and
dyskinesia, bipolar affective disorder, autism spectrum disorder (ASD) or
dysfunction.
Embodiment 74. The method according to any one of the preceding embodiments,
wherein the condition or
disease is selected from the group consisting of cystic fibrosis, progressive
pseudohypertrophic muscular
dystrophy, Becker muscular dystrophy, alpha-1 -antitrypsin deficiency, Pompe
disease, myotonic dystrophy,
Huntington's disease, fragile X syndrome, Friedreich ataxia, amyotrophic
lateral sclerosis, frontotemporal
dementia, hereditary chronic kidney disease, hyperlipidemia,
hypercholesterolemia, Leber congenital amaurosis,
sickle cell disease, and beta thalassemia.
Embodiment 75. The method according to any one of the preceding embodiments,
wherein the condition or
disease is caused by the presence of a pathogenic point mutation.
Embodiment 76. A kit comprising the CRISPR-Cas system according to any one of
the preceding embodiments;
preferably the components of the system are in the same container or in
separate containers.
Embodiment 77. A sterile container comprising the CRISPR-Cas system according
to any one of the preceding
embodiments; preferably the sterile container is a syringe.
Embodiment 78. An implantable device comprising the CRISPR-Cas system
according to any one of the
preceding embodiments; preferably the CRISPR-Cas system is stored in a
reservoir.
The disclosure also provides the following embodiments:
Item 1. An engineered, non-naturally occurring CRISPR-Cas system, comprising:
(1) a Cas12i protein or a polynucleotide encoding the Cas12i protein, wherein
the Cas12i protein comprises an
amino acid sequence having at least about 90% identity to any of SEQ ID NOs: 1-
3 and 6;
(2) a CRISPR RNA (crRNA) or a polynucleotide encoding the crRNA, the crRNA
comprising:
(i) a spacer capable of hybridizing to a target sequence of a target DNA, and
(ii) a Direct Repeat (DR) linked to the spacer and capable of guiding the
Cas12i protein to bind to the crRNA to
form a CRISPR-Cas complex targeting the target sequence.
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Item 2. The engineered, non-naturally occurring CRISPR-Cas system of item 1,
wherein the Cas12i protein
substantially lacks the spacer-specific endonuclease cleavage activity of the
corresponding parental Cas12i protein
of any of SEQ ID NOs: 1-3 and 6 against the target sequence of the target DNA.
Item 3. The engineered, non-naturally occurring CRISPR-Cas system of item 2,
wherein the Cas12i protein
comprises an amino acid substitution at one or more positions selected from
D700, D650, E875, and D1049 of the
parental Cas12i protein sequence of SEQ ID NO: 1.
Item 4. The engineered, non-naturally occurring CRISPR-Cas system of item 3,
wherein the amino acid
substitution is selected from the group consisting of D700A, D700V, D650A,
D650V, E875A, E875V, D1049A,
D1049V, D700A+D650A, D700A+E875A, D700A+D1049A, D650A+E875A, D650A+D1049A,
E875A+D1049A, D700A+D650A+E875A, D700A+D650A+D1049A, D650A+E875A+D1049A, and
D700A+D650A+E875A+D1049A.
Item 5. The engineered, non-naturally occurring CRISPR-Cas system of item 3,
wherein the Cas12i protein
comprises the amino acid sequence of any one of SEQ ID NOs: 79-82.
Item 6. The engineered, non-naturally occurring CRISPR-Cas system of item 2,
wherein the Cas12i protein is
fused to one or more functional domains to form a fusion protein.
Item 7. The engineered, non-naturally occurring CRISPR-Cas system of item 6,
wherein the functional domain
is selected from the group consisting of an adenosine deaminase catalytic
domain, a cytidine deaminase catalytic
domain, a DNA methylation catalytic domain, a DNA demethylation catalytic
domain, a transcription activation
catalytic domain, a transcription inhibition catalytic domain, a nuclear
export signal, and a nuclear localization
signal.
Item 8. The engineered, non-naturally occurring CRISPR-Cas system of item 7,
wherein the Cas12i protein is
fused to TadA8e or a functional fragment thereof to form the fusion protein.
Item 9. The engineered, non-naturally occurring CRISPR-Cas system of item 8,
wherein the fusion protein
comprises the amino acid sequence of SEQ ID NO: 85 or 184.
Item 10. The engineered, non-naturally occurring CRISPR-Cas system of item 1,
wherein the Cas12i protein
substantially lacks spacer non-specific endonuclease collateral activity of
the parental Cas12i protein of any of
SEQ ID NOs: 1-3 and 6 against a non-target DNA.
Item 11. The engineered, non-naturally occurring CRISPR-Cas system of item 1,
wherein the DR has a secondary
structure substantially identical to the secondary structure of the DR of any
one of SEQ ID NOs: 21-23, 26, and
101-106.
Item 12. The engineered, non-naturally occurring CRISPR-Cas system of item 11,
wherein the DR comprises a
stem-loop structure near the 3' end of the DR selected from any of SEQ ID NOs:
114-123, where N is any
nucleobase.
Item 13. The engineered, non-naturally occurring CRISPR-Cas system of item 1,
wherein the target sequence is at
the 3' end of a protospacer adjacent motif (PAM).
Item 14. The engineered, non-naturally occurring CRISPR-Cas system of item 13,
wherein the PAM is selected
from the group consisting of 5'-TTA, 5'-TTT, 5'-TTG, 5'-TTC, 5'-ATA, and 5'-
ATG.
Item 15. The engineered, non-naturally occurring CRISPR-Cas system of item 1,
wherein the engineered,
non-naturally occurring CRISPR-Cas system comprises a polynucleotide encoding
the Cas12i protein and a
polynucleotide encoding the crRNA located on the same or different vectors.
Item 16. The engineered, non-naturally occurring CRISPR-Cas system of item 15,
wherein the polynucleotide
encoding the Cas12i protein and the polynucleotide encoding the crRNA located
on the same vector are each
operably linked to a regulatory element.
Item 17. The engineered, non-naturally occurring CRISPR-Cas system of item 1,
wherein the spacer is at least
about 16 nucleotides in length.
Item 18. A method of modifying a target DNA, comprising contacting the target
DNA with the engineered,
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non-naturally occurring CRISPR-Cas system of item 1, wherein the crRNA
hybridizes to a target sequence of the
target DNA through the spacer of the crRNA, and wherein the Cas12i protein
binds to the crRNA to form a
CRISPR-Cas complex to modify the target sequence of the target DNA.
Item 19. The method of item 18, wherein the modification comprises one or more
of cleavage, single base editing,
and repairing of the target DNA.
Item 20. The method of item 19, wherein the modification comprises repairing
of the target DNA, and wherein
the method further comprises introducing a repair template DNA.
Item 21. The method of item 18, wherein the modification occurs in vitro, ex
vivo, or in vivo.
Item 22. A cell or descendant thereof obtained from the method of item 18.
Item 23. A non-human multicellular organism comprising the cell or descendant
thereof of item 22.
Item 24. A method of treating a condition or disease in a subject in need
thereof, comprising administering to the
subject an effective amount of the engineered, non-naturally occurring CRISPR-
Cas system of item 1, wherein the
condition or disease is associated with a mutation in a target DNA, wherein
the crRNA hybridizes to a target
sequence comprising the mutation of the target DNA through the spacer of the
crRNA, wherein the Cas12i protein
binds to the crRNA to form a CRISPR-Cas complex to modify the target sequence
of the target DNA, and wherein
the modification of the mutation in the target DNA treats the condition or
disease.
Item 25. The method of item 24, wherein the condition or disease is selected
from the group consisting of
transthyretin amyloidosis (ATTR), cystic fibrosis, hereditary angioedema,
diabetes, progressive
pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1 -
antitrypsin deficiency, Pompe
disease, myotonic dystrophy, Huntington's disease, fragile X syndrome,
Friedreich ataxia, amyotrophic lateral
sclerosis, frontotemporal dementia, hereditary chronic kidney disease,
hyperlipidemia, hypercholesterolemia,
Leber congenital amaurosis, sickle cell disease, and beta thalassemia.
Item 26. The method of item 25, wherein the condition or disease is ATTR.
Item 27. The method of item 24, wherein the engineered, non-naturally
occurring CRISPR-Cas system is
administered in a lipid nanoparticle.
Further embodiments are illustrated in the following Examples which are given
for illustrative purposes only and
are not intended to limit the scope of the disclosure.
EXAMPLES
Material and Methods
Unless otherwise specified, the experimental methods used in the Examples are
conventional.
Unless otherwise specified, the materials, reagents, etc., used in the
Examples are commercially available.
Unless otherwise specified, the following materials and experimental methods
were used in the Examples.
Plasmid vector construction.
Human codon-optimized Cas12i, TadA8e and human APOBEC3A genes were synthesized
by the GenScript Co.,
Ltd., and cloned to generate pCAG_NLS-Cas12i-NLS_pA_pUb_BpiI_pCMV_mCherry_pA
by Gibson Assembly.
crRNA oligos were synthesized by HuaGene Co., Ltd., annealed and ligated into
Bpil site to produce the
pCAG_NLS -C as12i-NLS_pA_pUb_crRNA_pCMV_mCherry_pA.
Cell culture, transfection, and flow cytometry analysis.
The mammalian cell lines used in this study were HEK293T and N2A. Cells were
cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% FBS,
penicillin/streptomycin and GlutMAX.
Transfections were performed using Polyetherimide (PEI). For variant
screening, HEK293T cells were cultured in
24-well plates, and after 12 hours 2 i.tg of the plasmids (1 i.tg of an
expression plasmid and 1 lag of a reporter
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plasmid) were transfected into these cells with 4 uL PEI. 48 hours after
transfection, BFP, mCherry, and EGFP
fluorescence were analyzed using a Beckman CytoFlex flow-cytometer. For assay
of mutations in target sites of
endogenous genes, 1 i.tg of expression plasmid was transfected into HEK293T or
N2A cells, which were then
sorted using a BD FACS Aria III, BD LSRFortessa X-20 flow cytometer, 48 hours
after transfection.
Detection of gene editing frequency.
Six thousand sorted cells were lysed in 20 tl of lysis buffer (Vazyme).
Targeted sequence primers were
synthesized and used in nested PCR amplification by Phanta Max Super-Fidelity
DNA Polymerase (Vazyme).
Targeted deep sequence analysis was used to determine indel frequencies. A-to-
G or C-to-T editing frequencies
were calculated by targeted deep sequence analysis or Sanger sequencing and
EditR. A-to-G editing purity were
calculated as A-to-G editing efficiency/ (A-to-T editing efficiency + A-to-C
editing efficiency + A-to-G editing
efficiency). C-to-T editing purity were calculated as C-to-T editing
efficiency/ (C-to-A editing efficiency + C-to-G
editing efficiency + C-to-T editing efficiency).
PEM-seq.
PEM-seq in HEK293 cells was performed as previously described23. Briefly, all-
in-one plasmids containing
LbCas12a, Ultra-AsCas12a, hfCas12Max, ABROO1 or Cas12i2HiFi with targeting
TTR.2 crRNA were transfected
into HEK293 cells by PEI respectively, and after 48 hrs, positive cells were
harvested for DNA extraction. The 20
jig genomic DNA was fragmented with a peak length of 300-700 bp by Covaris
sonication. DNA fragments was
tagged with biotin by a one-round biotinylated primer extension at 5'-end, and
then primer removal by AMPure
XP beads and purified by streptavidin beads. The single-stranded DNA on
streptavidin beads is ligased with a
bridge adapter containing 14-bp RMB, and PCR product was performed nested PCR
for enriching DNA fragment
containing the bait DSB and tagged with illumine adapter sequences. The
prepared sequencing library was
sequencing on an Hi-seq 2500, with a 2 x 150 bp.
RNP delivery and ex vivo editing.
RNP was complexed by mixing purified hfCas12Max proteins with chemically
synthesized RNA oligonucleotides
(Genscript) at a 1:2 molar ratio in 1X PBS. RNP was incubated at room
temperature for >15 min prior to
electroporation with Lonza 4DNucleofectorTM. 0.2 x 106 cells were resuspended
in 20 tL of Lonza buffer and
mixed with 5 tL RNP with different concentrations electroporated according to
Lonza specifications. HEK293 or
CD3+ T cells were harvested 72 hrs post-electroporation for targeted deep
sequence analysis.
LNP delivery and in vivo editing.
LNPs were formulated with ALC0315, cholesterol, DMG-PEG2k, DSPC in 100%
ethanol, carrying in vitro
transcription (IVT) mRNA and chemically synthesized RNA oligonucleotides
(Genscript) with a 1:1 weight ratio.
LNPs were formed according to the manufacturer's protocol, by microfluidic
mixing the lipid with RNA solutions
using a Precision Nano-systems NanoAssemblr Benchtop Instrument. LNPs diluted
in PBS were transfected into
N2a cells at 0.1, 0.3, 0.5, 1 ug RNA, or delivered into C57 mouse with
different dose by through tail intravenous
injection. Cells were harvested 48 hrs post-transfection for lysis and
targeted deep sequence analysis. For in vivo
editing, liver tissue was collected from the left or median lateral lobe of
each mouse 7 days post-injection for
DNA extraction and targeted deep sequence analysis.
Zygote Injection and Embryo Culturing.
Super ovulated female C57 mice (7-8 weeks old) by injecting 5 IU of pregnant
mare serum gonadotropin (PMSG),
followed by 5 IU of human chorionic gonadotropin (hCG) 48 hrs later were mated
to B6D2F1 males, and
fertilized embryos were collected from oviducts 20 hrs post hCG injection. For
zygote injection, hfCas12Max
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mRNA (100 ng/jiL) and sgRNA (100 ng/jiL) were mixed and injected into the
cytoplasm of fertilized eggs in a
droplet of HEPES-CZB medium containing 5 mg/ml cytochalasin B (CB) using a
FemtoJet microinjector
(Eppendorf) with constant flow settings. The injected zygotes were cultured in
KSOM medium with amino acids
at 37 C under 5% CO2 in air to blastocysts and harvested for targeted deep
sequence analysis.
Example 1 Identification of Cas12i proteins and evaluation of dsDNA cleavage
activity of
CRISPR-Cas12i systems comprising the Cas12i proteins
In order to identify more Cas12i, the applicant developed and employed a
bioinformatics pipeline to annotate
Cas12i proteins, CRISPR arrays, and predicted PAM preferences, and identified
10 CRISPR-Cas12i systems in
Table 1 below.
Table 1
SEQ ID NO:
Cas12i Cas12i Cas12i amino acid Corresponding Codon-optimized
Cas12i coding
protein protein sequence DR sequence Cas12i coding
sequence
sequences
SiCas12i Cas12i12
1 11 21 31
(xCas12i)
Si2Cas12i Cas12i3 2 12 22 32
WiCas12i Cas12i7 3 13 23 33
Wi2Cas12i Cas12i8 4 14 24 34
Wi3Cas12i Cas12i9 5 15 25 35
SaCas12i Cas12ill 6 16 26 36
Sa2Cas12i Cas12i4 7 17 27 37
Sa3Cas12i Cas12i5 8 18 28 38
WaCas12i Cas12i6 9 19 29 39
Wa2Cas12i Cas12i10 10 20 30 40
To evaluate the activity of these Cas12i in mammalian cells, the applicant
designed a dual plasmid fluorescent
reporter system, which detected the increased enhanced green fluorescent
protein (EGFP) signal intensity
activated by Cas-mediated dsDNA cleavage or double strand breaks (FIG. 3A).
This system relied on the
co-transfection of an expression plasmid encoding mCherry, a nuclear
localization signal (NLS) - tagged Cas
protein and its guide RNA (gRNA) or crRNA, and a reporter plasmid encoding BFP
and activatable EGxxFP
cassette, which is EGxx-target site-xxFP11. EGFP activation was carried out by
Cas mediated DSB and
single-strand annealing (SSA)-mediated repair.
Specifically, referring to FIG. 3A, the reporter plasmid comprised a
polynucleotide encoding, from 5' to 3', BFP -
P2A - activatable EGxxxxFP (SEQ ID NO: 41) (EGxx - insertion sequence (SEQ ID
NO: 42) (containing, from 5'
to 3', a protospacer adjacent motif (PAM)) of TTC for Cas12i, a protospacer
sequence (SEQ ID NO: 43) (which is
the reverse complementary sequence of a target sequence (SEQ ID NO: 44)), and
a protospacer adjacent motif
(PAM)) of GGG for Cas9 - xxFP), followed by a bGH polyA (SEQ ID NO: 448)
coding sequence, operably linked
to human CMV promoter (SEQ ID NO: 447). The protospacer sequence (SEQ ID NO:
43) contained a
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premature stop codon TAG that prevented the expression of EGFP and hence
emission of green fluorescent
signals. The BFP coding sequence expresses BFP to indicate the successful
transfection of the reporter plasmid
into host cells through blue fluorescence.
Most of the known Cas12i proteins recognize a 5'-T-rich PAM in dsDNA, while
Cas9 recognizes a 3'-G-rich PAM
in dsDNA. The co-existence of the 5' PAM of TTC for Cas12i and the 3' PAM of
GGG for Cas9 flanking the
protospacer sequence (SEQ ID NO: 43) allows the simultaneous comparison of
dsDNA cleavage activity of
Cas12i and Cas9.
Activatable EGxxxxFP coding sequence, SEQ ID NO: 41
atgagcgagctgattaaggagaacatgcacatgaagctgtatatggagggcaccgtggacaaccatcacttcaagtgca
catccgagggcgaaggcaagccctac
gagggcacccagaccatgagaatcaaggtggtcgagggcggccctctccccttcgccttcgacatcctggctactagct
tcctctacggcagcaagaccttcatcaa
ccacacccagggcatccccgacttcttcaagcagtccttccctgagggcttcacatgggagagagtcaccacatacgag
gacgggggcgtgctgaccgctacccag
gacaccagcctccaggacggctgcctcatctacaacgtcaagatcagaggggtgaacttcacatccaacggccctgtga
tgcagaagaaaacacteggctgggag
gccttcaccgagacactgtaccccgctgacggcggcctggaaggcagaaacgacatggccctgaagctcgtgggcggga
gccatctgatcgcaaacatcaagac
cacatatagatccaagaaacccgctaagaacctcaagatgcctggcgtctactatgtggactacagactggaaagaatc
aaggaggccaacaacgagacatacgtc
gagcagcacgaggtggcagtggccagatactgcgacctccctagcaaactggggcacaagctgaatgaattcgagggca
ggggcagcctgctgacctgcggcg
acgtggaggagaaccccggccccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagct
ggacggcgacgtaaacggccacaa
gttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaag
ctgcccgtgccctggcccaccctcgt
gaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgcc
atgcccgaaggctacgtccaggagcgc
accatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgca
tcgagctgaagggcatcgacttcaag
gaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcaga
agaacggcatcaaggtgaacttcaag
atccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccGGATCCGTGTCTTTCCCAT
TACAGTAGGA
GCATACGGGAGACAAGCTTTGgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgc
cctggcccaccctcg
tgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgc
catgcccgaaggctacgtccaggagcg
caccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgc
atcgagctgaagggcatcgacttcaa
ggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcag
aagaacggcatcaaggtgaacttcaa
gatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggcccc
gtgctgctgcccgacaaccactac
ctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccg
ccgggatcactctcggcatggacgag
ctgtacaagtaa
Insertion sequence, SEQ ID NO: 42
GGATCCGTGTCTTTCCCATTACAGTAGGAGCATACGGGAGACAAGCTTTG
Protospacer sequence (Reverse complementary sequence of the target sequence),
20bp, SEQ ID NO: 43
CCATTACAGTAGGAGCATAC
Target sequence for Cas12i, 20 nt, SEQ ID NO: 44
GTATGC TCC TAC TGTAATGG
EGxxxxFP-targeting spacer sequence, 20 nt, SEQ ID NO: 45
CCATTACAGTAGGAGCATAC
Non-targeting ("NT") spacer sequence, 20 nt, SEQ ID NO: 46
GGTCTTCGATAAGAAGACCT
PAM for Cas12i
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TTC
PAM for Cas9
GGG
Also referring to FIG. 3A, the expression plasmid comprised from 5' to 3' i) a
Cas12i coding sequence codon
optimized for expression in mammalian cells (SEQ ID NOs: 31-40) flanked by a
5V40 NLS (SEQ ID NO: 444)
coding sequence on the 5' end and a NP NLS (SEQ ID NO: 445) coding sequence on
the 3' end, followed by a
bGH polyA (SEQ ID NO: 448) coding sequence, operably linked to CAG promoter
(SEQ ID NO: 500), ii) a
sequence encoding a guide RNA (gRNA) in the configuration of 5' - DR sequence -
spacer sequence - 3' operably
linked to human U6 promoter (SEQ ID NO: 446); and iii) a coding sequence for
mCherry followed by a bGH
polyA (SEQ ID NO: 448) coding sequence operably linked to human CMV promoter
(SEQ ID NO: 447). The
mCherry coding sequence expresses mCherry to indicate the successful
transfection of the expression plasmid into
host cells through red fluorescence.
In the event that both the target sequence on the target strand and the
protospacer sequence on the nontarget strand
of the target dsDNA are successfully cleaved by a Cas12i polypeptide guided by
a gRNA to generate a
double-strand break (DSB), the subsequent DNA repairing such as single-strand
annealing (SSA)-mediated repair
trigged by the DSB would restore the EGFP coding sequence to express EGFP with
green fluorescence emission
indicative of dsDNA cleavage activity.
For test group, the spacer sequence comprised in the gRNA (SEQ ID NOs: 51-60)
for each tested Cas12i
polypeptide (SEQ ID NOs: 1-10) is a EGxxxxFP-targeting spacer sequence (SEQ ID
NO: 45) designed to target
and hybridize to the target sequence (SEQ ID NO: 44), and the DR sequence in
the gRNA (SEQ ID NOs: 51-60)
is a DR sequence (SEQ ID NOs: 11-20) corresponding to each tested Cas12i
polypeptide (SEQ ID NOs: 1-10), as
shown in Table 2.
Table 2
SEQ ID NO:
Cas12i protein DR sequence Spacer sequence Guide RNA
SiCas12i 11 45 51
Si2Cas12i 12 45 52
WiCas12i 13 45 53
Wi2Cas12i 14 45 54
Wi3Cas12i 15 45 55
SaCas12i 16 45 56
Sa2Cas12i 17 45 57
Sa3Cas12i 18 45 58
WaCas12i 19 45 59
Wa2Cas12i 20 45 60
For negative control ("NT") for each tested CRISPR-Cas system (Cas12i, SpCas9,
LbCas12a), a non-targeting
spacer sequence ("NT", SEQ ID NO: 46) incapable of hybridizing to the target
sequence (SEQ ID NO: 44) was
used in place of the EGxxxxFP-targeting spacer sequence (SEQ ID NO: 45), while
the other elements of each
CRISPR system remained.
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For positive control, CRISPR-SpCas9 and CRISPR-LbCas12a systems as shown in
Table 3 below were used in
place of the tested CRISPR-Cas12i systems, using the same EGxxxxFP-targeting
spacer sequence (SEQ ID NO:
45) and respective crRNA (SEQ ID NO: 48 or 50). In addition, for CRISPR-SpCas9
system, the gRNA was in
the configuration of 5' - spacer sequence - scaffold sequence - 3'.
Table 3
Control Cas Control Cas amino acid Guide RNA
protein sequence
SpCas9 SEQ ID NO: 47 SEQ ID NO: 48
LbCas12a SEQ ID NO: 49 SEQ ID NO: 50
HEI(293T cells were cultured in 24-well tissue culture plates according to
standard methods for 12 hours, before
the reporter and expression plasmids were co-transfected into the cells using
standard polyethyleneimine (PEI)
transfection. The transfected cells were then cultured at 37 C under 5% CO2
for 48 hours. Then the cultured cells
were analyzed by flow cytometry for BFP, EGFP, and mCherry fluorescent
signals. A "blank" control group was
also set up, where only the reporter plasmid was transfected, and no
expression plasmid was introduced.
The dsDNA cleavage activities of the Cas proteins were calculated as the
percentage of EGFP positive cells in
BFP & mCherry dual-positive cells ("EGFP", indicating dsDNA cleavage at the
indicated target site on the
reporter plasmid; "mCherry+ BFP", indicating successful co-transfection and co-
expression of the expression and
reporter plasmids). The higher the % EGFP + / mCherry+ BFP + is, the higher
the dsDNA cleavage activity would
be.
Using this dual plasmid fluorescent reporter system, it was observed that five
Cas12i (Cas12i3, Cas12i7, Cas12i10,
Cas12ill, and Cas12i12) exhibited targeted gRNA induced significant activation
of EGFP expression indicative
of significant dsDNA cleavage (FIG. 1A, FIG. 3B), and among them, Cas12i12
(also referred to as SiCas12i or
xCas12i herein) even exhibited a higher dsDNA cleavage than LbCas12a or SpCas9
as determined by
Fluorescence Activated Cell Sorter (FACS) analysis (FIG. 1A). The xCas12i was
smaller in size compared to
SpCas9 and LbCas12a (FIG. 4A).
Example 2 Evaluation of effective spacer sequence length for xCas12i
Using the dual plasmid fluorescent reporter system in Example 1, to test the
effective spacer sequence length for
xCas12i, spacer sequences of different lengths ranging from 10 to 50 nt (SEQ
ID NOs: 45 and 61-81 as shown in
Table 4 below) were designed to target and hybridize to the reverse
complementary sequence of a corresponding
protospacer sequence (also SEQ ID NOs: 45 and 61-81) of the insertion sequence
(SEQ ID NO: 42) of the
GFxxxxFP reporter plasmid in Example 1, and the 20 nt spacer sequence is
exactly the EGxxxxFP-targeting
spacer sequence (SEQ ID NO: 45) in Example 1. To evaluate the additional
spacer lengths, the EGxxxxFP
targeting spacer sequence (SEQ ID NO: 45) in the reporter plasmid was replaced
with the spacer sequence in
respective length (SEQ ID NOs: 61-81), while the other elements of the dual
plasmid fluorescent reporter system
remained.
Table 4
Protospacer / spacer sequence SEQ ID
NO:
10-nt CCATTACAGT 61
12-nt CCATTACAGTAG 62
14-nt CCATTACAGTAGGA 63
15-nt CCATTACAGTAGGAG 64
16-nt CCATTACAGTAGGAGC 65
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17-nt CCATTACAGTAGGAGCA 66
18-nt CCATTACAGTAGGAGCAT 67
19-nt CCATTACAGTAGGAGCATA 68
20-nt CCATTACAGTAGGAGCATAC 45
21-nt CCATTACAGTAGGAGCATACG 69
22-nt CCATTACAGTAGGAGCATACGG 70
23-nt CCATTACAGTAGGAGCATACGGG 71
24-nt CCATTACAGTAGGAGCATACGGGA 72
26-nt CCATTACAGTAGGAGCATACGGGAGA 73
27-nt CCATTACAGTAGGAGCATACGGGAGAC 74
28-nt CCATTACAGTAGGAGCATACGGGAGACA 75
30-nt CCATTACAGTAGGAGCATACGGGAGACAAG 76
32-nt CCATTACAGTAGGAGCATACGGGAGACAAGCT 77
35-nt CCATTACAGTAGGAGCATACGGGAGACAAGCTTTG 78
40-nt CCATTACAGTAGGAGCATACGGGAGACAAGCTTTGGCCAC 79
45-nt CCATTACAGTAGGAGCATACGGGAGACAAGCTTTGGCCACCTACG 80
50-nt CCATTACAGTAGGAGCATACGGGAGACAAGCTTTGGCCACCTACGGCAAG 81
By using the experimental procedure in Example 1, it was observed that a
spacer sequence length range of at least
16 nucleotides is effective for xCas12i's activity, and among that range, 17-
22 nt is optimal (FIG. 4B).
Example 3 Evaluation of PAM recognition for xCas12i
Considering the 5'-TTN PAM preference of Cas12i, the applicant performed a
NTTN PAM identification assay
using the dual plasmid fluorescent reporter system in Example 1, in which
various 5' PAM was used in place of
the original 5' PAM of TTC, while the other elements of the dual plasmid
fluorescent reporter system remained.
By using the experimental procedure in Example 1, it was observed that xCas12i
showed a consistent high
frequency of EGFP activation at target sites with 5'-NTTN PAM sequences,
wherein N is A, T, C, or G, while
LbCas12a had comparable activity at 5'-TTTN PAM, respectively (FIG. 4C).
Example 4 Effect of DR sequence on xCas12i's dsDNA cleavage activity
To test whether the original DR sequence (SEQ ID NO: 11) of xCas12i could
tolerate mutations, the applicant
truncated the original DR sequence to generate two functional fragments DR-Ti
and DR-T2 of SEQ ID NOs: 501
and 502, respectively, without destroying the secondary structure of the
original DR sequence, and then designed
five DR variants of DR-T2 to generate DR-A, DR-B, DR-C, DR-D, and DR-E
sequences of SEQ ID NOs:
503-507, respectively, each containing 5% to 30% mutations in the stem-loop
regions without destroying the
secondary structure of the original DR sequence (i.e. the secondary structures
of the DR variants were
substantially the same as that of the original DR sequence).
SEQ ID NO: 501 DR-T1, 30 nt
ATGACTCAGAAATGTGTCCCCAGTTGACAC
SEQ ID NO: 502 DR-T2 sequence, 23 nt
AGAAATGTGTCCCCAGTTGACAC
SEQ ID NO: 503 DR-A sequence, 23 nt
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AGAAATCCGTCCTTAGTTGACGG
SEQ ID NO: 504 DR-B sequence, 22 nt
AGACATGTGTCCCCAGTGACAC
SEQ ID NO: 505 DR-C sequence, 23 nt
AGAAATGTTTCCCCAGTTGAAAC
SEQ ID NO: 506 DR-D sequence, 23 nt
AGAAATGTGTTCCCAGTTAACAC
SEQ ID NO: 507 DR-E sequence, 23 nt
AGAAATTTGTCCCCAGTTGACAA
By using the dual plasmid fluorescent reporter system for xCas12i in Example 1
with the original DR sequence
replaced with each of the DR variants (DR-T1, DR-T2, DR-A, DR-B, DR-C, DR-D,
and DR-E), while the other
element of the reporter system remained, the results (HG. 21) show that
xCas12i still exhibited high dsDNA
cleavage activity mediated by gRNAs with various DR sequence variants. It can
be seen that under the condition
that the secondary structure of the DR sequence is maintained (i.e., the
secondary structures of the DR variants are
substantially the same as that of the original DR sequence), the CRISPR-
SiCas12i system can tolerate
mismatching or deletion on DR sequence without loss of dsDNA cleavage
activity, and has wide adaptability to
variations in the DR sequence. These data also demonstrated that the two
functionally truncated versions of
original xCas12i DR sequence of SEQ ID NO: 11(36 nt), i.e., DR-Ti (SEQ ID NO:
501, 30 nt) and DR-T2 (SEQ
ID NO: 502, 23 nt), could still mediate high dsDNA cleavage activity of
xCas12i.
Example 5 Evaluation of dsDNA cleavage activity of xCas12i at endogenous gene
To further verify the dsDNA cleavage activity of xCas12i at an endogenous gene
(genome cleavage) in
mammalian cells, the applicant transfected the expression plasmid (FIG. 3A,
FIG. 4D) in Example 1 encoding
NLS tagged xCas12i with gRNAs targeting 37 sites from human TTRI2 gene and
human PCSK913 gene in
HEI(293T (human embryonic kidney 293 cells) or mouse Ttr gene in N2a cells
(Neuro2a cells, a fast-growing
mouse neuroblastoma cell line). The EGxxxxFP targeting spacer sequence (SEQ ID
NO: 45) in Example 1 was
replaced with respective gene-targeting spacer sequence (SEQ ID NOs: 82-126 in
Table 5), the DR-Ti sequence
(SEQ ID NO: 501) was used in place of the original DR sequence (SEQ ID NO: 11)
(and also in the Examples
below unless otherwise specified), while the other elements of the CRISPR-
xCas12i system in Example 1
remained. The dsDNA cleavage activity, i.e., indel (insertion and/or deletion)
formation at these loci was
measured 48 hours after transfection using FACS and targeted deep sequencing.
It was observed that xCas12i mediated a high frequency, up to 90%, of indel
formation at most sites from Ttr, TER
and PCSK9, with a mean indel formation rate of over 50% (FIG. 4E-F). These
data indicate that xCas12i exhibits
a robust genome editing efficiency in mammalian cells, suggesting that it has
excellent potential for therapeutic
genome editing applications.
Table 5. Sequences for testing genome cleavage at target loci
SEQ ID
Genomic
Guide RNA PAM Protospacer / spacer sequences NO of Figure
loci
protospace
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r / spacer
sequence
FIG.1D, FIG. 6B-C,
DMD_sgl TTTG CAAAAACCCAAAATATTTTA 82
and FIG. 8D
FIG.1D, FIG. 6B-C,
DMD DMD_sg2 TTTA GCTCCTACTCAGACTGTTAC 83
and FIG. 8D
FIG.1D, FIG. 6B-C,
DMD_sg3 GTTG TGTCACCAGAGTAACAGTC 84
and FIG. 8D
Ttr_sgl TTTG CCTCGCTGGACTGGTATTTG 85 FIG. 4E-F
FIG.1D and FIG.
Ttr_sg2 TTTG TGTCTGAAGCTGGCCCCGCG 86
4E-F
FIG.1D and FIG.
Ttr_sg3 CTTC CCTTCGACTCTTCCTCCTTTG 87
4E-F, 19B
Ttr_sg4 CTTC CTCCTTTGCCTCGCTGGACTG 88 FIG. 4E-F
Ttr_sg5 TTTG ACCATCAGAGGACATTTGGA 89 FIG. 4E-F
Ttr_sg6 TTTG GATTCTCCAGCACCCTGGGC 90 FIG. 4E-F
Ttr_sg7 TTTA CAGCCACGTCTACAGCAGGG 91 FIG. 4E-F
Ttr_sg8 TTTT ACAGCCACGTCTACAGCAGG 92 FIG. 4E-F
Ttr_sg9 TTTT GAACACTTTTACAGCCACGT 93 FIG. 4E-F
Ttr_sg10 GTTC AAAAAGACCTCTGAGGGATC 94 FIG. 4E-F
Ttr_sgll TTTG AACACTTTTACAGCCACGTC 95 FIG. 4E-F
Ttr
FIG.1D, FIG.2F-H
Ttr_sg12 TTTG TAGAAGGAGTGTACAGAGTA 96
and FIG. 4E-F, 19B
Ttr_sg13 CTTG GCATTTCCCCGTTCCATGAA 97 FIG. 4E-F
Ttr_sg14 CTTC TCATCTGTGGTGAGCCCGTG 98 FIG. 4E-F
Ttr_sg15 TTTG GTGTCCAGTTCTACTCTGTA 99 FIG. 4E-F
Ttr_sg16 CTTC CAGTACGATTTGGTGTCCAG 100 FIG. 4E-F
Ttr_sg17 CTTC TACAAACTTCTCATCTGTGG 101 FIG. 4E-F
Ttr_sg18 TTTT CACAGCCAACGACTCTGGCC 102 FIG. 4E-F
Ttr_sg19 TTTC ACAGCCAACGACTCTGGCCA 103 FIG. 4E-F
CTGACGACAGCCGTGGTGCTG
Ttr_sg20 GTTG 104 FIG. 4E-F
T
AAAAAGACCTCTGAGGGATCC
Ttr_sg21 GTTC 105 FIG. 4E-F
T
AGAAAGGCTGCTGATGACACC FIG.1D and
FIG.
TTR_sgl GTTC 106
T 4E-F
TAGAAGGGATATACAAAGTGG FIG.1D and
FIG.
TTR_sg2 TTTG 107
A 4E-F,16
CACCACGGCTGTCGTCACCAA
TTR TTR_sg3 ATTC 108 FIG. 4E-F
T
TTR_sg5 TTTG AATCCAAGTGTCCTCTGATGGT 109 FIG. 4E-F
TTR_sg6 TTTC AATGTGGCCGTGCATGTGTTCA 110 FIG. 4E-F
TAGATGCTGTCCGAGGCAGTC
TTR_sg7 GTTC 111 FIG. 4E-F
C
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GCATGGGCTCACAACTGAGGA
TTR_sg8 ATTC 112 FIG. 4E-F
TATACAAAGTGGAAATAGACA
TTR_sg10 TTTG 113 FIG. 4E-F
CTGGAAGGCACTTGGCATCTC FIG.1D and
FIG.
TTR_sgll CTTA 114
4E-F
FIG.1D and FIG.
TTR_sg12 CTTG GCATCTCCCCATTCCATGAGCA 115
4E-F
ACAGCCAACGACTCCGGCCCC
TTR_sg14 ATTC 116 FIG. 4E-F
PCSK9_sg5 GTTG CCTGGCACCTACGTGGTGG 117 FIG. 4E-F
FIG.1D and FIG.
PC S K9_sg6 CTTC CATGGCCTTCTTCCTGGC 118
4E-F
PC S K9_sg7 CTTC TTCCTGGCTTCCTGGTGAAG 119 FIG. 4E-F
PCSK9
FIG.1D and FIG.
PC S K9_sg9 CTTG AAGTTGCCCCATGTCGACTA 121
4E-F
PC SK9_sgl FIG.1D and
FIG.
TTTG CCCAGAGCATCCCGTGGAAC 122
0 4E-F
TRAC_sgl TTTA CAGATACGAACCTAAACTTT 123 FIG.2B-C
TRAC TRAC_sg2 TTTA GAGTCTCTCAGCTGGTACAC 124 FIG.2B-C
TRAC_sg3 TTTG TCTGTGATATACACATCAGA 125 FIG.2B-C
Example 6 Development of xCas12i mutants and evaluation of their dsDNA
cleavage activity
To vary xCas12i's activity and expand its scope of PAM site recognition, the
applicant engineered xCas12i protein
via mutagenesis and screened for variants with higher efficiency and broader
PAM using a dual plasmid
fluorescent reporter system similar to the dual plasmid fluorescent reporter
system in Example 1, except that the
EGxxxxFP-targeting guide RNA (SEQ ID NO: 51) coding sequence was not on the
expression plasmid together
with the xCas12i coding sequence (SEQ ID NO: 31) but on the reporter plasmid
together with the BFP - P2A -
EGxxxxFP coding sequence (SEQ ID NO: 41) (referring to "On-Target Reporter" in
FIG. 1B). Combined with
predictive structural analysis of xCas12i, the applicant performed an arginine
(R) scanning mutagenesis approach
in the PI domain (amino acid residue position 173-291), REC-I domain (amino
acid residue position 427-473),
and RuvC-II domain (amino acid residue position 800-1082) of xCas12i,
generating a library of over 500 xCas12i
mutants with a single non-R amino acid substitution with R. The xCas12i (SEQ
ID NO: 1) coding sequence on
the expression plasmid was replaced with a sequence encoding each of the
xCas12i mutants, the DR-T1 sequence
(SEQ ID NO: 501) was used in place of the original DR sequence (SEQ ID NO:
11), while the other elements of
the reporter system remained. The applicant then individually transfected the
expression plasmid and the
reporter plasmid into HEK293T cells and analyzed them by FACS (FIG. 1B).
For negative control ("NT"), a non-targeting spacer sequence ("NT", SEQ ID NO:
46) incapable of hybridizing to
the target sequence (SEQ ID NO: 44) was used in place of the EGxxxxFP-
targeting spacer sequence (SEQ ID NO:
45) and used in combination with xCas12i (SEQ ID NO: 1), while the other
elements of the reporter system
remained.
For positive control ("WT"), the original xCas12i (SEQ ID NO: 1) was used.
Table 6
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dsDNA dsDNA dsDNA dsDNA
Mutant cleavage Mutant cleavage Mutant cleavage Mutant cleavage
activity activity activity activity
K109R 0.034 1(264R 0.671 S428R 0.075 M923R 1.044
N11OR 0.778 A265R 0.725 Y430R 0.359 S924R 0.351
Y111R 0.634 M266R 0.250 Y431R 0.856 S925R 1.276
L112R 0.041 I267R 0.933 E432R 0.670 H926R 1.440
M113R 0.062 S268R 0.959 D433R 0.605 Q927R 1.933
S114R 0.837 N269R 0.401 F434R 0.161 D928R 0.164
N115R 0.312 F270R 0.131 S435R 0.981 P929R 0.179
1116R 0.836 T271R 0.450 A436R 0.033 F930R 0.203
D117R 0.499 1(272R 0.383 K437R 0.880 V931R 1.547
S118R 1.481 N273R 1.652 N438R 0.309 H932R 0.229
D119R 1.337 A274R 0.207 F439R 0.010 M933R 1.827
F121R 1.356 A275R 0.713 L440R 1.379 Q934R 2.147
V122R 0.737 A276R 0.309 D441R 0.671 D935R 1.413
W123R 1.010 1(277R 0.282 G442R 0.051 K936R 1.489
V124R 0.119 A278R 0.471 A443R 0.033 K937R 1.442
D125R 0.040 A279R 0.683 K444R 0.547 T938R 1.452
C126R 0.051 1(280R 0.556 L445R 0.107 S939R 1.413
R127R 0.025 1(281R 0.671 N446R 0.410 V940R 1.333
K128R 0.844 P282R 0.575 V447R 0.004 L941R 0.988
F129R 0.802 I283R 0.390 L448R 1.369 P943R 0.812
A13OR 0.064 P284R 0.274 T449R 0.514 F945R 1.055
K131R 0.728 Y285R 0.287 E45OR 0.887 M946R 1.207
D132R 0.839 L286R 0.745 V451R 1.883 E947R 0.885
F133R 0.990 D287R 1.084 V452R 0.735 V948R 1.231
A134R 0.076 R288R 0.386 N453R 0.895 N949R 1.893
Y135R 0.863 L289R 0.400 Q455R 1.190 K95OR 1.640
Q136R 1.067 1(290R 0.403 K456R 0.887 D951R 2.347
M137R 0.128 E291R 0.363 A457R 0.004 S952R 1.500
E138R 1.010 M293R 0.019 H458R 0.008 I953R 0.382
L139R 0.194 V294R 0.665 P459R 0.008 D955R 1.221
G 1 4OR 0.957 S295R 1.172 T460R 0.009 Y956R 1.768
F141R 0.429 L296R 0.752 I461R 0.801 H957R 0.681
H142R 0.941 C297R 0.061 W462R 0.358 V958R 0.541
E143R 1.240 D298R 0.719 S463R 0.020 A959R 1.635
F144R 0.007 Y300R 0.168 E464R 1.127 G960R 1.840
T145R 0.951 N301R 0.359 1800R 0.596 L961R 0.152
V146R 1.106 V302R 1.517 S801R 0.204 L965R 0.443
L147R 0.038 Y303R 0.324 L802R 0.398 N966R 1.933
A148R 0.013 A304R 0.067 K803R 0.436 S967R 1.529
E149R 0.319 W305R 0.026 M804R 0.130 K968R 1.241
T15OR 0.686 A306R 0.187 1805R 0.325 S969R 1.548
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L151R 0.038 A307R 0.265 S806R 1.214 D970R 1.451
L152R 0.097 A308R 0.030 D807R 0.899 A971R 1.848
A153R 1.000 1309R 0.009 F808R 0.261 G972R 1.152
N154R 0.307 T31OR 0.163 K809R 0.905 T973R 0.641
S155R 1.577 N311R 0.120 G81OR 0.954 S974R 1.180
I156R 0.531 S312R 0.037 V811R 0.178 V975R 1.097
L157R 0.041 N313R 0.246 V812R 0.187 Y976R 1.148
V158R 1.990 A314R 0.030 Q813R 0.161 Y977R 0.007
L159R 0.085 D315R 0.046 S814R 0.023 Q979R 1.421
N16OR 0.860 V316R 0.007 Y815R 0.284 A980R 1.057
E161R 2.115 T317R 0.143 F816R 0.299 A981R 0.341
S162R 2.096 A318R 0.037 S817R 1.290 L982R 1.146
T163R 1.054 N320R 0.098 V818R 1.410 H983R 1.372
K164R 0.760 T321R 0.156 S819R 1.130 F984R 0.580
A165R 3.151 L324R 0.035 G820R 0.407 C985R 1.076
N166R 1.548 T325R 0.209 C821R 0.801 E986R 1.137
W167R 0.775 F326R 0.183 V822R 0.699 A987R 1.220
A168R 0.058 I327R 0.031 D823R 0.911 L988R 0.954
W169R 0.161 G328R 0.879 D824R 0.939 G989R 1.420
G17OR 0.572 E329R 0.249 A825R 0.884 V990R 1.094
T171R 0.211 Q330R 0.159 S826R 0.707 S991R 1.211
V172R 0.564 N331R 0.538 K827R 0.654 P992R 1.128
S173R 0.202 S332R 1.136 K828R 0.917 E993R 1.154
A174R 0.398 K335R 0.577 A829R 0.954 L994R 1.148
L175R 0.170 E336R 1.463 H830R 0.593 V995R 1.109
Y176R 0.215 L337R 0.613 D831R 0.318 K996R 1.038
G177R 0.135 S338R 1.505 S832R 1.010 N997R 1.211
G178R 1.920 V339R 1.183 M833R 1.088 K998R 1.171
G179R 0.737 L340R 0.419 L834R 0.835 K999R 1.348
D18OR 1.025 Q341R 0.766 F835R 1.280 T1000R 1.128
K181R 0.172 T342R 0.322 T836R 1.402 H1001R 1.209
E182R 0.235 T343R 0.710 F837R 1.270 A1002R 1.171
D183R 0.279 T344R 0.646 M838R 0.961 A1003R 1.241
S184R 0.987 N345R 0.218 C839R 1.700 E1004R 1.460
T185R 1.685 E346R 0.554 A840R 1.412 L1005R 0.665
L186R 0.641 K347R 0.684 A841R 0.245 G1006R 1.031
K187R 0.193 A348R 0.048 E842R 1.540 M1009R 0.980
S188R 0.234 K349R 0.461 E843R 1.710 G101OR 1.172
K189R 1.010 D35OR 0.474 K844R 1.520 S1011R 0.558
1190R 0.070 I351R 0.146 T846R 1.620 A1012R 1.098
L191R 0.118 L352R 0.023 N847R 1.180 M1013R 1.207
L192R 0.910 N353R 0.553 K848R 1.230 L1014R 1.044
A193R 1.566 K354R 0.681 E85OR 0.867 M1015R 0.535
F194R 0.194 N356R 0.542 E851R 0.977 P1016R 0.088
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V195R 0.019 D357R 0.472 K852R 0.337 W1017R 1.744
D196R 1.317 N358R 0.554 T853R 0.928 G1019R 0.387
A197R 0.791 L359R 0.398 N854R 1.031 G102OR 0.396
L198R 0.204 1360R 0.580 A856R 1.262 V1022R 1.260
N199R 1.354 Q361R 0.676 A857R 0.384 Y1023R 0.814
N200R 1.417 E362R 1.430 S858R 1.117 I1024R 0.296
H201R 0.183 V363R 0.696 F859R 0.000 A1025R 0.062
E202R 1.102 Y365R 0.016 1860R 0.146 S1026R 0.971
L203R 1.344 T366R 0.973 L861R 0.770 K1027R 0.978
1(204R 0.817 P367R 0.195 Q862R 1.882 K1028R 1.550
T205R 0.973 A368R 0.709 K863R 1.427 L1029R 0.444
1(206R 0.871 K370R 0.648 A864R 0.000 T103OR 0.824
E208R 0.279 H371R 0.068 Y865R 1.179 S1031R 0.000
1209R 0.108 L372R 0.006 L866R 1.417 D1032R 1.230
L21OR 0.346 G373R 0.430 H867R 0.000 A1033R 0.563
N211R 0.499 D375R 1.408 G868R 1.613 K1034R 1.301
Q212R 0.650 L376R 0.006 C869R 0.131 S1035R 0.790
V213R 0.114 A377R 1.097 K870R 1.510 V1036R 0.627
C214R 0.166 N378R 1.113 M871R 1.334 K1037R 1.750
E215R 0.329 L379R 0.008 I872R 0.163 Y1038R 0.666
S216R 0.591 F380R 0.087 V873R 0.306 C1039R 1.430
L217R 0.465 D381R 1.502 C874R 0.519 G104OR 1.077
1(218R 0.294 T382R 1.517 E875R 0.100 E1041R 0.920
Y219R 0.375 L383R 0.006 D876R 2.637 D1042R 0.928
Q220R 0.371 K384R 0.941 D877R 2.492 M1043R 0.930
S221R 1.150 E385R 1.424 L878R 0.132 W1044R 0.870
Y222R 0.417 K386R 0.980 P879R 0.132 Q1045R 1.560
Q223R 0.574 D387R 1.050 V880R 1.458 Y1046R 0.708
D224R 0.301 I388R 0.317 A881R 0.236 H1047R 1.430
M225R 0.099 N389R 0.895 D882R 0.356 A1048R 0.739
Y226R 0.000 N390R 1.066 G883R 1.303 D1049R 0.699
V227R 0.177 I391R 0.685 K884R 1.624 E105OR 0.788
D228R 0.168 E392R 0.996 T885R 0.464 11051R 0.678
F229R 0.190 N393R 0.662 G886R 1.856 A1052R 0.114
S231R 0.284 E394R 0.871 K887R 1.606 A1053R 0.035
V232R 0.559 E395R 1.144 A888R 2.077 V1054R 0.122
V233R 1.253 E396R 1.214 Q889R 0.720 N1055R 0.108
D234R 0.217 K397R 0.918 N890R 0.151 I1056R 0.078
E235R 1.727 Q398R 1.043 A891R 2.265 A1057R 0.285
N236R 1.242 N399R 1.050 D892R 1.417 M1058R 0.354
G237R 0.470 V400R 1.222 M894R 1.386 Y1059R 0.762
N238R 0.069 I401R 0.754 D895R 0.539 E106OR 0.623
1(239R 0.988 N402R 0.934 W896R 0.265 V1061R 0.947
1(240R 0.908 D403R 1.712 C897R 0.873 C1062R 0.699
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S241R 1.828 C404R 0.689 A898R 0.192 C1063R 1.137
P242R 0.167 I405R 0.048 A900R 1.324 Q1064R 0.948
N243R 3.606 E406R 1.758 L901R 0.376 T1065R 0.781
G244R 0.060 Q407R 1.735 A902R 0.621 G1066R 0.906
S245R 1.293 Y408R 0.064 K903R 1.115 A1067R 0.994
M246R 0.124 V409R 1.004 K904R 1.106 F1068R 0.010
P247R 0.240 D41OR 0.771 V905R 0.203 G1069R 1.067
I248R 0.962 D411R 1.447 N906R 1.606 K107OR 0.969
V249R 0.114 C412R 1.852 D907R 0.238 K1071R 0.833
T250R 0.140 L415R 0.650 G908R 0.244 Q1072R 0.879
1(251R 1.434 N416R 1.541 C909R 0.499 K1073R 0.464
F252R 0.009 N418R 1.292 V91OR 1.406 K1074R 0.286
E253R 0.321 P419R 0.171 A911R 0.222 S1075R 0.971
T254R 0.927 I420R 0.058 M912R 1.106 D1076R 0.777
D255R 1.182 A421R 0.910 S913R 1.471 E1077R 0.709
D256R 0.595 A422R 0.674 I914R 1.000 L1078R 0.915
L257R 1.162 L423R 0.092 C915R 1.663 P1079R 0.860
I258R 0.044 L424R 0.013 Y916R 1.356 G108OR 0.996
S259R 0.531 K425R 0.745 A918R 1.882 WT 1.000
D260R 0.293 H426R 0.742 P920R 0.831 NT 0.0084
N261R 0.484 I427R 0.005 A921R 0.338
Q262R 0.498 Y922R 0.446
Based on the fluorescence intensity of cells with activated EGFP, it was
observed that 192 xCas12i mutants
showed an increased dsDNA cleavage activity relative to wild type (WT) xCas12i
(SEQ ID NO: 1) (FIG. 5A,
Table 6), and among them, one mutant, xCas12i-N243R, referred to as Cas12Max,
showed about 3.6-fold
improvement (FIG. 5A). In addition, 51 xCas12i mutants has no more than 5%
dsDNA cleavage activity relative
to WT xCas12i (SEQ ID NO: 1).
The applicant then performed saturation mutagenesis of N243 and observed that
the mutation to R indeed showed
the highest dsDNA cleavage activity (FIG. 6A).
The applicant next targeted DMD or Ttr sites using the fluorescent reporter
system (replacing the insertion
sequence (SEQ ID NO: 42) with an insertion sequence containing DMD or Ttr
protospacer and corresponding 5'
PAM as listed in Table 5) and observed that Cas12Max displayed a markedly
increased frequency of EGFP
activation, relative to WT xCas12i (FIG. 1C, FIG. 6B-C).
To further test the efficacy of Cas12Max in targeting genomic loci, the
applicant designed a total of eight gRNAs
to target sites TER and PCSK9 in HEK293T cells and three more to target Ttr in
N2a cells (Table 5), and DR-T2
(SEQ ID NO: 502) was used. Consistent with the previous results, Cas12Max
exhibited a significantly increased
frequency of indels compared to WT xCas12i (FIG. 1D).
Example 7 Further development of mutants based on Cas12Max and evaluation of
their off-target
dsDNA cleavage activity
To examine the specificity of Cas12Max, the applicant transfected a construct
designed to express it with a gRNA
targeting TTRI2 (with TTR-targeting (on-target) spacer sequence of SEQ ID NO:
130), and performed indel
frequency analysis of on- and off-target (OT) sites predicted by Cas-
OFFinder17.
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Table 7
Off-target protospacer sequence (with 5' PAM of TTTG) SEQ ID NO:
TTR off-target.3 (0T.3) CAGCAGGCTTCTACAAAGTGGA 127
TTR off-target.2 (0T.2) TAAAAGGGATATACAATATGTA 128
TTR off-target.1 (0T.1) TAGAAGGGATATAGAAAGTATC 129
On-target protospacer / spacer sequence (with 5' PAM of
TTTG)
TTR on-target.1 (ON.1) TAGAAGGGATATACAAAGTGGA 130
A dual plasmid fluorescent reporter system for evaluation of off-target dsDNA
cleavage activity (off-target
reporter system; referring to "Off-Target Reporter" in FIG. 1B) was
established, which is similar to the dual
plasmid fluorescent reporter system in Example 5 for evaluation of dsDNA
cleavage activity, except that the
insertion sequence of the EGxxxxFP coding sequence contains an TTR off-target
protospacer sequence (SEQ ID
NOs: 127-129) containing one or more mismatches (bold, underlined) with the
TTR-targeting spacer sequence
(SEQ ID NO: 130), rather than containing the TTR protospacer sequence (also
SEQ ID NO: 130), and DR-Ti
sequence (SEQ ID NO: 501) was used.
Using the off-target reporter system (FIG. 7A) or targeted deep sequence
analysis on endogenous gene (FIG. 7B),
the applicant observed that Cas12Max efficiently edited the target site
("ON.1"), while resulting in indel
formation at 2 ("OT.1" and "OT.2") of 3 predicted off-target sites ("OT.1",
"OT.2", and "OT.3"), indicating
off-target dsDNA cleavage activity.
To eliminate the off-target activity of Cas12Max, the applicant selected those
mutants in Example 5 with a single
mutation in the REC and RuvC domains18 and undiminished on-target cleavage
activity (comparable to WT), and
then tested their off-target dsDNA cleavage activity by using two off-target
reporter systems above with TTR OT1
and 0T2, respectively (FIG. 1B).
It was observed that four xCas12i mutants (xCas12i-V880R (v4.1), xCas12i-M923R
(v4.2), xCas12i-D892R
(v4.3), and xCas12i-G883R (v4.4)) maintained a high level of on-target dsDNA
cleavage activity and showed
substantially no off-target dsDNA cleavage activity at both TTR OT1 and 0T2
(FIG. 8A).
The applicant further combined one or more of these four amino acid
substitutions with N243R or N243R+E336R
(FIG. 8B) and it was observed that the variant v6.3 (N243R+E336R+D892R) showed
the lowest off-target EGFP
activation at OT.1 and OT.2 sites and high on-target at the ON.1 site (FIG. 8B-
C). Targeted deep sequencing
analysis of endogenous TTR.2 site and its off-target sites in HEI(293T showed
that v6.3 (N243R+E336R+D892R)
significantly reduced off-target indel frequencies at six OT sites and
retained on-target at ON site, compared to
Cas12Max (FIG. 1E). In addition, relative to Cas12Max (v1.1), v6.3
(N243R+E336R+D892R) retained
comparable or even higher on-target activity at DMD.1, DMD.2 and DMD.3 sites
(HG. 8D). Therefore, the
applicant named v6.3 as high-fidelity Cas12Max (hfCas12Max).
Table 8
Mutant Version ON OT-1 OT-2 OT-3
N243R v1.1 73.80 60.17 47.50 0.11
N243R+V880R v5.1 71.60 3.82 0.24 0.15
N243R+M923R v5.2 76.10 4.90 0.92 0.15
N243R+D892R v5.3 75.85 6.66 5.46 0.21
N243R+G883R v5.4 77.30 16.80 1.36 0.15
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N243R+E336R+V880R v6.1 75.70 2.04 0.44 0.15
N243R+E336R+M923R v6.2 75.57 2.41 2.90 0.05
N243R+E336R+D892R v6.3 77.73 1.55 0.25 0.13
(hfCas12Max)
N243R+E336R+G883R v6.4 74.75 6.65 0.64 0.03
N243R+E336R+D892A v6.7 77.30 54.80 51.50
N243R+E336R+G883A v6.8 78.50 44.00 36.40
NT 0.028 0.048 0.067 0.014
Additionally, to investigate hfCas12Max's PAM preference, the applicant
performed a 5'-NNN PAM recognition
assay by designing reporter plasmids with the same target sequence but
different PAM, similar to Example 3.
Besides showing a consistent or higher cleavage activity at sites with a 5'-
TTN PAM, hfCas12Max and
Cas12Max showed a similarly high cleavage activity for targets with TNN, ATN,
GTN and CTN PAM sites,
compared with the commonly used Cas127' 19 (LbCas12a, Ultra-AsCas12a) and
recently reported improved
Cas12i22 ' 21 (ABRO01, Cas12i2HIFI) (FIG. 1F). Taken together, these results
demonstrate that hfCas12Max
exhibits high-efficiency editing activity with highly flexible 5'-TN or 5'-TNN
PAM recognition.
Example 8 Verification and comparison of hfCas12Max's on- and off-target dsDNA
cleavage activity at
TTR gene
To comprehensively evaluate the performance of hfCas12Max in human cells, the
applicant designed large
number of target sites in the exons of TTR for various Cas nucleases. DR-T2
(SEQ ID NO: 502) was used in this
and subsequent Example unless otherwise specified.
In total, editing activity was monitored at 43 sites for hfCas12Max with TTN
PAMs, 43 sites for ABROO1
(engineered Cas12i2 from Prof. ZHANG Feng) with TTN PAMs, 43 sites for
Cas12i2HIFI (Prof. LI Wei) with TTN
PAMs, 45 sites for SpCas9 with NGG PAMs, 12 sites for LbCas12a with TTTN PAMs,
12 sites for Ultra
AsCas12a with TTTN PAMs, and 20 sites for KKH-saCas9 with NNNRRT PAMs (Table
9). Indel analysis
showed that hfCas12Max exhibited an average on-target dsDNA cleavage activity
of 70%, which is higher than
other Cas nucleases and Cas12Max (FIG. 1G, FIG. 9).
Table 9. Sequence of target loci for indel frequency (FIG. 1G, FIG. 9)
Genomic Cas SITE 573'PAM Protospacer / Spacer Sequence
SEQ ID
loci
NO of
protospacer
/
spacer
sequence
TTR LbCas12a TTTN.1 TTTG TGTCTGAGGCTGGCCCTACGGTG 131
TTTN.2 TTTG ACCATCAGAGGACACTTGGATTC 132
TTTN.3 TTTC TGAACACATGCACGGCCACATTG 133
TTTN.4 TTTG CCTCTGGGTAAGTTGCCAAAGAA 134
TTTN.5 TTTG GCAACTTACCCAGAGGCAAATGG 135
TTTN.6 TTTC ACACCTTATAGGAAAACCAGTGA 136
TTTN.7 TTTC CTATAAGGTGTGAAAGTCTGGAT 137
TTTN.8 TTTT CCTATAAGGTGTGAAAGTCTGGA 138
TTTN.9 TTTG TAGAAGGGATATACAAAGTGGAA 139
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TTTN.10 TTTG TATATCCCTTCTACAAATTCCTC 140
TTTN.11 TTTC CACTTTGTATATCCCTTCTACAA 141
TTTN.12 TTTG GTGTCTATTTCCACTTTGTATAT 142
UltraCas12a TTTN.1 TTTG
TGTCTGAGGCTGGCCCTACGGTG 143
TTTN.2 TTTG
ACCATCAGAGGACACTTGGATTC 144
TTTN.3 TTTC
TGAACACATGCACGGCCACATTG 145
TTTN.4 TTTG
CCTCTGGGTAAGTTGCCAAAGAA 146
TTTN.5 TTTG
GCAACTTACCCAGAGGCAAATGG 147
TTTN.6 TTTC
ACACCTTATAGGAAAACCAGTGA 148
TTTN.7 TTTC
CTATAAGGTGTGAAAGTCTGGAT 149
TTTN.8 TTTT
CCTATAAGGTGTGAAAGTCTGGA 150
TTTN.9 TTTG
TAGAAGGGATATACAAAGTGGAA 151
TTTN.10 TTTG TATATCCCTTCTACAAATTCCTC 152
TTTN.11 TTTC CACTTTGTATATCCCTTCTACAA 153
TTTN.12 TTTG GTGTCTATTTCCACTTTGTATAT 154
KKH-S aC as9 NNGRRT.1 ACAGAT CCACCTATGAGAGAAGACAG 155
NNGRRT.2 AGGAAT GGCTGTCGTCACCAATCCCA 156
NNGRRT.3 AGGAGT GACGACAGCCGTGGTGGAAT 157
NNGRRT.4 ATTGAT CTGAACAC ATGCACGGCC AC 158
NNGRRT.5 CCAAGT CACCCAGGGCACCGGTGAAT 159
NNGRRT.6 CGGAGT AATGGTGTAGCGGCGGGGGC 160
NNGRRT.7 GCAAAT CTTTGGCAACTTACCCAGAG 161
NNGRRT.8 GTGAGT TGTCTGAGGCTGGCCCTACG 162
NNGRRT.9 TACGGT TTTGTGTCTGAGGCTGGCCC 163
NNGRRT.10 TGGAAT ATTGGTGACGACAGCCGTGG 164
NNGRRT.11 TGGGAT AGGAGAAGTC CCTCATTC CT 165
NNGRRT.12 TTTGGT CCAAGTGCCTTCCAGTAAGA 166
SpCas9 NGG.1 AGG ACACAAATACCAGTCCAGCA 167
NGG.2 AGG CCAGTCCAGCAAGGCAGAGG 168
NGG.3 AGG GAAGTCCACTCATTCTTGGC 169
NGG.4 AGG AAAGTTCTAGATGCTGTCCG 170
NGG.5 AGG CCCAGAGGCAAATGGCTCCC 171
NGG.6 AGG TTCTTTGGCAACTTACCCAG 172
NGG.7 AGG ACTGAGGAGGAATTTGTAGA 173
NGG.8 AGG CCCATTCCATGAGCATGCAG 174
NGG.9 AGG GCATGGGCTCACAACTGAGG 175
NGG.10 AGG AATAGGAGTAGGGGCTCAGC 176
NGG.11 AGG GACGACAGCCGTGGTGGAAT 177
NGG.12 AGG GGCTGTCGTCACCAATCCCA 178
NGG.13 AGG GTCACCAATCCCAAGGAATG 179
NGG.14 CGG TGTGTC TGAGGCTGGCCC TA 180
NGG.15 CGG AGCCTTTCTGAACACATGCA 181
NGG.16 CGG CAGAGGACACTTGGATTCAC 182
NGG.17 CGG CATTGATGGCAGGACTGCCT 183
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NGG.18 CGG CTTCTC TAC ACC CAGGGCAC 184
NGG.19 CGG AATGGTGTAGCGGCGGGGGC 185
NGG.20 CGG CCCCTACTCCTATTCCACCA 186
NGG.21 CGG GCAGGGCGGCAATGGTGTAG 187
NGG.22 CGG GGAGTAGGGGCTCAGCAGGG 188
NGG.23 CGG GTATTCACAGCCAACGACTC 189
NGG.24 GGG TCACAGAAACACTCACCGTA 190
NGG.25 GGG AAAGGCTGCTGATGACACCT 191
NGG.26 GGG CTTGGATTCACCGGTGCC CT 192
NGG.27 GGG GCCGTGGTGGAATAGGAGTA 193
NGG.28 GGG GCGGCAATGGTGTAGCGGCG 194
NGG.29 GGG GGAGAAGTCCCTCATTCCTT 195
NGG.30 GGG GGCGGCAATGGTGTAGCGGC 196
NGG.31 GGG TCACCAATCCCAAGGAATGA 197
NGG.32 TGG GCAACTTACCCAGAGGCAAA 198
NGG.33 TGG AAGTGCCTTCCAGTAAGATT 199
NGG.34 TGG ACCTCTGCATGCTCATGGAA 200
NGG.35 TGG TACTCACCTCTGCATGCTCA 201
NGG.36 TGG TGTAGAAGGGATATACAAAG 202
NGG.37 TGG AGGAGAAGTC CCTCATTC CT 203
NGG.38 TGG ATTGGTGACGACAGCCGTGG 204
NGG.39 TGG GCGGCGGGGGCCGGAGTC GT 205
NGG.40 TGG GGGATTGGTGACGACAGCCG 206
NGG.41 TGG GGGGCTCAGCAGGGCGGCAA 207
Cas12Max TTTN.1 TTTG
TGTCTGAGGCTGGCCCTACGGTG 208
TTTN.2 TTTG
ACCATCAGAGGACACTTGGATTC 209
TTTN.3 TTTC
TGAACACATGCACGGCCACATTG 210
TTTN.4 TTTG
CCTCTGGGTAAGTTGCCAAAGAA 211
TTTN.5 TTTG
GCAACTTACCCAGAGGCAAATGG 212
TTTN.6 TTTC
ACACCTTATAGGAAAACCAGTGA 213
TTTN.7 TTTC
CTATAAGGTGTGAAAGTCTGGAT 214
TTTN.8 TTTT
CCTATAAGGTGTGAAAGTCTGGA 215
TTTN.9 TTTG
TAGAAGGGATATACAAAGTGGAA 216
TTTN.10 TTTG TATATCCCTTCTACAAATTCCTC 217
TTTN.11 TTTC CACTTTGTATATCCCTTCTACAA 218
TTTN.12 TTTG GTGTCTATTTCCACTTTGTATAT 219
VTTN.1 CTTA CTGGAAGGCACTTGGCATCT 220
VTTN.2 CTTA TAGGAAAACCAGTGAGTCTG 221
VTTN.3 CTTC TCATCGTCTGCTCCTCCTCT 222
VTTN.4 ATTC TTGGCAGGATGGCTTCTC AT 223
VTTN.5 ATTC ACCGGTGCCCTGGGTGTAGA 224
VTTN.6 GTTC AGAAAGGCTGCTGATGACAC 225
VTTN.7 GTTC TAGATGCTGTCCGAGGCAGT 226
VTTN.8 CTTC TCTACACCCAGGGCACCGGT 227
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VTTN.9 GTTC TTTGGCAACTTACCCAGAGG 228
VTTN.10 CTTC CAGTAAGATTTGGTGTCTAT 229
VTTN.11 ATTC CATGAGCATGCAGAGGTGAG 230
VTTN.12 ATTC CTCCTCAGTTGTGAGCCCAT 231
VTTN.13 CTTC TACAAATTCCTCCTCAGTTG 232
VTTN.14 ATTC ACAGCCAACGACTCCGGCCC 233
VTTN.15 ATTC CACCACGGCTGTCGTCACCA 234
VTTN.16 ATTC CTTGGGATTGGTGACGAC AG 235
VTTN.17 CTTC TCTCATAGGTGGTATTCAC A 236
VTTN.18 CTTG CTGGACTGGTATTTGTGTCT 237
VTTN.19 CTTG GCAGGATGGCTTCTCATCGT 238
VTTN.20 ATTG ATGGCAGGACTGCCTCGGAC 239
VTTN.21 CTTG GATTCACCGGTGCCCTGGGT 240
VTTN.22 CTTG GCATCTCCCCATTCCATGAG 241
VTTN.23 GTTG TGAGCCCATGCAGCTCTCCA 242
VTTN.24 ATTG CCGCCCTGCTGAGCCCCTAC 243
VTTN.25 GTTG GCTGTGAATACCACCTATGA 244
VTTN.26 CTTG GGATTGGTGACGACAGCCGT 245
VTTN.27 ATTG GTGACGACAGCCGTGGTGGA 246
VTTN.28 ATTT GTGTCTGAGGCTGGCCCTAC 247
VTTN.29 CTTT GACCATCAGAGGACACTTGG 248
VTTN.30 ATTT GCCTCTGGGTAAGTTGCC AA 249
VTTN.31 CTTT GGCAACTTACCCAGAGGCAA 250
VTTN.32 ATTT GGTGTCTATTTCCACTTTGT 251
VTTN.33 CTTT GTATATCCCTTCTACAAATT 252
hfCas12Max TTTN.1 TTTG
TGTCTGAGGCTGGCCCTACGGTG 253
TTTN.2 TTTG
ACCATCAGAGGACACTTGGATTC 254
TTTN.3 TTTC
TGAACACATGCACGGCCACATTG 255
TTTN.4 TTTG
CCTCTGGGTAAGTTGCCAAAGAA 256
TTTN.6 TTTC
ACACCTTATAGGAAAACCAGTGA 257
TTTN.7 TTTC
CTATAAGGTGTGAAAGTCTGGAT 258
TTTN.8 TTTT
CCTATAAGGTGTGAAAGTCTGGA 259
TTTN.9 TTTG
TAGAAGGGATATACAAAGTGGAA 260
TTTN.10 TTTG TATATCCCTTCTACAAATTCCTC 261
TTTN.11 TTTC CACTTTGTATATCCCTTCTACAA 262
TTTN.12 TTTG GTGTCTATTTCCACTTTGTATAT 263
VTTN.1 CTTA CTGGAAGGCACTTGGCATCT 264
VTTN.2 CTTA TAGGAAAACCAGTGAGTCTG 265
VTTN.3 CTTC TCATCGTCTGCTCCTCCTCT 266
VTTN.4 ATTC TTGGCAGGATGGCTTCTC AT 267
VTTN.5 ATTC ACCGGTGCCCTGGGTGTAGA 268
VTTN.6 GTTC AGAAAGGCTGCTGATGACAC 269
VTTN.7 GTTC TAGATGCTGTCCGAGGCAGT 270
VTTN.9 GTTC TTTGGCAACTTACCCAGAGG 271
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VTTN.10 CTTC CAGTAAGATTTGGTGTCTAT 272
VTTN.11 ATTC CATGAGCATGCAGAGGTGAG 273
VTTN.12 ATTC CTCCTCAGTTGTGAGCCCAT 274
VTTN.13 CTTC TACAAATTCCTCCTCAGTTG 275
VTTN.14 ATTC ACAGCCAACGACTCCGGCCC 276
VTTN.15 ATTC CACCACGGCTGTCGTCACCA 277
VTTN.16 ATTC CTTGGGATTGGTGACGAC AG 278
VTTN.17 CTTC TCTCATAGGTGGTATTCAC A 279
VTTN.18 CTTG CTGGACTGGTATTTGTGTCT 280
VTTN.19 CTTG GCAGGATGGCTTCTCATCGT 281
VTTN.20 ATTG ATGGCAGGACTGCCTCGGAC 282
VTTN.21 CTTG GATTCACCGGTGCCCTGGGT 283
VTTN.22 CTTG GCATCTCCCCATTCCATGAG 284
VTTN.23 GTTG TGAGCCCATGCAGCTCTCCA 285
VTTN.24 ATTG CCGCCCTGCTGAGCCCCTAC 286
VTTN.25 GTTG GCTGTGAATACCACCTATGA 287
VTTN.26 CTTG GGATTGGTGACGACAGCCGT 288
VTTN.27 ATTG GTGACGACAGCCGTGGTGGA 289
VTTN.28 ATTT GTGTCTGAGGCTGGCCCTAC 290
VTTN.29 CTTT GACCATCAGAGGACACTTGG 291
VTTN.30 ATTT GCCTCTGGGTAAGTTGCC AA 292
VTTN.31 CTTT GGCAACTTACCCAGAGGCAA 293
VTTN.32 ATTT GGTGTCTATTTCCACTTTGT 294
VTTN.33 CTTT GTATATCCCTTCTACAAATT 295
ABROO1 TTTN.1 TTTG
TGTCTGAGGCTGGCCCTACGGTG 296
TTTN.2 TTTG
ACCATCAGAGGACACTTGGATTC 297
TTTN.3 TTTC
TGAACACATGCACGGCCACATTG 298
TTTN.4 TTTG
CCTCTGGGTAAGTTGCCAAAGAA 299
TTTN.6 TTTC
ACACCTTATAGGAAAACCAGTGA 300
TTTN.7 TTTC
CTATAAGGTGTGAAAGTCTGGAT 301
TTTN.8 TTTT
CCTATAAGGTGTGAAAGTCTGGA 302
TTTN.9 TTTG
TAGAAGGGATATACAAAGTGGAA 303
TTTN.10 TTTG TATATCCCTTCTACAAATTCCTC 304
TTTN.11 TTTC CACTTTGTATATCCCTTCTACAA 305
TTTN.12 TTTG GTGTCTATTTCCACTTTGTATAT 306
VTTN.1 CTTA CTGGAAGGCACTTGGCATCT 307
VTTN.2 CTTA TAGGAAAACCAGTGAGTCTG 308
VTTN.3 CTTC TCATCGTCTGCTCCTCCTCT 309
VTTN.4 ATTC TTGGCAGGATGGCTTCTC AT 310
VTTN.5 ATTC ACCGGTGCCCTGGGTGTAGA 311
VTTN.6 GTTC AGAAAGGCTGCTGATGACAC 312
VTTN.7 GTTC TAGATGCTGTCCGAGGCAGT 313
VTTN.9 GTTC TTTGGCAACTTACCCAGAGG 314
VTTN.10 CTTC CAGTAAGATTTGGTGTCTAT 315
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VTTN.11 ATTC CATGAGCATGCAGAGGTGAG 316
VTTN.12 ATTC CTCCTCAGTTGTGAGCCCAT 317
VTTN.13 CTTC TACAAATTCCTCCTCAGTTG 318
VTTN.14 ATTC ACAGCCAACGACTCCGGCCC 319
VTTN.15 ATTC CACCACGGCTGTCGTCACCA 320
VTTN.16 ATTC CTTGGGATTGGTGACGAC AG 321
VTTN.17 CTTC TCTCATAGGTGGTATTCAC A 322
VTTN.18 CTTG CTGGACTGGTATTTGTGTCT 323
VTTN.19 CTTG GCAGGATGGCTTCTCATCGT 324
VTTN.20 ATTG ATGGCAGGACTGCCTCGGAC 325
VTTN.21 CTTG GATTCACCGGTGCCCTGGGT 326
VTTN.22 CTTG GCATCTCCCCATTCCATGAG 327
VTTN.23 GTTG TGAGCCCATGCAGCTCTCCA 328
VTTN.24 ATTG CCGCCCTGCTGAGCCCCTAC 329
VTTN.25 GTTG GCTGTGAATACCACCTATGA 330
VTTN.26 CTTG GGATTGGTGACGACAGCCGT 331
VTTN.27 ATTG GTGACGACAGCCGTGGTGGA 332
VTTN.28 ATTT GTGTCTGAGGCTGGCCCTAC 333
VTTN.29 CTTT GACCATCAGAGGACACTTGG 334
VTTN.30 ATTT GCCTCTGGGTAAGTTGCC AA 335
VTTN.31 CTTT GGCAACTTACCCAGAGGCAA 336
VTTN.32 ATTT GGTGTCTATTTCCACTTTGT 337
VTTN.33 CTTT GTATATCCCTTCTACAAATT 338
Cas12i2H1F1 TTTN.1 TTTG
TGTCTGAGGCTGGCCCTACGGTG 339
TTTN.2 TTTG
ACCATCAGAGGACACTTGGATTC 340
TTTN.3 TTTC
TGAACACATGCACGGCCACATTG 341
TTTN.4 TTTG
CCTCTGGGTAAGTTGCCAAAGAA 342
TTTN.6 TTTC
ACACCTTATAGGAAAACCAGTGA 343
TTTN.7 TTTC
CTATAAGGTGTGAAAGTCTGGAT 344
TTTN.8 TTTT
CCTATAAGGTGTGAAAGTCTGGA 345
TTTN.9 TTTG
TAGAAGGGATATACAAAGTGGAA 346
TTTN.10 TTTG TATATCCCTTCTACAAATTCCTC 347
TTTN.11 TTTC CACTTTGTATATCCCTTCTACAA 348
TTTN.12 TTTG GTGTCTATTTCCACTTTGTATAT 349
VTTN.1 CTTA CTGGAAGGCACTTGGCATCT 350
VTTN.2 CTTA TAGGAAAACCAGTGAGTCTG 351
VTTN.3 CTTC TCATCGTCTGCTCCTCCTCT 352
VTTN.4 ATTC TTGGCAGGATGGCTTCTC AT 353
VTTN.5 ATTC ACCGGTGCCCTGGGTGTAGA 354
VTTN.6 GTTC AGAAAGGCTGCTGATGACAC 355
VTTN.7 GTTC TAGATGCTGTCCGAGGCAGT 356
VTTN.9 GTTC TTTGGCAACTTACCCAGAGG 357
VTTN.10 CTTC CAGTAAGATTTGGTGTCTAT 358
VTTN.11 ATTC CATGAGCATGCAGAGGTGAG 359
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VTTN.12 ATTC CTCCTCAGTTGTGAGCCCAT 360
VTTN.13 CTTC TACAAATTCCTCCTCAGTTG 361
VTTN.14 ATTC ACAGCCAACGACTCCGGCCC 362
VTTN.15 ATTC CACCACGGCTGTCGTCACCA 363
VTTN.16 ATTC CTTGGGATTGGTGACGACAG 364
VTTN.17 CTTC TCTCATAGGTGGTATTCACA 365
VTTN.18 CTTG CTGGACTGGTATTTGTGTCT 366
VTTN.19 CTTG GCAGGATGGCTTCTCATCGT 367
VTTN.20 ATTG ATGGCAGGACTGCCTCGGAC 368
VTTN.21 CTTG GATTCACCGGTGCCCTGGGT 369
VTTN.22 CTTG GCATCTCCCCATTCCATGAG 370
VTTN.23 GTTG TGAGCCCATGCAGCTCTCCA 371
VTTN.24 ATTG CCGCCCTGCTGAGCCCCTAC 372
VTTN.25 GTTG GCTGTGAATACCACCTATGA 373
VTTN.26 CTTG GGATTGGTGACGACAGCCGT 374
VTTN.27 ATTG GTGACGACAGCCGTGGTGGA 375
VTTN.28 ATTT GTGTCTGAGGCTGGCCCTAC 376
VTTN.29 CTTT GACCATCAGAGGACACTTGG 377
VTTN.30 ATTT GCCTCTGGGTAAGTTGCCAA 378
VTTN.31 CTTT GGCAACTTACCCAGAGGCAA 379
VTTN.32 ATTT GGTGTCTATTTCCACTTTGT 380
VTTN.33 CTTT GTATATCCCTTCTACAAATT 381
To further evaluate the specificity of hfCas12Max on endogenous genes in human
cells, the applicant determined
indel frequencies of P2RX5 and NLRC4 on-target and their corresponding in
silico predicted off-target sites22.
Targeted deep sequence analysis showed that hfCas12Max had a higher on-target
editing efficiency and similarly
almost no indel activity at potential off target sites, compared to Ultra
AsCas12a and LbCas12a (FIG. 10A-B;
protospacer / spacer sequence of SEQ ID NOs: 382-390 from upside to downside
in FIG. 10A; protospacer /
spacer sequence of SEQ ID NOs: 391-397 from upside to downside in FIG. 10B).
To sufficiently detect off-target of hfCas12Max and to compare to other Cas
proteins, the applicant used
PEM-5eq23 to quantify germline events (uncut or perfect rejoining) and editing
events including indels and
translocations events of TTR.2 libraries. Overall, these results demonstrate
that hfCas12Max has high efficiency
and specificity and is superior to SpCas9 and other Cas12 nucleases.
Example 9 Development and evaluation of base editor based on dead xCas12i
The applicant further explored the base editing of xCas12i by generating a
nuclease-deactivated xCas12i (dead
xCas12i, dxCas12i). This was done by first introducing single mutations
(D650A, D700A, E875A, or D1049A) in
the conserved active site of xCas12i based on alignment to Cas12i18 and
Cas12i21 (FIG. 12A-B).
Then, dxCas12i-D1049A was C-terminally fused to TadA8evi 6w (SEQ ID NO: 439,
TadA8e.1) via a GS linker
containing a XTEN linker (SEQ ID NO: 442) or a GS linker containing a BP NLS
(SEQ ID NO: 443) to form an
adenine base editor TadA8e.1-dxCas12i, and dxCas12i-D1049A was C-terminally
fused to human
APOBEC3Aw1114A (SEQ ID NO: 440, hA3A.1) via a GS linker containing a XTEN
linker (SEQ ID NO: 442) or a
GS linker containing a BP NLS (SEQ ID NO: 443), and one UGI (SEQ ID NO: 441),
to form a cytidine base
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editor hA3A.1-dxCas12i24 26 (FIG. 1H and 1J). For the adenine base editor, it
contained a N-terminal SV40 NLS
(SEQ ID NO: 444) and a C-terminal BP NLS (SEQ ID NO: 443). For the cytidine
base editor, it contained a
N-terminal BP NLS (SEQ ID NO: 443) and a C-terminal BP NLS (SEQ ID NO: 443).
TadA8evi 6w, SEQ ID NO: 439
SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLV
MQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILA
DECAALLCDFYRMPRQVFNAQKKAQSSIN
hAPOBEC3w1a4A, SEQ ID NO: 440
MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYG
RHAELRFLDLVPSLQLDPAQIYRVTWFISYSPCFSAGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEAL
QMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN
UGI, SEQ ID NO: 441
TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQD
SNGENKIKML
XTEN linker, SEQ ID NO: 442
SGSETPGTSESATPES
bpNLS (also known as BP NLS or bpSV40 NLS), (doi: 10.1038/nature20565.), SEQ
ID NO: 443
KRTADGSEFESPKKKRKV
5V40 NLS, from Betapolyomavirus macacae, SEQ ID NO: 444
PKKKRKV
NP NLS (also known as Xenopus laevis Nucleoplasmin NLS or nucleoplasmin NLS),
(doi:
10.1126/science.abj6856.), also a bipartite NLS, SEQ ID NO: 445
KRPAATKKAGQAKKKK
human U6 promoter, 241 bp, SEQ ID NO: 446
gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttga
ctgtaaacacaaagatattagtacaaaatacgtg
acgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgta
acttgaaagtatttcgatttcttggctttatatatcttgt
ggaaaggac
human CMV promoter, 204 bp, SEQ ID NO: 447
gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattg
acgtcaatgggagtttgttttggcaccaaaat
caacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtct
atataagcagagct
bGH polyA signal, 208 bp, SEQ ID NO: 448
ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccac
tgtcctttcctaataaaatgaggaaattgcatcg
cattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaata
gcaggcatgctgggga
T5 EXO, SEQ ID NO: 449
MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKG
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KSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLI
GHLYDHVWLISTDGDWDTLLTDKVSRFSETTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGV
EGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKF
TKDILEIAEQ
CAG promoter (human CMV enhancer+ chicken 13-actin promoter) (containing a
hybrid intron), SEQ ID NO: 500
cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgcca
atagggactttccattgacgtcaatgggt
ggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaat
gacggtaaatggcccgcctggcattGtgcc
cagtacatgaccttatgggacMcctacttggcagtacatctacgtattagtcatcgctattaccatggtegaggtgagc
cccacgttctgettcactctecccatctecce
cccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggggggggggggggg
cgcgcgccaggcggggcggggcggggcg
aggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggc
gaggcggcggcggcggcggccc
tataaaaagcgaagcgcgcggcgggcgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcg
cgccgcccgccccggctctgactg
accgcgttacteccacaggtgagegggegggacggccettctectccgggctgtaattagetgagcaagaggtaagggt
ttaagggatggttggttggtggggtatta
atgtttaattacctggagcacctgcctgaaatcactttttttcag
The initial versions of TadA8e.1-dxCas12i and hA3A.1-dxCas12i showed low base
editing activity with
frequencies of 8% A-to-G and 2% C-to-T, respectively (FIG. 11, 1K). To address
this, the applicant introduced
single and combined mutations for high cleavage activity into the PI and Rec
domains of dxCas12i, which
resulted in significantly increased A-to-G editing activity (FIG. 13A). Among
the improved variants,
TadA8e.1-dxCas12i-v2.2 (N243R+E336R) achieved 50% activity at A9 and All sites
of the KLF4 locus,
markedly higher than the 30% activity of TadA8e.1-dLbCas12a (FIG. 11, FIG. 13B-
C). At target sites within
PCSK9 and TTR, TadA8e.1-dxCas12i-v2.2 showed a similarly increased efficiency
to mediate A-to-G transitions,
and higher than TadA8e.1-dLbCas12a at PCSK9 site (FIG. 15). To test whether
the orientation of deaminase
fusion affects the base editing efficiency, the applicant constructed dxCas12i-
ABE by fusing the TadA8e.1 to N or
C terminus of dxCas12i, and found that TadA8e.1 at C terminus of dxCas12i
showed slightly higher activity than
N terminus (FIG. 14). The applicant then further engineered the NLS, linker,
and TadA8e.1 protein (return back to
TadA8e) (FIG. 13A) to produce v3.1-v3.8 and v4.1-v4.4, where TadA8e-dxCas12i-
v4.3 exhibited a nearly 80%
A-to-G editing efficiency and >95% editing purity, while the editing
activities of other dxCas12i-ABE versions
were unchanged (FIG. 1H-I, FIG. 13D-E). The applicant named TadA8e-dxCas12i-
v4.3 as dCas12Max-ABE.
To further characterize the base editing activity of dCas12Max-ABE, the
applicant performed 21 sites with TTN
PAM, 13 sites with ATN PAMs and 13 sites with CTN PAMs (Table 10). It was
observed that dCas12Max-ABE
exhibited significant A-to-G activity at sites with TTN PAM (FIG. 16).
In addition, hA3A.1-dxCas12i-v1.2 (N243R), hA3A.1-dxCas12i-v2.2 (N243R+E336R),
and
hA3A.1-dxCas12i-v4.3 (N243R+E336R-bpNLS) showed consistently elevated C-to-T
editing efficiency along
with >95% editing purity, at C7 and C10 sites of RUNX1, DYRK1A, and SITE4
locus, even higher than
hA3A.1-dLbCas12a at RUNX1 and DYRK1A (FIG. 1J-K).
These results together demonstrate that engineered dxCas12i-based editors
exhibit the high base editing activity in
mammalian cells.
Table 10. Sequence of target loci for A to G frequency at different sites
(FIG. 16)
Genomic loci ABE SITE 573'PAM Protospacer /Spacer Sequence SEQ
ID NO
of
Protospacer
/Spacer
Sequence
TTR TTN sitel CTTC AGCACCACCACGTAGGTGCC 398
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site2 CTTC CTGGTGAAGATGAGTGGC GA 399
site3 CTTG AAGTTGCCCC ATGTCGAC TA 400
site4 GTTG CCCCATGTCGACTACATCGA 401
site5 TTTG CCCAGAGCATCCCGTGGAAC 402
site 6 TTTC CCGGTGGTCACTCTGTATGC 403
site7 GTTG AGCACGCGCAGGCTGCGCAT 404
site8 GTTA GCGGCACCCTCATAGGTGAG 405
site9 GTTG GGGCCACCAATGCCCAGGAC 406
site 10 ATTG GTGGCCCCAACTGTGATGAC 407
site 11 ATTG GTGCCTCCAGCGACTGCAGC 408
site 12 ATTC ACCCCTGCACCAGGCATTGC 409
sitel3 GTTC CCTGAGGACCAGCGGGTACT 410
sitel4 GTTG GTGGCAGTGGACACGGGTCC 411
sitel5 GTTG TCTACGGCGTAGGCCCCCAG 412
ATN sitel AATC CAAGTGTCCTCTGATGGTCA 413
site2 GATG GTCAAAGTTCTAGATGCTGT 414
site3 GATG CTGTCCGAGGCAGTCCTGCC 415
site4 AATG TGGCCGTGCATGTGTTCAGA 416
site5 CATG TGTTCAGAAAGGCTGCTGAT 417
site6 GATG ACACCTGGGAGCCATTTGCC 418
site7 GATT CACCGGTGCCCTGGGTGTAG 419
site8 CATC AGAGGACACTTGGATTCACC 420
site9 CATC TAGAACTTTGACCATCAGAG 421
site 10 GATG GCAGGACTGCCTCGGACAGC 422
site 11 CATT GATGGCAGGACTGCCTCGGA 423
site 12 CATG CACGGCCACATTGATGGC AG 424
site 13 CATC AGCAGCCTTTCTGAACAC AT 425
C TN sitel CCTC TGATGGTCAAAGTTCTAGAT 426
site2 TCTG ATGGTCAAAGTTCTAGATGC 427
site3 GCTG TCCGAGGCAGTCCTGCCATC 428
site4 GCTG ATGACACCTGGGAGCCATTT 429
site5 CCTG GGAGCCATTTGCCTCTGGGT 430
site 6 CCTC TGGGTAAGTTGCCAAAGAAC 431
site7 ACTT GGATTCACCGGTGCCCTGGG 432
site8 ACTT TGACCATCAGAGGACACTTG 433
site9 TC TA GAACTTTGACCATCAGAGGA 434
sitel0 CCTC GGACAGCATCTAGAACTTTG 435
sitell ACTG CCTCGGACAGCATCTAGAAC 436
sitel2 GCTC CCAGGTGTCATCAGCAGC CT 437
site 13 ACTT ACCCAGAGGCAAATGGCTCC 438
Example 10 Evaluation of RNP delivery of hfCas12Max in T cells
To explore the therapeutic potential application of hfCas12Max, the applicant
delivered hfCas12Max RNP
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targeting TRAC in CD3+ T cells19 (FIG. 2A). Beforehand, the applicant tested
hfCas12Max RNP targeting TTR
and TRAC in HEK293 cells, and it was found that gene editing efficiency was
increased following increasing
dose of RNPs, with unaffected cellular viability and proliferation (FIG. 18A-
C). The applicant achieved about 90%
dsDNA cleavage activity and >95% viability at 3.2 i.t1\4 dose for TRAC (FIG.
18A-C) in HEK293 cells. Three
guides were designed to target TRAC (Table 5), and both TRAC sg.2 and sg.3
generated ¨90% editing at both 1.6
and 3.2 I.LM dose along with ¨80% viability (FIG. 2B) in CD3+ T cells. Flow
cytometric analysis showed that
TRAC expression was detected to be reduced to a level of 2-3% in CD3+ T cells
post 5 days post electroporation
treated with RNPs targeting sg.2 or sg.3, compared to 96.6% with untreated
cells (FIG. 2C). The guide RNA
used in this Example was in the configuration of 5' DR-Ti - spacer sequence -
DR-T2 - spacer sequence -3'.
Example 11 Evaluation of LNP delivery of hfCas12Max in vivo
To assess the feasibility of hfCas12Max or its base editor of in vivo gene
editing, the applicant delivered a guide
RNA and a mRNA encoding hfCas12Max by LNP packaging to the liver of C57 mouse
via tail intravenous
injection27 (FIG. 2D). The applicant targeted the exon 3 in the murine
transthyretin (Ttr) gene (Ttr_5g12 in Table 5)
by gene editing (dsDNA cleavage) and base editing (FIG. 2E). Robust editing
efficiencies were detected at four
concentration and nearly 100% at 1 lag dose in N2a cells (FIG. 2F). Similarly,
targeted deep sequence analysis
indicated that the editing efficiencies of murine liver were approximately 70%
at the dose of 0.3 and 0.5
milligrams per kilogram (mpk), equivalent to saturation (FIG. 2G). Further,
through the LNP packaging delivery,
TadA8e-dxCas12i-v4.3 (dCas12Max-ABE) achieved approximately 25% A-to-G
efficiency of Al3 in Ttr locus in
murine liver at 3 mpk dose (FIG.2H). The guide RNA used in this Example was in
the configuration of 5'
DR-Ti - spacer sequence - DR-T2 - spacer sequence -3'.
In addition, the applicant injected hfCas12Max mRNA with two gRNAs (Ttr_5g3
and 12 in Table 5) targeting Ttr
gene into murine zygotes, which were cultured to blastocyst stage for
genotyping analysis (FIG. 19A). Targeted
deep sequence analysis showed that most zygotes were edited and some up to
100% (FIG. 19B). These results
indicate that hfCas12Max mediates robust ex vivo and in vivo gene editing,
showing significant potential for
disease modeling and therapies.
Mis-folding and aggregation of transthyretin (TTR) is associated with amyloid
diseases, including
transthyretin-related wild-type amyloidosis (ATTRwt), transthyretin-related
hereditary amyloidosis (ATTRm),
familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy
(FAC). Gene silencing of TTR to
reduce TTR protein production may have therapeutic effects in TTR-associated
amyloid diseases. The
high-efficiency cleavage of TTR target sites in mice in this example
demonstrates that the SiCas12i-crRNA
system of the present invention has very promising prospects for the treatment
of TTR-related amyloid diseases,
such as ATTR (e.g., ATTRwt or ATTRm).
Example 12: Screening of xCas12i mutant with nickase activity
To screen xCas12i mutant with nickase activity (i.e., having ssDNA cleavage
activity and substantially lacking
dsDNA cleavage activity), xCas12i mutant in Tables 11-14 were designed and
tested for their nickase activity and
dsDNA cleavage activity, by using the reporter system for dsDNA cleavage
activity in Example 1 and a reporter
system for nickase activity established based on the reporter system for dsDNA
cleavage activity in Example 1
wherein the insertion sequence was replaced with an insertion sequence
containing, from 5' to 3', a 5' PAM, a
protospacer sequence (SEQ ID NO: 43), a linker, a target sequence (SEQ ID NO:
44), a reverse complementary
sequence of the 5' PAM.
When the xCas12i mutant has only nickase activity, it does not generate green
fluorescence with the reporter
system for dsDNA cleavage activity but can generate green fluorescence with
the reporter system for nickase
activity. When the xCas12i mutant has dsDNA cleavage activity, it can generate
green fluorescence with both
the reporter systems for nickase activity and dsDNA cleavage activity. So the
reporter system for nickase
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activity indicates the sum of the dsDNA cleavage activity and nickase
activity. The nickase activity is calculated
as green fluorescence from the reporter system for nickase activity minus
green fluorescence from the reporter
system for dsDNA cleavage activity. Nickase preference was calculated as
nickase activity / dsDNA cleavage
activity.
It was observed that xCas12i-W896R, xCas12i-S924R, and xCas12i-S925R exhibited
significant nickase activity
relative to WT xCas12i.
Table 11
Nickase (ssDNA Nickase activity /
cleavage) activity dsDNA cleavage dsDNA cleavage
Mutant (%) activity (%) activity
NT 0.000 0.020 0.000
Blank 0.000 0.020 0.000
SiCas12i -0.300 76.100 -0.004
xCas12i-W896R 30.130 4.970 6.062
xCas12i-S924R 22.300 26.800 0.832
xCas12i-S925R 6.650 5.350 1.243
Further mutagenesis was conducted at W896, S924, or S925 of xCas12i to
generate the mutants in Tables 12-14. It
was observed that eight xCas12i mutants, W896R, W896P, W896K, S924F, S924D,
S924E, S924H, and S925T,
achieved more significant nickase preference (Nickase activity / dsDNA
cleavage activity >1.0) and higher
nickase activity (higher than 20%).
Table 12: xCas12i-W896 mutants
Nickase
(ssDNA Nickase activity /
cleavage) dsDNA cleavage dsDNA cleavage
Mutant activity (%) activity (%) activity
W896G -3.100 72.900 -0.043
W896A 6.500 75.700 0.086
W896V -0.300 64.300 -0.005
W896L 13.900 61.300 0.227
W8961 -0.600 74.700 -0.008
W896M 0.500 76.800 0.007
W896F 5.800 74.100 0.078
W896W -0.400 80.300 -0.005
W896P 32.170 8.030 4.006
W896S 0.000 72.000 0.000
W896T 0.600 67.200 0.009
W896C 2.200 72.800 0.030
W896Y 2.300 67.700 0.034
W896N 0.700 63.700 0.011
W896Q 1.500 69.800 0.021
W896D -1.900 49.200 -0.039
W896E 11.900 58.400 0.204
W896K 37.500 14.700 2.551
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Nickase
(ssDNA Nickase activity /
cleavage) dsDNA cleavage dsDNA cleavage
Mutant activity (%) activity (%) activity
W896H 3.100 68.000 0.046
Table 13: xCas12i-S924 mutants
Nickase
(ssDNA Nickase activity /
cleavage) dsDNA cleavage dsDNA cleavage
Mutant activity (%) activity (%) activity
S924G 0.100 70.900 0.001
S924A 18.000 53.400 0.337
S924V 11.100 53.500 0.207
S924L 2.800 54.500 0.051
S9241 14.900 41.800 0.356
S924M 8.100 49.600 0.163
S924F 26.600 15.500 1.716
S924W 3.530 8.670 0.407
S924P 15.500 10.100 1.535
S924S -5.000 82.200 -0.061
S924T 2.800 78.200 0.036
S924C 2.700 70.700 0.038
S924Y 11.000 11.000 1.000
S924N 8.400 71.800 0.117
S924Q 23.400 29.200 0.801
S924D 29.000 12.700 2.283
S924E 22.800 15.400 1.481
S924K 14.600 41.600 0.351
S92411 36.000 25.300 1.423
Table 14: xCas12i-S925 mutants
Nickase
(ssDNA Nickase activity /
cleavage) dsDNA cleavage dsDNA cleavage
Mutant activity (%) activity (%) activity
S925G 28.700 40.900 0.702
S925A -0.600 12.700 -0.047
S925V 3.000 3.560 0.843
S925L 6.650 5.750 1.157
S9251 9.000 5.800 1.552
S925M 5.350 5.150 1.039
S925F 7.530 6.870 1.096
S925W 3.330 9.770 0.341
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Nickase
(ssDNA Nickase activity /
cleavage) dsDNA cleavage dsDNA cleavage
Mutant activity (%) activity (%) activity
S925P 4.700 9.700 0.485
S925S -0.300 76.300 -0.004
S925T 32.000 21.200 1.509
S925C 7.600 8.000 0.950
S925Y 7.780 5.820 1.337
S925N 1.300 12.300 0.106
S925Q 6.230 5.970 1.044
S925D 9.320 6.180 1.508
S925E 11.690 6.610 1.769
S925K 6.700 10.800 0.620
S925H 6.100 10.600 0.575
* * *
Various modifications and variations of the described products, methods, and
uses of the disclosure will be
apparent to those skilled in the art without departing from the scope and
spirit of the disclosure. Although the
disclosure has been described in connection with specific embodiments, it will
be understood that it is capable of
further modifications and that the disclosure as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the disclosure that are
obvious to those skilled in the art are intended to be within the scope of the
disclosure. This application is intended
to cover any variations, uses, or adaptations of the disclosure following, in
general, the principles of the disclosure
and including such departures from the present disclosure come within known
customary practice within the art to
which the disclosure pertains and may be applied to the essential features
herein before set forth.
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EXAM PI ARY SEQUENCES
SEQ ID NO: 1 >SiCas12i protein
MSSDVVRPYIsITKLLPDNRKHNMFLQTFKRLNSISLNHFDLLICLYAAITNKKAEEYKSEKEAHVTADSLCAINWFRP
MSKRYSKYATTFFNMLELFKEYSGHEPDAYSKNYLM
SNIDSDRFVWVDCRKFAKDFAYQMELGFHEFTVLAETLLANSILVLNESTKANWAWGTVSALYGGGDKEDSTLKSKILL
AFVDALNNHELKTKREILNQVCESLKYQSYQDM
YVDFRSVVDENGNKKSPNGSMPIVTKFETDDLISDNQRKAMISNFTKNAAAKAAKKPIPYLDRLKEHMVSLCDEYNVYA
WAAAITNSNADVTARNTRNLTFIGEQNSRRKEL
SVLQTTTNEK A
KDILNKINDNLIQEVRYTPAPKHLGRDLANLFDTLKEKDINNIENEEEKQNV1NDCIEQYVDDCRSLNRNPIAALL K
H1SRYYEDFSAK NFLDGAKLNVI,TEVV
NRQKAHPTIWSEKAYTWISKFDKNRRQANSSLVGWVVPPEEVHKEIUAGQQSMMWVTLTLLDDGKWVKHHIPFSDSRYY
SEVYAYNPNLPYLDGGIPRQSKFGNKPTTNLTA
ESQALLANSKYKKANKSFLRAKENATHNVRVSPNTSLCIRLLKDSAGNQMFDKIGNVLFGMQINHKITVGKPNYKIEVG
DRFLGFDQNQSENHTYAVLQRVSESSHDTHHFNG
WDVKVLEKGKVTSDVIVRDEVYDQLSYEGVPYDSSKFAEWRDKRRRFVLENLSIQLEEGKTFLTEFDKLNKDSLYRWNM
NYLKLLRKAIRAGGKEFA K1AKTEIFELAVERFG
PINLGSLSQISLKMIASFKGVVQSYFSVSGCVDDASKKAHDSMLFFFMCAAEEKRTNKREEKTNRAASFILQKAYLHGC
KMIVCEDDLPVADGKTGKAQNADRMDWCARAL
AKKVNDGCVAMSICYRAIPAYMSSHQDPFVHMQDKKTSVLRPRFMEVNKDSIRDYHVAGLRRMLNSKSDAGTSVYYRQA
ALHFCEALGVSPELVKNKKTHAAELGKHMGS
AM LMPWRGGRVYLASICKLTSDAKSVKYCGEDMWQYHADEIAAVNIAMYEVCCQTGAFGKKQKKSDELPG
SEQ ID NO: 2 >Si2Cas 12i protein
MSSDVVRPYIsITKLLPDNRKYNMFLQTFKRLNLJSSNHFDLLVCLYAAITNKKAEEYKSEKEDHVTADSLCAIN
WFRPMSK RY 1K YATITFKMLELFKEYSGHEPDTYSKNYLM
SNIVSDRFVWVDCRKFAKDFANQMELSFHEFTTLSETLLANSILVLNE,STKANWAWGAVSALYGGGDKEDSTLKSKIL
LAFVDALNNPELKTRREILNHVCESLKYQSYQDMY
VDFRSVVDDKGNKKSPNGSMPIVTICFESDDLIGDNQRKTMISSFTKNAA AK ASK
KPIPYLDILKDHMISLCEEYNVYAWAAAITNSNADVTARNTRNLTFIGEQNTRRICELSVL
QISTNEKAKDILNKINDNIIPEVRYTPAPKHLGRDLANLFEMFKEKDINQIGNEEEKQNVINDCIEQYVDDCRSLNRNP
VAALLKHISGYYEDFSAKNFLDGAKLNVI,TEVVNR
QKAHRTICSEKAYTWISKIDKNRRQANSSLVGWVVPPEEV H KEK 1
AGQQSMMWVTLTLLDDGKWVKHRIPFADSRYYSEVYAYNPNLPY LEGGIPRQSKFGNKFITNLTAESQ
ALLANSKHKKANKTFLRAKENITHNVRVSPNTSLCIRPLKDSAGNQMFDNIGNMLFGMQINHRIFVGKPNYKIEVGDRF
LGFDQNQSENHTYAVLQRVSESSHGTHHFNGWD
VKVIEKGKVTSDVVVRDEVYDQLSYEGVPYDSPKFTEWREKRRKFVLENMSIQIEEGKTFLTEFDKLNKDSLYRWNMNY
MKLLRKAIRAGGKEFAKITKAEIFELGVMRFGP
MNLGSLSQVSLKM1 A AFKGV1QSYFSVSGCIDDASKKAHDSMLFAFLCSADEKRTNK
REEKTNRAASF1LQKAYSHGCKMIVCEDDLPIADGKVGKAQN ADRMDWCARSLA
KIO/NDGCVAMSICYRAIPAYMSSHQDPFTHMQDKKTSVLRPRFMEVGKDSIRDHHVAGLRRMLNSKGNTGISVYYREA
ALRFCEALGVLPELVKNK KTHASELGK HMGSA
MLMPWRGGRIYVASICKLTSDAKSIKYCGEDMWQYHADEIAAINIAMYEV
SEQ ID NO: 3 >WiCas 12i protein
MGISISRPYGTKLRPDARKKEMLDKFFTTLAKGQRVFADLGLCIYGSLTLEMVK R
LEPESDSELVCAIGWFRLVDKVIWSENEIKQENLVRQYETYSGKEASEVIKTYLSSPSSD
KYVWIDCRQKFLRFQRDLGTRNLSEDFECMLFEQYLRLTKGELDGHTAMSNMFGTKTKEDRATKLRYAARMKEWLEANE
ETTWEQYHQALQDKLDANTLEEAVDNYKGK
AGGSNPFFSYTLLNRGQIDKKTHEQQLKKFNKVLKTKSKNLNFPNKEKLKQYLETAIGIPVDAQVYGQMFNNGVSEVQP
KTTRNMSFSMEKLELLNELKSLNKTDGFERANE
VLNGFFDSELHTTEDKFNITSRYLGGDRNNRLPKLYELWKKEGVDREEGIQQFSQAIQDKMGQIPVKNVLRITWEFRET
VSAEDFEAAAKANQLEEKITRTK A HPVVISNRYW
TFGSSALVGNIMPADKMHKDQYAGQSFKMWLEAELHYDGKKVKHHLPFYNARFFEEVYCYHPSVAEVTPFKTKQFG
YAIGKD1PADVSVVLKDNPYKKATICRFLRAISNPVA
NTVDVNKPTVCSFMIKRENDEYKLVINRKIGVDRPKRIKVGRKVMGYDRNQTASDTYWIGELVPHGTTGAYRIGEWSVQ
YIKSGPVLSSTQGVNDSTTDQLIYNGMPSSSERF
KAWKKSRMSF1RKLIRQLNAEGLESKGQDYVPENPSSFDVRGETLYVFNSNYMKALVSKHRK AK
KPVEGILEE1EALTSKAKDSCSLMRLSSISDAAMQGIASLKSLINSYFNK
NGCKTIEDKEKFNPDLYVKLVEVE,QKRTNKRKEKVGRIAGSLEQLALLNGVDVVIGEADLGEVKKGKSKKQNSRNMDW
CAKQVAERLEYKLTFHCIGYFGVNPMYTSHQDP
FEHRRVADHLVMRARFEEVNVSNVSEW
HMRNFSNYLRADSGTGLYYKQATLDFLKHYDLEEHADDLEKQNIKFYDFRIGLEDKQLTSVIVPKRGGRIYMATNPVTS
DSTPVTY
AGKTYNRCNADEVAAANIAISVLAPHSKK EEKEDKIPHSKKPKSKNTPKARKNLK1SQLPQK
SEQ ID NO: 4 >Wi2Cas12i protein
MASKHVVRPFNGKVTATGKRLAYLEETFHYLEKAAGGVSTLFAALGSYLDATTISNLINKNQDLAVVIFRYHVVPKGEA
HTLPVGTDMVSRFVADYGMEPNEFQRAYLDSPID
QEKYCWQDNRDVGCWLGEQLGVSEADMRALAVTFYNNQMLYDCVKGTGSGNAVSLLFGSGKKSDYSMKGVIAGKAASVL
AKYRPATYQDARKMILEANGFTSVKDLVTS
YGITGRSSALQIFMEGIESGPISSKTLDARIKKFTEDSERNGRKNLVPHAGAIRNWLIEQAGSSVENYQMAWCEVYGNV
SADWNAKVESNFNFVAEKVKALTELSNIQKSTPDL
GKALKLFEEYLTTCQDEFA 1 APY HFSVMEEVRMEM
ATGREFNDAYDDALNSLDMESKQPIQPLCKFLIERGGSISFDTFKSAAKYLKTQSKIAGRYPHPFVKGNQGFTFGSKNI
WAAINDPMMEYADGRIAGGSAMMWVTATLLDGKKWVRHHIPFANTRYFEEVYASKKGLPVLPCARDGKHSFKLGNNLSV
ERVEKVKEGGRTKATKAQERJLSNLTHNVQFD
SSTTFHRRQEESFVICVNHRHPAPLMKKEMEVGDKHGIDQNVTAPTFYAIVERVASGGIER NG
KQYKVTAMGAISSVQKTRGGEVDVISYMGVELSDSKNGFQSLWNICCLDF
VTKHGTENDVKYYNNTAVWANKLYVWHKMYFRLLKQLMRRAKDLKPFRDHLQHLLFHPNLSPLQRHSLSL3SLEATIUV
RNCIHSYFSLLGLKTLDERKAADINLLEVLEKL
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YAGLVERR KERTK LTAGLLVRLCNEHG ISFA A GDLPV VGEGKSK AANNTQQDW TARE I, KR LSEM
AEV VG IK V I AV LP HYTS HQDPFVYSKNTK KMR(awNwRTTKTim)
RDALSIRR1LSKPETGTNLYYQKGLKAFAEK HG I
DLAEMKKRKDAQWYLEIUQDKNFLVPMNGGRVYLSSVKLAGKETIDMGGEILYLNDADQVAALNVLLVKI
SEQ ID NO: 5 >Wi3Cas12i protein
MAKKEHERPFKGTLPLRGDRLRYLQDTMKYM KK
VEDTITELCAAVIAYAKPITIQQ1LGEEIETTSTFCSFRLVGIHENFFMPLTFNMIKHFQKTFNINPSEKQAIYISSGF
DSDK
YRWQDTSEVSRNFANKCRLTNQEFQEFA EQALLN MCFIGCSGSPGATNAVSQIFGTGEKSDYQRKSQIA K I
AADTLENHKPSTYESARLMVINTLG HKTIEDCVNDYGAIGAK
SAFRLFMESKEIGPITSE,QMIUK KFREDHK
KNSIKKQLPHVEKVRNALLSQFKE,QYLPSAWAEAWCNIMGEFNSKLSNNNNFIDQKTKMVNDCDNIK
KSMPQLDKAVNMLD
EWKYKNWDDNSAIHPYRIGDLK K LM ARNINNEGTFDERFSASWE,QFSTSLEYGEKPPVRDLL AHIIK
NDLTYTDVINAAKFLKLQDNIRNKYPHPFVMPNKGCTFGKDNL
WGE1NDPTAKIKSTEEVAGQRPMM
WLTAKLLDNGKWVEHH3PFASSRYFAEVYYTNPALPTLPIARDGKHSYKLTKTIDANTAKTLVNNPRDKAAKLIARTKA
NTTHNVKWIK
PTYRIQKENNQFVITINHRHPCTFPPKEIILGDRILSFDQNETAPTAFSILEKTTKGTEFCGHHIKVLKTGMLEAKIKT
SKKSIDAFTYMGPMEDDHASGFPTLLNICEKFISENGDE
K DKSFSSRKLPFKRSLYFFHGSHFDLLKKM IR K A
KNDPKKLKLVRRIINEILFNSNLSPIKLHSLSIHSMENTKK V I
AAISCYMNVHEWKTIDE,QKNADITLYNAKEKLYNNLVNR
RKERVINTAGMLIRLARENNCRFMVGEAELPTQQQGKSKINNNSKQDWCARDIAQRCEDMCEVVGIKWNGVTPHNTSHQ
NPFIY KNTSGQQMRCRYSLVKKSEMTDKMA
EIORNILHAEPVGITAYYREGILEFAKHHGLDLGMMKKRRDAKYYDNLPDEFLLPTRGGRIYLSENQLGGNETIVINGK
KYFVNQADQVAAVNIGLLYLLPKKIsIQS
SEQ ID NO: 6 > SaCas I2i protein
MSEKKFHIRPYRCSISPNARKADMIKATISYLDSLTSVFRSGFTALLAGIDPSTVSRLAPSGAVGSPDLWSAVNWFRIV
PLAEAGDARVGQASLINLFRGYAGHEPDEEASIYME
SRVDDKRHAWVDCRAMFRAMALECGLEEAQLASDVFALASREVIVFKDGEINGWGIASLLFGEGEKADSQKKVALLRSV
RLALEGDYATYEELSGLMLAKTGASSGSDLLD
EYKRSEKGGSSGGRHPFFDEVFRRGGRVKQEERERLLKSCDTAIQKQGQALPLSHVASWRQWFLRRVTLLRNRRQESFA
VCJTNALMDLQPKNLRNVHYVTNPKSEKDKGVL
ELRVDVKNNEGPDVAGAQAVFDAYMARLAPDLRFSVM PR
HLGSLKDLYALWAKLGRDEAIEEYLEGYEGPFSKRPIAGILQDHAHRGKVGHDSLLRAARLNRAMDRLERKR
AHACAAGNKGYVYGKSSMVGRINPQSLEVGGRKSGRSPMMWVTLDLVDGDRFAQHHLPFQSARFFSEVYCHGDGLPATR
VPGMVRNRRNGLAIGNGLGEGGISALRAGSD
RR K RANKRTLRALENITHNVEIDPVISFFLREDGIIISHRIE K IEPK
LVAFGDRALGFDLNQTGAHTFAVLQKVDSGGLDVGHSRVSIVLTGTVRSICKGNQASGGRDYDLLSYDG
PERDDGAFTAWRSDRQAFLMSAIRELPTPAEGEKDYKADLLSQMASLDHYRRLYAYNRICCLGIYIGALRRATRNAVAA
FKDERSIANHRCGPLMRGSLSVNGMESLANLKG
LATAYLSKFK DSKSEDLLSK DEEM
ADLYRACARRMTGKRKERYRRAASEIVRLANEHGCLFVFGEKELPTTSKGNKSKQNQRNTDWSARAIVKAVKEACEGCG
LGFKPVW K
EYSSLTDPFERDGDGRPALRCRFAKVAAPDSELPPRLTKAVGSYVKNALKADKAEKKQTCYQRGA1EFCSR HG IDV
R K ATDK AIRKAVRGSSDLLVPFDGGRTFLISTRISPESR
KVEWAGRTLYERSDMVAAINIACRGLEPRKA
SEQ ID NO: 7 >Sa2Cas12i protein
MDEQAVVSSGSDKTLIUVRPYRAKVTATGIRLEGIKIsITLNYLKRTEICLSRLNAACGAFLTPAIVEQICKDDPALVC
ALARFQLVPVGSEATLSDSGLMRHFKAALGELTPLQEAY
LNSSYNDELYAWQDTLVLARQDAFTGLTEDQFRAFAHACFKNGNIIGCAGGPGASNAISGIFGEGIKSDYSLRSEMTAA
VAKVFEEKRPITYEEARALALEATGHASVQSFVEAF
GKQGRKGTLILFMEDTKTGAFPSNEFDYKLKKL
KEDAERVGRKGDPHRDVIASYLRNQTGADIEYNSKAWCESYCCAVSEYNSKMSNNVRFATEKSLDLTK
LDETIRETPK ISE
AMLVFENYMARIDADLRFIVSKHHLGNLAKFRQTM M HVSASEFEEAFK A
MWADYLAGLEYGEKPAICELVRYVLTHGNDLITEAFYAACKFLSLDDKJK IYRYPHPFVPG NK
G YTFG A KNLWAE1NDPFKPIRQGNPEVAGQRPM M WATADLLDNNKWVLH
HIPFASSRYFEEVYYTDPSIPTAQKARDGKHG YRLGKVLDEAARERLKANNRQR KAAK AIERI
KANCEHNVAWDPTFTFMLQLDSEGNVKMTINHRHL4YRAPKEIGVGDRVIGIDQNETAPTTYAILERTENPRDLEYNGK
YYRVVKMGSVTSPNVSKYRTVDALTYDGVSLSDD
ASGAVNFVVLCREFFAAHGDDEGR K Y LERTLGWSSSLYSFHGNYFKCLTQM M R RSARSGGDLTVYRA H
LQQILFQHNLSPLRMHSISLRSMESTMKVISCMKSYMSLCGWK
TDADRIANDRSLFEAARKLYTSLVNRRTERVRVTAGILMRLCLEHNVRFIHMEDELPVAETG
KSKKSNGAKMHWCARELAVRISQMAEVTSVKITGVSPHYTSHQDPFVHSK
TSKVMRARWSWRNRADFTDKDAERJRTILGGDDAGTKAYYRSALAEFASRYGLDMEQMRKRRDAQWYQERLPEFFDPQR
GGRVYLSSHDLGSGQKVDGIYGGRAFVNHA
DEVAALNVALVRL
SEQ ID NO: 8 >Sa3Cas12i protein
MKTETLIRPYPGKLNLQPRRAQFLEDSIQYHQKMTEFFYQFLQAVGGATTHQNISDFIDNICATDEHQATLLFQVVSKD
STTPECPAEELLARFAQYTGKQPNEAVTHYLTSRINT
DKYRWQDNRLLAQNIASQLNISETQFQE1AHAILSNNLYIGQTASNAAANFISQVTGTGQICAPKAARLDVLFQTNQAL
AKTQPTITGQLQQDVQACGESTTDAVLAKFGNKG
AATSLQLALKTDPNITLDQKKYEALQKKFAEDETK YRN K V
DIPHKTQLRNLILNTSNQFCNWHTKPAIEAFKCALADIQSKVSNNLRIMQEKAKLYEAFRNVDPQVQIAVQAL
ENHMNTLEEPYAPYAHSFGSVKDFYEDLNNGSNLDEAIQTI V
HDSDNFNRKPDPNWLRDAPLHSSHSASQIMEAVKYLSSKQDYELRKPFPFVATNLPATYGKFNIPGTLNPPTD
SLHGRLNGSHSNMWLTALLLDGRDWKN H HLCFASSRYFEEVYFFNISLPTFDKVRSPKCGFFLKSVLDSEAKDR
IRN A PKSRTK AV KA IERIKANSTHNVAWNPETSFQMQKR
NDEFYITINHRJEM
EKIPGQKKTDDGFTIHPKGLFAILKEGDRJLSQDLNQTAATHCAVYEVAKPDQNTFNHHGIHLKLIATEELKMPLKTKK
STIPDALSYQGRIAHDRENGLQQ
LKDACGAFISPRLDPKQKATWDNSVSKKENLYPFITAYM KLLKK VM
KAGRQELKLFRTHLDH3LFKHNLSPLKLHGVSMIGLESSRATKSVINSFFNLQNAKTEQQQIALDRPL
86
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
FEAGKTLINNQTRRRQERVRLETSLTMRLAHKYNAKAIIIEGELPHSSTGTSQYQNNVRLDWSAKKSAKLKTESANCAG
IAICQIDPCHTSHQNPFRHTPINPDLRPRFAQVKK
GKMFQYQLNGLQRLLNPRSKSSTAIYYRQAVQSFCAHHNLTERDITSAKEPSDLEKKIKDDTYLIPQRGGRIYISSFPV
TSCARPCTSNHYFGGGQFECNADAVAAVNIMLKVHP
SEQ ID NO: 9 >WaCas12i protein
MPIRGYKCTVVPNVRKKKLLEKTYSYLQEGSDVFFDLFLSLYGGIAPKMIPQDLGINEQVICAANWFKIVEKTKDCIAD
DALLNQFAQYYGEKPNEKVVQFLTASYNKDKYV
WVDCRQKFYTLQKDLGVQNLENDLECLIREDLLPVGSDKEVNGWHSISKLFGCGEKEDRTIKAKILNGLWERIEKEDIL
TEEDARNELLHSAGVLITKEFRKVYKGAAGGRDC
YHTLLVDGRNFTFNLKTLIKQTKDKLKEKSVDVEIPNKEALRLYLEKRIGRSFEQKPWSEMYKTALSAVMPKNTLNYCF
AIDRHAQYTKIQTLKQPYDSAITALNGFFESECFT
GSDVEVISPSHLGKTLKKLYNYKDVESGISEIVEDEDNSLRSGVNVNLLRYIFTLKDMESAEDFIKAAEYNVVFERYNR
QKVIIPTVKGNQSFTEGNSALSGKVIPPSKCLSNLPG
QMWLAINILLDQGEWKEHHIPFHSAREYEEIYATSDNQNNPVDLRTKREGCSLNKITSAADIEKVKESAKKKHGKAAKR
ILRAKNTNTAVNWVDCGEMLEKTEVNEKITVNYK
LPDQKLGKEEPIVGTKILAYDQNQTAPDAYAILEICDDSEAFDYKGYKIKCLSTGDLASKSLTKQTEVDQLAYKGVDKT
SNEYKKWKQQRRLFVKSLNIPDALKSFENINKEYL
YGENNSYLKLLKQILRGKEGPILVDIRPELIEMCQGIGSIMRLSSLNHDSLDAIQSLKSLLHSYFDLKVKEEIKTEELR
EKADKEVFKLLQQVIQKQKNKRKEKVNRTVDAILTLA
ADEQVQVIVGEGDLCVSTKGTKKRQNNRTIDWCARAVVEKLEKACKLHGLHEKEIPPHYTSHQDCFEHNKDIENPKEVM
KCRENSSENVAPWMIKKFANYLKCETKYYVQG
MQDFLEHYGLVEYKDHIKKGKISIGDFQKLIKLALEKVGEKEIVFPCKGGRIYLSTYCLTNESKPIVFNGRRCYVNNAD
HVAAINVGICLLNFNARAKVAEKTP
SEQ ID NO: 10 >Wa2Cas12i protein
MAKKDFIARIWNSFLLINDRKLAYLEETWTAYKSIKTVLHRFLIAAYGAIPFQTFAKTIENTQEDELQLAYAVRMERLV
PKDFSKNENNIPPDMLISKLASYTNINQSPTNVLSYV
NSNYDPEKYKWIDSRNEAISLSKEIGIKLDELADYATTMLWEDWLPLNKDTVNGWGTTSGLFGAGKKEDRTQKVQMLNA
LLLGLICNNPPKDYKQYSTILLKAFDAKSWEEA
VKIYKGECSGRTSSYLTEKHGDISPETLEKLIQSIQRDIADKQHPINLPKREEIKAYLEKQSGITYNLNLWSQALHNAM
SSIKKTDTRNENSTLEKYEKEIQLKECLQDGDDVELL
GNKFFSSPYHKTNDVFVICSEHIGTNRKYNVVEQMYQLASEHADFETVFTLLKDEYEEKGIKTPIKNILEYIWNNKNVP
VGTWGRIAKYNQLKDRLAGIKANPTVECNRGMTF
GNSAMVGEVMRSNRISTSTKNKGQILAQMHNDRPVGSNNMIWLEMTLLNNGKWQKHHIPTHNNKFFEEVHAFNPELKQS
VNVRNRMYRSQNYSQLPTSLTDGLQGNPKAK
IFKRQYRALNNMTANVIDPKLSFIVNKKDGRFEISIIHNVEVIRARRDVLVGDYLVGMDQNQTASNTYAVMQVVQPNTP
DSHEFRNQWVKFIESGKIESSTLNSRGEYIDQLSH
DGVDLQEIKDSEWIPAAEKFLNKLGAINKDGITISISNTSKRAYTTNSIYEKILLNYLRANDVDLNLVREEILRIANGR
ESPMRLGSLSWTTLKMLGNFRNLIHSYFDHCGFKEMP
ERESKDKTMYDLLMHTITKLTNKRAERTSRIAGSLMNVAHKYKIGTSVVHVVVEGSLSKTDKSSSKGNNRNTTDWCSRA
VVKKLEDMCVFYGFNLKAVSAHYTSHQDPLVH
RADYDDPKLALRCRYSSYSRADFEKWGEKSFAAVIRWATDKKSNTCYKVGAVEFFKNYKIPEDKITKKLTIKEFLEDAC
AESHYPNEYDDILIPRRGGRIYLTTKKLLSDSTHQR
ESVHSHTAVVKMNGKEYYSSDADEVAAINICLHDWVVPLNWINHCLPAGWCSDHLKECVQCHTPDPVRISM
SEQ ID NO: 11 >SiCas12i Direct Repeat
CTAGCAATGACTCAGAAATGTGTCCCCAGTTGACAC
SEQ ID NO: 12>Si2Cas12i Direct Repeat
ATCGCAACATCTTAGAAATCCGTCCTTAGTTGACGG
EQ ID NO: 13 >WiCas12i Direct Repeat
TCTCAACGATAGTCAGACATGTGTCCCCAGTGACAC
SEQ ID NO: 14>W12Cas12i Direct Repeat
CTCAAAGTGTCAAAAGAATGTCCCTGCTAATGGGAC
SEQ ID NO: 15 >W13Cas12i Direct Repeat
TCCCAAAGTGGCAAAAGAATCTCCCTGTTAATGGGAG
SEQ ID NO: 16>SaCas12i Direct Repeat
GTCTAACTGCCATAGAATCGTGCCTGCAATTGGCAC
SEQ ID NO: 17 >Sa2Cas12i Direct Repeat
87
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
TCGGGGCACCAAAATAATCTCCTTGGTAATGGGAG
SEQ ID NO: 18>Sa3Cas12i Direct Repeat
CCACAACAACCAAAAGAATGTCCCTGAAAGTGGGAC
SEQ ID NO: 19 >WaCas12i Direct Repeat
GTAACAGTGGCTAAGTAATGTGTCTTCCAATGACAC
SEQ ID NO: 20>Wa2Cas12i Direct Repeat
GAGAGAATGTGTGCAAAGTCACAC
SEQ ID NO: 21 >SiCas12i coding sequence
ATGTCTAGTGATGTCGTTCGTCCATATAACACCAAACTGCTTCCAGATAATCGCAAACACAATATGTTTTTGCAAACTT
TCAAGCGACTTAATTCTATTTCTCTTAATCATTT
TGATCTCTTAATTTGTCTTTATGCTGCCATTACCAACAAGAAGGCAGAAGAATATAAGTCTGAAAAAGAAGCTCATGTA
ACCGCTGATAGCCTTTGTGCTATCAATTGGTTC
CGTCCTATGTCCAAGCGTTACAGCAAATACGCAACTACAACTTTCAATATGCTTGAATTGTTCAAAGAATACTCTGGGC
ATGAACCAGATGCTTATTCCAAGAATTATCTTA
TGTCCAATATTGACTCAGACAGGTTTGTCTGGGTTGATTGCCGTAAATTTGCCAAAGATTTTGCGTATCAAATGGAACT
TGGTTTCCATGAATTTACAGTCTTGGCAGAAA
CCTTGTTGGCAAATAGTATTCTTGTACTCAACGAATCAACTAAGGCAAATTGGGCATGGGGCACCGTTTCTGCACTTTA
CGGTGGAGGCGATAAGGAAGATTCTACGCTG
AAGTCGAAAATCCTTTTGGCTTTTGTTGATGCACTCAATAACCACGAACTTAAAACTAAGCGTGAAATTCTCAATCAAG
TTTGTGAATCACTAAAATATCAATCATACCAA
GACATGTATGTTGATTTCCGTTCTGTTGTTGACGAAAATGGAAACAAGAAGTCTCCCAATGGCTCAATGCCAATCGTCA
CCAAGTTTGAAACAGATGATTTGAM CTGAT
AATCAACGCAAAGCAATGATTTCTAATTTCACAAAGAATGCTGCTGCTAAAGCGGCTAAAAAACCTATTCCCTACCTAG
ACAGACTCAAGGAACATATGGTTTCCTTGTG
CGATGAATATAATGTTTATGCTTGGGCAGCAGCTATCACTAACTCTAATGCCGATGTAACAGCTAGGAATACTCGCAAT
TTAACATTCATCGGGGAACAAAATTCTCGAAGG
AAAGAACTATCGGTTTTACAAACTACAACAAACGAAAAAGCAAAAGATATCTTGAATAAGATTAATGACAATCTTATTC
AAGAAGTAAGGTATACCCCTGCCCCCAAGCA
CTTGGGGCGTGATCTTGCCAATCTTTTTGATACTCTGAAAGAAAAAGATATCAATAATATTGAAAACGAAGAAGAGAAG
CAGAATGTAATTAATGATTGCATTGAGCAATA
TGTTGATGATTGCCGTTCACTGAACCGCAATCCCATTGCTGCTTTGCTCAAGCACATTAGCCGATACTATGAAGATTTT
TCAGCCAAGAATTTCTTGGATGGTGCCAAGTT
GAATGTCTTGACTGAAGTTGTAAATCGTCAAAAGGCACATCCAACTATTTGGTCTGAAAAGGCTTATACTTGGATTTCC
AAGTTTGACAAGAATAGGCGACAAGCAAACT
CTTCYTTGGTTGGATGGGTTGTTCCACCAGAAGAAGTCCATAAAGAGAAGATTGCTGGTCAACAAAGCATGATGTGGGT
CAC Ell GACTCTGCTTGATGATGGCAAGTGG
GTAAAGCACCATATTCCTTTTTCAGATTCCAGATATTATTCTGAAGTCTATGCCTACAATCCAAATTTGCCATATCTTG
ATGGTGGTATTCCACGCCAGTCAAAGTTTGGCAA
TAAACCAACCACTAATCTGACTGCTGAAAGTCAAGCGTTACTTGCAAACAGCAAGTATAAAAAGGCAAATAAGTCATTT
CTCCGTGCCAAGGAAAATGCTACTCACAAT
GTCCGTGTTAGTCCAAACACTTCCTTGTGCATTCGTTTGCTCAAGGATAGTGCTGGTAATCAAATGTTTGATAAGATTG
GCAATGTTCTGTTTGGAATGCAGATCAACCATA
AAATCACCGTTGGCAAGCCCAACTACAAGATCGAAGTTGGTGATAGGTTCCTTGGTTTCGACCAGAACCAAAGTGAAAA
CCACACTTATGCTGTCTTGCAACGAGTCTC
TGAAAGCTCTCATGACACTCATCATTTTAATGGATGGGATGTCAAGGTTCTTGAAAAGGGCAAAGTAACAAGTGATGTC
ATCGTTAGAGATGAGGTCTATGACCAACTTA
GCTATGAGGGCGTTCCTTATGATTCTTCAAAGTTTGCAGAATGGAGAGACAAGAGGAGAAGGYTTGTTTTGGAAAACTT
GTCTATCCAGTTGGAAGAAGGCAAAACATT
CTTGACTGAATTCGACAAATTAAATAAAGATTCTCTTTATCGTTGGAATATGAATTATCTGAAACTGCTCAGGAAAGCT
ATTCGTGCCGGTGGCAAGGAATTTGCCAAGAT
TGCTAAGACTGAGATTTTTGAATTGGCAGTTGAAAGGYTTGGACCAATCAACCTTGGTAGTTTGTCACAAATTAGCTTG
AAGATGATTGCATCTTTCAAGGGAGTGGTTC
AGTCTTACTTTTCTGTATCTGGTTGTGTTGATGACGCATCCAAGAAGGCACATGATTCCATGCTCTTCACTTTCATGTG
TGCAGCAGAAGAAAAAAGGACAAACAAAAGA
GAAGAAAAGACTAATCGTGCAGCATCTTTTATCTTGCAGAAAGCATATTTGCATGGCTGCAAGATGATTGTTTGCGAAG
ACGATCTTCCTGTTGCTGATGGAAAAACAGG
CAAGGCACAAAATGCGGATCGTATGGACTGGTGTGCCCGTGCTTTGGCAAAGAAAGTCAACGATGGTTGTGTGGCAATG
TCTATCTGCTATCGTGCCATTCCAGCTTATAT
GTCTAGCCACCAAGATCCATTTGTTCACATGCAAGACAAAAAGACTTCTGTTTTGCGTCCAAGGTTCATGGAAGTTAAC
AAGGATAGCATCAGGGATTATCATGTTGCTG
GTTTGCGGAGAATGCTGAACAGCAAGAGTGATGCAGGCACTTCCGTTTACTATCGTCAGGCAGCTTTGCATTTCTGCGA
AGCGTTGGGCGTGTCTCCAGAATTAGTCAAG
AACAAAAAGACTCATGCTGCCGAATTAGGAAAGCATATGGGTTCTGCCATGTTGATGCCTTGGCGGGGTGGCAGGGTTT
ATATTGCCAGCAAGAAGTTGACTTCGGATGC
TAAAAGTGTAAAATACTGTGGAGAAGATATGTGGCAGTATCATGCTGATGAGATTGCTGCTGTCAATATCGCAATGTAT
GAAGTTTGCTGCCAGACAGGTGCGTTTGGCAA
GAAGCAAAAGAAGAGTGATGAACTACCGGGATAA
SEQ ID NO: 22>Si2Cas12i coding sequence
CATGTCTAGTGATGTTGTTCGTCCATATAACACTAAGCTGCTTCCTGATAATCGCAAATACAATATGTTTTTGCAAACT
TTCAAAAGACTCAATTTGATTTCATCAAATCATT
88
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
TTGATCTCTIGGTTTGICTTTATGCTGCTATCACCAACAAAAAAGCTGAAGAATATAAGTCAGAAAAAGAAGATCATGT
AACCGCTGATAGCCTITGCGCCATCAATTGGT
TCCGTCCTATGTCCAAGCGITATATCAAATACGCAACCACTACTITTAAGATGCTTGAATMITTAAGGAGTACTCTGGT
CATGAACCAGATACTTATFCCAAGAATTATCTC
ATGTCCAATATCGTCTCAGATAGGTTFGTFTGGGTFGATMCCGCAAATTMCCAAAGATTFPGCCAATCAAATGGAACTT
AGTTFCCACGAATTFACCACFnuTCAGAGA
CTITGITGGCAAATAGTATCCTTGTACTCAATGAGTCAACCAAGGCAAATTGGGCATGGGGTGCTGTFTCAGCACTITA
TGGTGGAGGCGACAAAGAAGATFCTACGCTG
AAGTCCAAAATCCTITTGGCTITFMTGATGCTCTCAATAATCCTGAACTFAAAACTAGGCGGGAAAWCTCAATCAT6
__ n 1111 GAATCACTAAAATATCAATCATACCAAG
ATATGTATMTGATITFCGATCTGTCGITGATGATAAGGGAAACAAGAA=CCCAATGGCTCAATGCCAATCGTCACTAAG
TTFGAATCAGATGATTTGATTGGTGACAA
TCAACGCAAAACTATGATTFCTAGITFCACAAAAAACGCCGCTGCCAAAGCGTCTAAGAAGCCCAITCC.ATATCTAGA
CATTCTAAAAGACCACATGATTFCCITGTGCGA
GGAATACAATGTCTATGCTTGGGCAGCAGCTATFACCAATFCCAATGCTGATGTAACTGCTAGAAACACTCGCAATCTG
ACATTCATCGGGGAACAAAATACCCGAAGGA
AAGAACTATCGGITITACAAACFFCTACAAACGAAAAAGCAAAAGATATCTFAAATAAGATFAACGACAATCTTATFCC
AGAAGTAAGGTACACCCCTGCTCCCAAGCAC
TTGGGGCGTGATCTTGCCAATCTITITGAAATGITCAAAGAAAAAGATATAAATCAGATFGGAAATGAAGAAGAAAAGC
AAAATGTGATCAATGATTGCATTGAGCAATA
TGTCGATGATTGCCGTFCATTGAACCGCAATCCTGITGCAGCTITGCTCAAGCATATTAGCGGATATTATGAAGATTFT
TCAGCCAAGAAn ibGATGGTGCCAAGITG
AATGTCITGACGGAAGTFGTCAATCCFCAAAAGGCACATCCAACTATFTGTFCTGAAAAGGCTTATACTFGGATTTCCA
AGATTGACAAGAATAGGCGACAAGCAAACTC
EHJI II
GGITGGATGGGTFMTCCACCGGAGGAAGTCCATAAGGAAAAAATFGCCGGTCAACAAAGCATGATGTGGGTCALTi
__ RACTITGCTTGATGACGGCAAGTGG
GTAAAGCATCATATFCCITTTGCAGACTCAAGATATFATTCTGAAGTCTATGCCTATAATCCAAATTFGCCATATCITG
AAGGTGGTATTCCACGACAATCAAAGITTGGCAA
TAAACCAACAACTAATFTGACCGCTGAAAGCCAAGCATTACTTGCCAACAGTAAGCACAAGAAAGCCAACAAGACATFT
CTCCGTGCCAAGGAGAATATCACTCACAAT
GTFCGTGITAGTCCAAATACFFCATFGTGCATTCGTCCCCTCAAGGATAGTGCTGGTAATCAAATGITTGACAACATFG
GTAATATGITGTITGGAATGCAGATCAATCACA
GAATTACTGTCGGCAAGCCAAACTACAAGATCGAAGITGGTGATCGGTFCCTTGGTITTGACCAGAACCAAAGCGAAAA
CCACACCTATGCAGTFCTFCAACGAGTATC
CGAAAGCTCTCATGGCACTCATCATFTCAATGGITGGGATGTCAAAGTGATTGAGAAGGGCAAGGTGACAAGTGATGTC
GTCGTCAGAGATGAAGTCTATGATCAATTAA
GCTACGAGGGTGTCCCTTACGATFCTCCAAAGITTACAGAATGGAGAGAGAAGAGGCGAAAGITTGTCITGGAAAATAT
GTCAATCCAGATFGAAGAAGGCAAAACATF
CTTGACTGAATTFGACAAGTFAAACAAAGACTCTFTGTATCGITGGAACATGAATTACATGAAATTGCTTAGGAAGGCA
ATTCGTGCTGGTGGCAAGGAATTFGCCAAGA
TTACAAAGGCTGAGATITITGAACTAGGAGITATGAGATTFGGACCAATGAACTFGGGCAGCTFGTCGCAAGTCAGCTT
GAAGATGATTGCTGCTITFAAGGGAGTFATT
CAGTCITACTITTCCGTATCTGGITGCATTGATGACGCATCCAAGAAAGCTCATGATTCGATGITATFCGCTITCTTGT
GTFCAGCAGATGAGAAAAGGACAAACAAGAGG
GAAGAAAAGACAAATCGTGCAGCATCTFTCATATTGCAGAAAGCATACTCGCATGGTTGCAAGATGATTGTTTGCGAGG
ATGATCTFCCCAITGCCGATGGCAAGGTGGG
CAAGGCACAAAATGCGGATCGCATGGACTGGTGCGCCCGITCATFGGCAAAGAAAGTCAACGATGGITGTGTGGCTATG
TCCATATGTFATCGTGCCATTCCAGCATATAT
GTCAAGCCATCAAGATCCATTFACTCATATGCAAGATAAAAAGACFFCTGTFTTGCGTCCAAGGITCATGGAAGTCGGC
AAGGATAGCATFAGGGATCATCATMTGCTGG
TCTGCGGAGAATGCTGAACAGTAAAGGTAATACTGGCACFHJI __________________________________
GTTFACTATCG'PGAGGCAGCTITGCGTFFCTGCGAAGCMTGGGTGTGCTFCCCGAATFAGTCAAGA
ACAAAAAGACTCATGCTFCGGAATTAGGAAAGCATATGGlinuiuCCATMTGATGCCITGGCGGGGTGGCAGGATCTAT
GTCGCCAGCAAGAAATTGACTTCGGATGCC
AAGAGTATAAAATATTGTGGAGAAGATATGTGGCAATATCATGCTGATGAGATTGCTGCTATCAATATCGCAATGTATG
AGGTCTGCTGTCAGACAGGTGCTTITGGCAAA
AAACAAAAGAAGAGTGATGAACTACCGGGATAA
SEQ NO: 23>WiCas12i coding sequence
ATGGGTATFACCATITCACGTCCGTACCGTACAAACTMCCFCCTGATGCTCGTAAGAAGGAAATGITGGATAAGI __
ITI itACCACGCTAGCAAAAGGTCAGCGTh n 111
GCGGATCTGGGACTGTGCATTFACGGCAGCCITACTITAGAAATGGTAAAGCGGCTTGAGCCAGAATCCGATFCTGAAC
TTGTCTGTGCAATFGGITGGTFTCGTCTTGTA
GATAAGGTAACTTGGTCTGAGAATGAAATTAAACAAGAGAACCTGGITAGACAATATGAGACCFATTCAGGAAAAGAAG
CGTCTGAGGITATCAAGACTTACCTAAGCT
CFCCAAGTTCAGACAAGTATGTGTGGATAGACTGCCGACAAAAGIllul _________________________
IAGGTTFCAAAGGGATCTGGGAACACGTAATCTGTCTGAAGACTITGAGTGCATGL niii
GAACAGTACCTCAGACTCACAAAGGGAGAGCITGATGGGCATACCGCTATGTCCAACATGITTGGAACAAAAACAAAAG
AAGATCGCGCCACAAAACTGAGATATGCC
GCAAGGATGAAAGAATGGCTCGAGGCTAACGAAGAAATTACTFGGGAACAATATCACCAAGCCITGCAAGATAAATTAG
ACGCCAATACTITAGAGGAGGCTGITGATA
ATTACAAAGGCAAAGCGGGAGGCTCTAATCCATITITTAGITACACGCFITFAAACAGAGGTCAGATTGATAAAAAAAC
TCACGAGCAGCAATFAAAGAAATFCAACAA
AGITCTAAAAACCAAATCCAAAAAITTAAATITFCCAAACAAAGAGAAGTFAAAACAATATTFAGAAACAGCAATTGGT
ATTCCiuI ibATGCTCAGGTCTACGGTCAGA
TGTITAATAACGGCGTFTCTGAAGITCAACCAAAGACAACGCGCAACATGTCITITTCTATGGAGAAGCTTGAGCTITF
AAACGAGTTGAAAAGTCTCAACAAGACTGA
CCU __________________________________________________________________________
ITI
ibAACGCGCTAATGAAGTCTTGAATGG1TFCTFTGATFCTGAACTTCACACTACTGAAGACAAGTTCAACATCACTTCC
AGGTATTTGGGTGGAGACAGAAACA
ATCGGCTACCAAAGCTGTACGAGCTITGGAAAAAGGAAGGAGTAGATCGTGAGGAAGGTATCCAGCAATFCAGCCAAGC
AATCCAAGATAAGATGGGTCAGATACCTGT
TAAGAATGTCCITAGGTATATTFGGGAA1TFCGTGAGACT6 ____________________________________
II ICI GCCGAAGALTi RJAAGCGGCAGCGAAAGCGAATCAMTGGAAGAAAAAATCACGCCFACCAAA
GCGCACCCCG7TG7TATATCTAACAGGTATTGGACATTFGGCTCTFCGGCTCTFG7TGGTAATATCATGCCAGCAGACA
AGATGCACAAAGACCAGTACGCAGGTCAAAGT
TFCAAGATGTGGCTTGAAGCCGAACPGCACTACGACGGTAAGAAAGTCAAACATCACTTGCCGTFCTACAACGCCAGG
__ nurnuAAGAGGTCTACTGCTATCACCCGA
GCCFAGCTGAAGITACACCATTCAAAACCAAGCAGTFTGGITATGCAATTGGAAAAGATATTCCAGCTGACGITFCGGI
TGTACTGAAAGACAATCCITATAAAAAGGCA
89
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
ACCAAGCGCTTCCTTCGGGCTATCAGCAATCCAGTCGCCAACACAGTGGATGTAAACAAGCCTACAGTTTGCTCATTCA
TGATTAAACGAGAAAATGACGAATACAAACT
AGTCATTAATCGAAAGATCGGTGTTGATCGCCCAAAGCGTATTAAAGTAGGTAGGAAGGTCATGGGCTATGACCGTAAC
CAAACTGCTTCTGATACTTACTGGATTGGAG
AGCTTGTTCCACATGGAACAACCGGAGCGTACCGTATTGGAGAATGGAGCGTCCAGTATATCAAGAGCGGTCCCGTGTT
GTCTTCTACGCAAGGCGTAAATGACAGTACT
ACGGATCAACTTATATACAACGGAATGCCGAGCTCCAGCGAACGTTTTAAAGCTTGGAAGAAATCTAGGATGTCTTTCA
TTCGTAAGTTGATACGCCAACTGAACGCCGA
AGGCTTGGAAAGTAAAGGACAGGACTATGTTCCTGAAAATCCAAGTAGCTTTGATGTTAGGGGCGAAACACTTTACGTA
TTCAACAGCAACTATATGAAAGCTTTGGTGT
CTAAGCATCGAAAAGCCAAGAAACCTGTTGAAGGTATTCTTGAAGAAATAGAAGCCTTGACAAGCAAAGCTAAAGATTC
TTGTTCGTTGATGCGTTTGAGTTC Ill GTCT
GATGCGGCTATGCAAGGTATTGCTTCGTTGAAGAGTTTGATCAACTCATACTTCAACAAGAATGGTTGCAAAACAATTG
AAGACAAAGAAAAGTTTAACCCAGATCTGTA
TGTGAAACTTGTTGAAGTTGAGCAAAAGAGAACTAACAAGAGAAAAGAAAAAGTTGGTCGAATCGCCGGTTCTCTTGAA
CAGTTAGCTTTGCTTAACGGTGTTGACGT
TGTTATCGGTGAAGCTGATCTTGGCGAAGTCAAGAAAGGCAAATCCAAAAAACAAAATAGTCGAAACATGGACTGGTGT
GCCAAGCAAGTCGCTGAGCGGCTTGAGTA
CAAGCTGACCTTCCATTGTATTGGTTATTTTGGTGTCAACCCGATGTATACGTCTCATCAAGATCCATTTGAACATCGT
CGCGTTGCTGACCACCTAGTAATGCGTGCGAGG
TTTGAAGAAGTGAATGTAAGTAATGTTTCGGAATGGCACATGCGAAACTTCTCAAACTATCTGCGTGCGGACTCAGGTA
CTGGYTTGTATTACAAACAAGCTACCTTGGAT
TTCCTCAAGCATTATGATTTGGAAGAGCACGCCGATGATTTGGAAAAGCAGAATATCAAATTCTATGACTTCAGGAAAA
TTCTTGAAGACAAACAATTGACTTCTGTTATT
GTTCCAAAACGTGGCGGTCGCATTTACATGGCGACTAACCCGGTAACTTCCGATAGTACGCCTGTCACTTATGCCGGTA
AAACTTACAACCGGTGTAATGCTGACGAAGT
GGCTGCGGCTAACATCGCTATCAGCGTCTTAGCTCCTCACTCTAAGAAAGAAGAAAAGGAAGATAAGATCCCGATTATT
TCTAAGAAGCCTAAGTCTAAGAATACTCCCA
AGGCCCGGAAGAATTTAAAGACTTCTCAACTTCCTCAGAAA
SEQ ID NO: 24>Wi2Cas121 coding sequence
ATGGCTAGCAAACATGTAGTGCGTCCCTTTAATGGCAAAGTAACAGCTACTGGCAAGCGTTTGGCATACTTGGAAGAAA
CTTTTCATTATTTGGAAAAAGCTGCTGGTGG
TGTTAGTACTTTGTTTGCTGCCCTTGGTTCTTATCTTGATGCAACCACAATAAGCAATTTAATTAATAAAAATCAAGAT
TTAGCCGTTGTAATATTTCGTTATCATGTGGTTCC
CAAAGGTGAGGCTCATACTTTACCTGTAGGTACAGACATGGTTAGTCGTTTTGTTGCCGACTATGGTATGGAGCCGAAT
GAGTTTCAGAGAGCTTATTTGGACAGTCCGAT
TGACCAAGAAAAGTATTGTTGGCAGGATAATAGGGATGTTGGTTGTTGGTTGGGTGAGCAATTGGGTGTTAGCGAAGCG
GACATGCGGGCAATAGCAGTAACTTTTTATA
ACAATCAGATGCTTTATGATTGTGTAAAAGGTACTGGGAGTGGTAATGCTGTGAGTCTTTTGTTTGGCAGTGGTAAAAA
GTCTGATTACAGTATGAAGGGCGTTATAGCAG
GTAAGGCTGCTTCAGTACTGGCAAAATATCGCCCAGCTACCTATCAAGATGCCCGAAAGATGATTTTGGAAGCTAATGG
YTTCACCTCAGTAAAAGATTTGGTTACTTCTT
ATGGAATAACTGGAAGGTCTAGTGCTTTGCAGATATTTATGGAAGGGATTGAAAGTGGTCCTATTAGCAGCAAGACATT
AGATGCTCGTATTAAGAAGTTCACAGAGGATT
CGGAGCGCAATGGCAGGAAGAATCTAGTCCCTCATGCTGGGGCTATACGAAATTGGCTGATTGAGCAAGCTGGTAGTAG
TGTAGAAAACTATCAGATGGCATGGTGCGA
GGTTTACGGTAATGTGTCTGCCGACTGGAATGCCAAAGTAGAAAGTAATTTCAATTTCGTAGCGGAGAAAGTAAAGGCA
TTAACAGAATTATCCAACATTCAGAAATCGA
CTCCTGATTTGGGTAAGGCTTTGAAATTATTTGAAGAATATTTGACTACTTGTCAGGATGAATTTGCTATTGCGCCTTA
TCATTTTAGCGTCATGGAAGAGGTGCGAATGGA
AATGGCAACAGGCAGGGAATTCAATGATGCTTATGATGACGCCCTAAATAGCTTGGACATGGAGTCTAAGCAGCCCATT
CAGCCTTTGTGTAAGTTTTTGATTGAGCGTGG
AGGTAGTATCAGTTTTGATACTTTCAAGAGTGCAGCCAAGTATTTGAAAACACAGAGCAAGATTGCTGGTCGATATCCA
CATCCATTTGTAAAAGGTAATCAGGGATTTAC
TTTTGGTTCCAAAAACATTTGGGCAGCCATCAACGATCCTATGATGGAGTATGCAGATGGTCGTATTGCTGGTGGTTCT
GCAATGATGTGGGTGACGGCTACATTGTTGGA
TGGGAAAAAGTGGGTTCGCCATCATATCCCATTTGCCAATACTCGATACTTTGAGGAGGTTTATGCTAGCAAGAAAGGG
TTGCCTGTATTGCCTTGTGCTAGAGATGGCAA
ACACTCATTTAAATTGGGCAATAATTTGAGTGTAGAGAGAGTTGAAAAGGTCAAAGAAGGCGGTAGAACTAAAGCAACC
AAGGCACAAGAGCGTATTTTAAGCAACTTG
ACTCACAATGTGCAGTTTGACAGTTCGACAACTTTTATTATTCGTCGTCAGGAAGAAAGTTTTGTAATTTGCGTGAATC
ATCGACATCCAGCTCCGCTCATGAAGAAGGA
GATGGAAGTTGGCGACAAAATCATTGGTATCGACCAGAATGTGACGGCACCCACAACCTATGCCATAGTTGAGCGTGTG
GCTTCTGGCGGCATTGAGCGTAACGGCAAG
CAGTACAAAGTGACGGCGATGGGAGCCATTTCCAGCGTTCAGAAGACCAGAGGCGGTGAGGTGGATGTTTTGAGTTATA
TGGGGGTTGAACTTTCTGACAGCAAAAATG
GATTTCAAAGCTTGTGGAATAAATG ____________________________________________________
Ell
GGACTTTGTTACCAAACATGGCACTGAAAATGATGTTAAATATTATAACAACACTGCTGTCTGGGCCAACAAGCTGTAT
GTGT
GGCACAAGATGTATTTCCGGCTTTTGAAGCAGTTGATGCGTCGGGCAAAGGACTTGAAACCTTTCAGGGACCATTTACA
GCATCTATTATTCCATCCTAATCTTAGTCCCTT
GCAACGCCATAGCTTGTCCTTAACAAGTCTGGAAGCAACTAAGATAGTGCGGAATTGCATTCATTCGTATTTCAGTCTA
TTGGGGTTGAAGACCTTGGATGAACGCAAAG
CCGCTGACATCAATTTATTGGAAGTTTTGGAAAAGCTGTATGCTGGTTTGGTTGAGAGGCGAAAAGAAAGAACCAAACT
AACCGCTGGGCTATTGGTTCGCTTATGTAAT
GAGCATGGGATTTCTTTTGCAGCTATTGAGGGTGATTTGCCGGTCGTTGGAGAGGGCAAATCTAAAGCTGCCAACAATA
CACAACAGGATTGGACAGCCAGAGAGTTAG
AGAAGCGATTATCTGAGATGGCGGAGGTGGTTGGCATCAAGGTAATAGCTGTTTTGCCCCACTATACCAGTCATCAGGA
CCCATTTGTTTATAGTAAAAATACCAAGAAAA
TGAGATGTCGTTGGAACTGGAGGACCACCAAGACCTTCACTGATCGTGATGCTTTGAGTATACGCAGGATATTAAGCAA
GCCTGAGACGGGTACAAATTTGTATTATCAG
AAGGGCTTGAAAGCATTTGCTGAAAAGCATGGTCTGGATTTGGCAGAGATGAAGAAGCGCAAGGATGCTCAATGGTATC
TTGAGCGCATTCAAGACAAGAATTTTTTGG
TGCCAATGAATGGTGGTAGAGTTTATTTGAGTTCTGTCAAATTAGCCGGGAAAGAAACAATTGACATGGGTGGCGAAAT
TTTATATCTTAACGATGCCGATCAAGTCGCAG
CGTTGAATGTTTTGTTAGTGAAGATTTGA
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
SEQ ID NO: 25 >Wi3Cas12i coding sequence
ATGGCTAAGAAAGAACATATTATAAGACCATIVAAAGGAACACTACCACITCGTGGTGATAGACTAAGGTATCITCAAG
ATACCATGAAATATATGAAAAAGGTTGAAGAT
ACTATCACAGAAeltiltiCGCCGCTGITATCGCCTA'PGCCAAACCCACCATCATFCAACAAATACTMGCGAAGAAAT
FGAAACCACCAGCACATrTPGTAGCTFCCGCTFA
GTAGGCATTCATGAAAACTITACCATGCCACTAACCACAAATATGATAAAACAMCCAGAAAACCITTAACATAAACCCA
TCAGAAAAACAAGCAATCTATCTCTCCAGT
GGATFCGATIVAGATAAATATCGCTGGCAAGATACITCCGAAGTATCCAGAAACTIVGCCAACAAATGCCGACTIACTA
ATCAAGAATIVCAAGAATITGCCGAACAAGC
ACTACIVAATATGTGCTTCATAGGITGCFCTGGTAGCCCCGGTGCAACTAATGCCGTCTCACAAATCTITGGCACAGGC
GAAAAAAGCGATTACCAACGCAAAAGCCAAA
TCGCTAAAATTGCTGCTGATACCCTCGAAAACCACAAACCTAGCACCTATGAGTCTGCTAGATTAATGGTIVITAATAC
ACTMGACACAAAACAATAGAAGATFGTGTCA
ATGACTATGGCGCAATAGGAGCCAAATCCGCCITCCGACTATFCATGGAATCAAAAGAAATAGGACCAATFACATCTGA
ACAACFCACAACCAAAATFAAGAAGTFCAGA
GAAGATCATAAAAAGAACTCCATCAAGAAACAACrFCCACATGTAGAAAAACITCGTAACGCMuCTATCACAATTCAAA
GAACAATACCTGCCCTCAGCATGGGCAG
AAGCATGGTGCAATATCATGGGCGAATITAACFCCAAATTATCAAATAATAATAACITCATCGACCaaaaaacaaaaaT
GGTCAATGACTGCGATAATATTAAAAAATCTAATCCA
CAACTAGACAAAGCTGTFAATATGCTCGATGAATGGAAATATAAAAACTGGGATGATAATTCTGCTATACACCCATATC
ATATTGGCGATCTFAAAAAACFCATGGCAATATF
CAATATCAATAACGAAGGAACMCGACGAAAGATTITCAGCTAGCTGGGAACAATIVTCCACATCACTAGAATACGGGGA
GAAACCACCCGITCGTGATCTACTAGCCC
ATATCATCAAAAATATGAATGACCTCACCTACACAGACGTAATCAACGCCGCAAAATITCTCAAACTFCAAGATAATAT
AAGAAATAAATACCCACACCCTITCGITATGCC
AAATAAAGGATGTACCTITGGTAAAGATAACCITTGGGGCGAAATIAATGACCCCACAGCCAAAATCAAATCAACAGAA
GAAGTTGCTGGACAAAGACCTATGATGTGG
CTGACAGCCAAACrFCFCGATAATGGAAAATGGGTAGAACACCACATCCCMCGCCTCCAGTAGATALTFRiCCGAAGTT
FATTATACCAATCCAGCACTCCCCACTCTA
CCAATAGCTAGAGATGGAAAACAT1VATACAAATTAACAAAAACTATAGATGCCAATACTGCAAAAACTCTAGTAAATA
ATCCTAGAGATAAAGCAGCTAAACTAATCGCA
CGAACTAAAGCCAATACTACACACAATGTAAAATGGATTAAACCTACATACAGAATCCAAAAAGAAAATAACCAATIVG
ITATTACTATCAATCATCGACACCCATGCATA
ACACCACCAAAGGAAATCATACTCGGAGATCGTATCCTATCCITCGACCAAAACGAAACAGCCCCCACAGCATI'CTCC
ATFCTCGAAAAAACAACCAAAGGTACAGAAT
TCTGTGGCCACCACATTAAAGTGCTAAAGACTGGTATGCTAGAAGCTAAAATIAAAACCAGTAAGAAATCAATAGATGC
ATIVACATACATGGGACCAATGGAAGATGAT
CATGCGTCTGGCMCCAACACTACIVAACATATGTGAAAAATIVATATCAGAGAATGGAGATGAAAAAGACAAAAGTITC
TCTFCTCGTAAATI'GCCCTITAAAAGGTCT
TTGTACITCTITCATGGCTCACACITCGATTTACTAAAGAAAATGATCAGAAAGGCCAAAAATGACCCCAAGAAATMAA
GITAGTAAGAATTCATATCAATGAAATTCTA
TIVAATIVCAATITGTCACCAATAAAACTACACAGTCTGTCTATTCACAGCATGGAAAATACCAAAAAAGTTATAGCTG
CTATFAGCTGCTATATGAATGTTCATGAATGGA
AAACTATCGATGAACAAAAGAATGCTGATATAACATMTATAATGCTAAAGAAAAACTATACAACAACCTTGTFAACCGC
CGTAAAGAAAGAGTAAAAGTAACTGCAGGT
ATGTTGATFCGATFAGCTAGAGAAAACAATMCAGATIVATGGTCGGGGAAGCAGAATTACCCACCCAACAACAAGGCAA
ATCAAAAAAGAACAATAACFCCAAACAGG
ATMGTGCGCCAGAGATATAGCACAACGATGTGAAGATATGTGCGAAGTCGTAGGTATAAAATGGAATGGCGITACFCCG
CATAATACCAGCCATCAAAACCCATTCATCT
ATAAAAATACTAGTGGACAACAAATGCGATGCCGITATAGTCTCGTAAAGAAGTCAGAAATGACAGACAAGATGGCAGA
AAAAATTAGAAATATTITACACGCTGAACCT
GTAGGCACTACAGCATACTACCGTGAAGGCATTITGGAATFCGCCAAACATCATGGATFAGATCTGGGAATGATGAAAA
AACGAAGAGATGCTAAGTATTATGATAATCIT
CCAGATGAG ____________________________________________________________________
MtiltiCTFCCTACTAGAGGTGGTAGAATCTATCTGTCCGAAAATCAACTAGGCGGAAACGAAACCATPGTFATTAATG
GGAAAAAATATTITGTCAATCAG
GCAGATCAAGTCGCTGCCGTAAATATTGGCCTGCTITATCTTCTGCCGAAGAAAAACCAGAGTFAAG
SEQ ID NO: 26>SaCas12i coding sequence
ATGTCCGAGAAGAAGTTCCACATCAGGCCCTACCGCTGCTCGATAAGCCCGAACGCCCGCAAGGCCGATATGCTCAAGG
CGACGATCTCCTACC1TGACFCCCTGACCTC
CGTGTIVAGGTCGGGATTCACCGCACTACTTGCGGGCATAGACCCGTCGACGGTGAGCCGCCTGGCGCCITCGGGGGCC
GTCGGCAGCCCGGACCTGTGGAGCGCCGT
CAACTGGTFCCGCATCGTGCCGCTCGCAGAGGCCGGCGACGCCCGAGTCGGCCAGGCATCGCFCAAGAACCTCTTCCGT
GGCTACGCAGGCCACGAGCCCGACGAAGA
GGCGTCGATCTATATGGAGTCGAGAGTGGACGATAAGAGGCACGCGTGGGTGGACTGCCGTGCCATGTTCAGGGCGATG
GCGCTCGAGTGCGGGCTGGAGGAGGCCCA
GCTCGCCTCCGACGTGTFCGCCCTCGCCTCAAGGGAGGTCATAG _________________________________
ltirltAAGGACGGCGAGATCAACGGCPGGGGCATAGCCTCCCTGCTGTIVGGCGAGGGCGAGAA
GGCCGACTCGCAAAAGAAGGTCGCCCTGCMCGCMCGTGAGGCTGGCCCITGAGGGGGACTACGCGACCTACGAGGAACF
CTCCGGGCTCATGCTGGCCAAGACCGG
AGCCTCCAGCGGCFCCGACCTCCITGACGAGTACAAGAGGAGCGAGAAGGGCGGCAGCAGCGGCGGCAGGCACCCCTTM
CGACGAGGTMCCGGAGGGGCGGCA
GGGTCAAGCAGGAGGAGCGCGAGAGGCTGCTGAAGAGCMCGACACAGCGATCCAGAAGCAGGGGCAGGCGCTGCCGCTG
TCGCACGTCGCATCITGGAGGCAATGG
TTCCTGCGCAGGGTCACGCTGCTGCGCAACCGCAGGCAAGAGTCGITCGCAGTCTGCATCACCAACGCCCTCATGGACC
TACAGCCCAAGAACCTACGCAACGTCCACT
ACGTGACGAACCCCAAGAGCGAGAAGGACAAGGGCGTGCTCGAGCMCGCGTCGACGTCAAGAACAACGAGGGGCCGGAC
GTGGCGGGCGCGCAGGCGGTCITCGA
CGCCTACATGGCGAGGCTGGCACCCGACCTGCGCITCTCCGTGATGCCACGGCACCTCGGCFCCCTCAAGGACCTCTAC
GCCCTTI'GGGCCAAGCTCGGGCGGGACGAG
GCCATCGAGGAGTACCTCGAGGGCTACGAGGGACCATTCAGCAAGAGGCCCATCGCAGGCATFCTACAAATCATCCACG
CACACCGTGGCAAGGTGGGCTACGATAGCC
TGTMCGTGCGGCGAGGCTCAACAGGGCGATGGACAGGCTGGAGAGGAAGAGGGCCCACGCCTGCGCAGCCGGCAACAAG
GGITACGTCTACGGCAAGAGCTCGATG
GTCGGCCGCATCAACCCGCAGAGCCTCGAGGTCGGCGGCCGCAAGTCGGGCCGAAGCCCGATGATGTGGGTGACCCTCG
ACCTGGTGGACGGCGACAGGITCGCGCA
GCACCACCITCCMCCAGAGCGCCCGCITCITCTCCGAGGTCTACTGCCACGGCGACGGGCTCCCGGCCACCCGTGTCCC
CGGCATGGTCAGGAACCGTCGCAACGGG
91
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
CTGGCGATAGGGAACGGGCTCGGGGAGGGTGGACTCTCAGCGCTGCGCGCAGGCAGCGACAGGAGGAAGAGGGCCAACA
AGAGGACGCTGCGCGCCCTCGAGAACA
TCACGCACAACGTGGAGATCGACCCCAGCACCTCCTTCACGCTGCGGGAGGACGGGATAATCATTTCGCACAGGATCGA
GAAGATTGAGCCGAAGCTTGTCGCCTTCGG
GGACAGGGCGCTCGGCTTCGACCTCAACCAGACAGGGGCTCATACGTTTGCGGTGCTCCAGAAGGTGGACTCGGGCGGC
CTAGACGTCGGCCACTCTCGCGTGTCGAT
CGTGCTCACCGGCACTGTTCGCAGCATCTGCAAGGGCAACCAGGCGAGCGGCGGACGGGACTACGACCTGCTTTCCTAC
GACGGCCCCGAGCGCGACGACGGGGCGTT
CACGGCATGGAGGTCGGACAGGCAGGCCTTCCTGATGTCTGCCATACGGGAGCTGCCCACGCCCGCCGAGGGGGAAAAG
GACTACAAGGCAGACCTCCTCTCCCAGAT
GGCGAGCCTTGACCACTACAGGCGACTGTACGCGTACAACAGGAAGTGCCTCGGCATCTACATCGGGGCCTTGAGACGC
GCGACCAGGAGGCAGGCCGTGGCCGCATT
CAAGGACGAGATACTCTCGATCGCGAATCACCGCTGCGGGCCTCTCATGCGTGGGAGCCTTTCGGTGAACGGCATGGAG
TCCCTCGCGAACCTCAAGGGCCTAGCCACG
GCATACCTGAGCAAGTTCAAGGACAGCAAGTCCGAGGACCTGCTGTCGAAGGACGAGGAGATGGCCGACCTGTACAGGG
CTTGCGCGCGCAGAATGACTGGCAAGCG
CAAGGAGAGGTACAGGAGGGCGGCTAGCGAGATCGTCCGGCTGGCCAACGAGCACGGCTGCCTGTTCGTCTTCGGCGAG
AAAGAGCTGCCCACCACCAGCAAGGGCA
ACAAGAGCAAGCAGAACCAGAGGAACACCGACTGGTCGGCCCGTGCCATAGTGAAGGCGGTCAAGGAGGCCTGCGAGGG
CTGCGGTCTCGGCTTCAAGCCCGTGTGG
AAGGAGTACTCGAGCCTCACGGACCCGTTCGAGAGGGACGGGGACGGAAGGCCTGCCCTCCGCTGCCGGTTCGCCAAGG
TGGCCGCACCCGACTCCGAACTCCCGCC
TCGCCTGACGAAGGCCGTCGGCTCCTATGTGAAGAACGCCCTCAAGGCCGACAAGGCGGAGAAGAAGCAGACCTGCTAC
CAGCGTGGCGCCATCGAGTTCTGCTCAAG
GCACGGCATCGACGTCCGGAAGGCGACCGACAAGGCCATTCGCAAGGCAGTCCGTGGCTCCTCCGACCTGCTTGTGCCG
TTCGACGGGGGGAGGACCTTCCTGCTCTC
GACGAGGCTGTCCCCGGAGTCGCGAAAGGTGGAGTGGGCCGGGCGCACCCTGTACGAGTTCCCCAGCGACATGGTCGCC
GCAATCAACATCGCCTGCAGGGGCCTAGA
GCCACGCAAGGCCTAG
SEQ ID NO: 27 >Sa2Cas12i coding sequence
ATGGACGAGCAAGCTGTTGTTTCCTCTGGTTCCGACAAGACCCTCAAGATCGTACGCCCTTACAGGGCAAAAGTAACCG
CTACTGGAATTCGCCTTGAGGGAATTAAAA
ATACCCTGAATTACCTGAAGCGTACAGAAATTTGTCTGTCACGCCTGAATGCAGCTTGTGGAGCTTTTCTCACTCCTGC
CATCGTGGAGCAGATCTGTAAGGACGATCCTG
CCCTAGTTTGTGCCATTGCTCGCTTTCAATTGGTTCCGGTTGGTAGTGAAGCCACTTTGTCCGACAGTGGGCTAATGCG
TCATTTTAAGGCTGCTCTCGGTGAATTGACCC
CGCTACAAGAAGCCTACCTGAATAGCAGCTATAACGACGAATTGTACGCATGGCAGGATACTCTTGTCTTAGCGCGACA
GATTATTGCTGAAACCGGATTGACTGAAGAT
CAATTCCGCGCCTTTGCTCATGCCTGTTIVAAGAACGGCAATATTATCGGGTGCGCTGGTGGTCCCGGTGCCAGCAACG
CCATCTCTGGCATTTTTGGCGAGGGAATTAAA
TCCGATTATTCACTCCGAAGTGAAATGACCGCTGCCGTTGCAAAGGTGTTTGAAGAGAAACGTCCTATCACTTACGAAG
AAGCTCGGGCTCTCGCTCTGGAAGCAACTG
GACACGCCAGCGTTCAGTCTTTCGTGGAAGCATTTGGTAAACAGGGGCGTAAAGGCACTCTGATTCTTTTCATGGAAGA
TACCAAGACAGGCGCATTCCCAAGCAATGA
ATTCGATTACAAGCTCAAGAAACTGAAGGAGGATGCAGAGCGTGTCGGGCGTAAGGGTATCATCCCGCACCGCGATGTG
ATTGCTTCTTATCTCCGCAATCAGACTGGTG
CTGATATTGAATACAACTCCAAGGCATGGTGCGAGTCCTACTGTTGTGCCGTGAGCGAATACAACTCAAAGATGAGCAA
CAATGTTCGATTTGCCACGGAAAAAAGTCTT
GATTTGACCAAGCTTGATGAAACGATCAGGGAAACGCCCAAGATCAGTGAAGCCATGCTTGTTTTTGAAAACTACATGG
CGCGAATTGATGCCGATCTCCGGTTCATTGT
GAGCAAGCATCATCTCGGCAATCTCGCCAAATTCCGTCAGACCATGATGCATGTCTCTGCATCAGAATTTGAAGAGGC
__ Ell TAAGGCGATGTGGGCTGATTACTTGGCTGG
TCTGGAATACGGTGAAAAACCCGCGATCTGTGAACTGGTGCGGTATGTCCTGACCCATGGCAACGATTTGCCTGTCGAA
GCGTTTTACGCTGCGTGCAAGTTCCTTAGCT
TGGATGACAAGATCAAGAATCGTTACCCTCACCCATTTGTTCCGGGTAACAAAGGCTACACCTTTGGCGCGAAAAACTT
GTGGGCAGAAATCAATGATCCCTTCAAGCCC
ATCCGTCAAGGCAACCCAGAGGTTGCTGGTCAACGCCCCATGATGTGGGCTACCGCCGACCTTCTGGACAACAACAAAT
GGGTCTTGCATCACATCCCCTTTGCCTCCAG
CAGGTATTTCGAGGAAGTGTACTACACCGATCCCTCGCTTCCTACGGCTCAAAAGGCGCGAGACGGCAAGCATGGCTAT
CGGTTGGGCAAAGTGCTGGATGAGGCTGCT
CGGGAGCGTTTAAAAGCAAATAATCGCCAGCGCAAGGCAGCTAAAGCCATCGAGCGGATCAAAGCCAACTGTGAGCACA
ATGTGGCTTGGGATCCGACCACCACCTTC
ATGCTIVAGTTGGATTCTGAGGGTAATGTGAAAATGACGATCAATCATCGTCACATTGCCTATCGCGCACCCAAGGAAA
TTGGTGTTGGGGACAGGGTGATTGGCATCGA
CCAAAACGAGACTGCTCCTACAACCTACGCCATTCTTGAGCGCACGGAAAATCCTCGCGATCTTGAATACAACGGCAAG
TATTACCGTGTAGTCAAGATGGGTAGTGTGA
CTTCACCGAATGTCAGCAAGTATCGCACGGTGGACGCTTTGACTTACGATGGCGTGTCCTTGTCGGATGATGCTIVTGG
TGCTGTGAACTTTGTGGTATTGTGTCGCGAGT
TTTTTGCAGCACATGGCGACGATGAGGGTCGCAAGTACCTTGAGAGGACTTTGGGGTGGAGTTCAAGCCTGTATTCCTT
CCATGGAAACTATTTCAAGTGCCTTACGCAG
ATGATGCGTCGATCCGCTCGTTCTGGTGGTGATTTGACGGTCTATCGCGCCCATTTGCAGCAGATCCTGTTCCAACACA
ATCTGTCGCCCTTGAGGATGCACAGCTTGTCT
TTAAGGAGCATGGAATCGACGATGAAGGTCATCAGTTGCATGAAGAGCTACATGTCTCYTTGTGGCTGGAAGACCGACG
CGGATCGGATTGCCAATGATAGGTCGCTGTT
TGAGGCTGCTCGTAAGCTTTACACCAGTTTGGTAAATCGTCGGACGGAGCGGGTTCGTGTGACTGCTGGCATTCTGATG
CGTCTGTGCTTGGAGCACAACGTTAGGTTTA
TTCACATGGAGGATGAACTTCCTGTGGCTGAAACGGGCAAAAGCAAGAAAAGCAATGGCGCGAAGATGCATTGGTGTGC
CCGGGAGCTTGCCGTTCGTTTGTCCCAGAT
GGCAGAGGTGACGAGCGTCAAGTTCACAGGTGTGTCACCGCATTACACTAGCCATCAAGACCCATTTGTGCATTCCAAG
ACTAGTAAGGTAATGCGTGCCCGTTGGAGT
TGGCGGAATCGTGCCGATTTCACGGACAAGGATGCGGAGCGTATTCGGACGATTCTGGGTGGTGATGACGCAGGGACGA
AGGCTTATTATCGCTCGGCGTTGGCTGAATT
TGCCTCGCGCTATGGTCTGGACATGGAGCAGATGCGGAAGAGGCGCGATGCTCAGTGGTATCAAGAGAGACTGCCAGAA
ACCTTTATTATTCCTCAGCGGGGTGGTAGA
GTGTACTTGTCTTCTCACGATCTGGGATCAGGTCAAAAAGTTGACGGGATTTATGGTGGTCGTGCTTTCGTGAATCACG
CTGACGAGGTTGCTGCGCTGAATGTGGCGTT
GGTCAGGCTGTGA
92
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
SEQ ID NO: 28>Sa3Cas12i coding sequence
ATGAAGACTGAAACHJI ____________________________________________________________
lATCCGTCCCTACCCCGGCAAACFCAACCTCCAACCCCGTCGAGCACAATTCCTCGAAGACTCCATIVAATATCACCAG
AAAA'PGACGGAATF
T1TCTACCAATFCCTCCAAGCAGTCGGCGGTGCCACCACGCACCAAAACATCAGCGATITCATCGACAATAAAGCCACC
GATGAACACCAAGCCACIVTCCTCITCCAAG
TAGTCTCCAAAGACAGCACAACACCAGAATGCCCCGCAGAAGAACIVCTAGCCCGATITGCCCAATACACCGGCAAACA
ACCCAATGAGGCTGTCACCCACTACCTGAC
CAGCAGAATCAATACAGATAAATACCGCTGGCAGGACAATCGACIVCTCGCCCAAAACATCGCTTCACAACTGAACATC
TCCGAAACIVAATFCCAAGAGATCGCFCAC
GCAATCCTGTCCAACAACCTATACATCGGTCAAACTGCATCCAACGCAGCAGCCAACTTCATCAGCCAAGTCACAGGCA
CAGGCCAGAAAGCCCCCAAGGCAGCACGG
CTCGATGTCCTGTFCCAGACCAACCAAGCCCTCGCCAAAACACAACCCACAACCITCGGCCAACFCCAACAGATCATCG
TACAAGCCTGCGGTGAATCCACCACCGATG
CAGTCCTCGCCAAATTCGGCAACAAAGGCGCTGCAACCAGCCTFCAACTGGCCCITAAAACCGACCCCAACACAACGCT
GGATCAGAAGAAGTACGAAGCCCTGCAAA
AGAAAT1TGCAGAGGACGAAACCAAATATCGCAACAAGGTCGATATCCCCCACAAGACCCAACTGCGCAACCTCATCCT
CAACACCTCAAACCAATTCTGCAACTGGCA
CACCAAGCCAGCCATCGAAGCCTITAAGTGCGCCATCGCTGACATCCAGTCCAAAGTCAGCAACAACCTCCGCATCATG
CAGGAAAAGGCCAAACTCTACGAGGCATTC
AGAAATGTCGATCCACAAGTCCAGATCGCCGTCCAAGCFCITGAAAACCACATGAACACACTTGAGGAACCCTACGCAC
CCTACGCCCACFCGTTCGGCAGCGTCAAAG
ACITCTACGAAGACCTCAACAACGGCFCCAACTTAGATGAGGCCATTCAAACCATCGTCCACGATFCCGACAACTTCAA
CAGGAAGCCAGACCCCAACTGGCFCCGCAT
CATCGCACCTCTCCACFCATCCCATFCCGCAAGCCAAATCATGGAGGCAGTAAAATACCTGTCCAGCAAACAGGATTAC
GAACIVCGTAAACCMCCCAITCGTCGCCA
CTAACCTGCCAGCAACCTACGGGAAATTTAACATTCCCGGCACCCTCAACCCACCCACCGACAGCCTTCACGGCAGACT
GAACGGTAGCCACTCCAATATGTGGCFCAC
AGCCCTGCFCCTCGACGGCAGGGA1TGGAAAAACCACCACCTITGCITCGCCTCAAGCCGCTACITCGAGGAGGTCTAC
ITCACAAACCCCAGCCTGCCCACTACAGAC
AAAGTCCGTAGCCCCAAATGCGCCITCACACIVAAGAGCGTGCTCGACFCCGAAGCCAAAGACAGGATTCGCAACGCFC
CCAAATCCCGCACCAAGGCCGTGAAAGCC
ATCGAACGCATCAAGGCCAACFCCACCCACAATGTGGCGTGGAACCCCGAAACCTCT1TCCAGATGCAGAAAAGAAACG
ATGAGTTCTACATCACCATCAACCACCGCA
TCGAAATGGAAAAAATCCCCGGTCAGAAAAAGACCGATGACGGITTCACAATCCACCCCAAAGGTCI'MCGCCATCCTC
AAGGAAGGCGACAGAATCCTGTCACAAG
ACCTCAACCAGACCGCAGCCACACATTGCGCCGTCTATGAAGTCGCCAAACCCGACCAGAACACCITCAACCACCACGG
CATIVACCTCAAGCTGAITGCCACAGAAG
AACIVAAAATGCCCCTCAAGACCAAAAAGTCCACAATCCCAGATGCCCTCTCCTACCAAGGCATCCACGCCCACGACCG
TGAAAACGGCTFACAACAACIVAAAGATGC
CTGCGGAGCTITCATCAGCCCCAGACTCGATCCCAAACAAAAGGCTACTTGGGACAACFCCGTCTCCAAGAAGGAGAAT
CTCTATCCATTCATCACCGCCTACATGAAAC
TCCTCAAGAAGGTCATGAAGGCAGGTCGTCAAGAACTGAAM-Trl ________________________________
11,AGGACACACCITGACCACATCCTCTITAAACACAACCTCAGCCCCCTCAAGCTGCACGGTGT
GTCCATGATCGGTCTGGAATCATCCAGAGCAACCAAATCCGTCATCAACACCITCTIVAACCITCAGAACGCCAAGACG
GAACAGCAGCAGATCGCCCTCGACCGACCC
CTGT7TGAGGCCGG1AAAACCCTCATCAACAACCAAACCCGCCGACGACAGGAAAGGGTCAGGTTAGAAACCAGTCTCA
CCATGAGACTGGCACACAAATACAACGCC
AAGGCAATCATCATCGAGGGTGAACTGCCACACFCCAGCACCGGAACCTCGCAGTACCAGAACAATGTCCGTCTGGACT
GGTCTGCCAAGAAATCCGCAAAGCTGAAA
ACCGAATCAGCCAACTGTGCAGGCATTGCCATATGCCAGATCGATCCGTGCCACACAAGCCACCAAAATCCC1TCCGGC
ACACFCCAACTAACCCAGACCTCAGACCAC
GATITGCGCAAGTCAAAAAGGGCAAAATGITCCAGTATCAACIVAATGGACTACAGAGGCTGCTCAACCCCAGAAGCAA
ATCCIVAACTGCCATCTACTACAGGCAGGC
AGTCCAAAG ____________________________________________________________________
MtiltiCGCCCACCACAACCTGACGGAGAGGGACATCACCTCTGCCAAATTCCCCAGCGATCTGGAGaaaaaaaTCAAG
GATGACACCTATCTGATFCCCCAG
AGAGGTGGTAGAATATACATCAGCAGCITCCCCGTCACTAGCMCGCCCGTCCCTGCACCAGCAACCATTATITCGGGGG
TGGACAAITCGAGTGCAATGCTGACGCTGT
CGCAGCCGTCAACATCATGCTGAAGG'ITCACCCGTAA
SEQ ID NO: 29>WaCas12i coding sequence
ATGCCCATTCGCGGATATAAATGCACTGTFGTCCCAAACGTACGCAAAAAGAAACiuriuGAAAAAACCTATAGCTACI
TACAAGAGGla ILA GATGTATITITTGATM
riunuAGTCTGTATGGTGGGATCGCCCCAAAAATGATTCCACAAGACCTGGGGATCAATGAACAAGTAATTTGTGCTGC
CAAITGGTFCAAAATTGTTGAAAAAACGAA
AGATTGCATCGCTGATGATGCGTTGITGAATCAATFTGCTCAATAITATGGGGAAAAACCCAATGAAAAGGITG'FTCA
ATFTFTGACGGCATCTTACAATAAAGACAAATAT
GMGGGTCGATTGI'CGI'CAAAAATMACACTCTGCAAAAGGATTTGGGAGI'CCAAAACCTAGAAAACGACCTGGAGTG
ITTGATFCGAGAAGATTTGTTGCCCGTAGG
AAGCGACAAAGAAGTFAATGGATGGCACTCGATATCAAAATFGTTFGGITGTGGAGAAAAAGAAGACAGAACAATFAAG
GCTAAAATFCTGAATGGCCTATGGGAAAGA
ATFGAGAAAGAAGATAITCTAACAGAAGAAGACGCAAGAAATGAACTATFGCACFCTGCTGGGGTGTTGACTCCAAAAG
AAMAGAAAAGTATATAAAGGGGCTGCTG
GTGGGCGTGATFGITATCACACGITGCTGGTAGATGGGAGAAAMCACFFTFAACCTTAAAACACFCATTAAGCAGACCA
AGGATAAATFAAAAGAAAAGTCTG'FTGAT
GITGAAATCCCCAATAAAGAAGCATMCGTCTATATCTCGAAAAACGAATFGGACGGTCMCGAGCAAAAGCCATGGAGCG
AAATGTATAAAACGGCCCTCTCAGCCGT
TATGCCAAAAAATACGCTAAATTATTGTITCGCCATFGATAGGCACGCCCAATATACAAAAATIVAAACACTAAAGCAG
CCATATGATFCGGCAATTACTGCCCTAAATGGG
ITIT1 _________________ apAGTCTGAATGCMACAGGCTCAGATG __________________________
I ITI IbTFATFFCFCCCFCCCATFFGGGGAAAACIUI
iAAAAAACTITATAATTACAAAGATMTGAATCTGGCATFAG
CGAAATMITGAAGATGAAGACAATAGMGCGATCTGGGGTAAATGTAAATITACTFAGATATATFITTACFCITAAAGAT
ATGTFITCTGCTGAGGATITCATCAAAGCG
GCAGAATATAATG1TGTATITGAACGCTACAACAGGCAAAAAGTCCACCCTACAGTAAAAGGGAATCAATCGITCACIT
FCGGCAATFCCGCATFGAGCGGTAAAGITATF
CCTCCATCAAAATGCTTGTCCAATFTGCCTGGACAAATGTGGCTGGCCATFAATCTACTFGACCAGGGCGAATGGAAAG
AACATCACATTCCTTFTCACAGTGCAAGATTC
93
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
TATGAAGAAATCTATCCAACAAGTGACAATCAAAATAATCCCGTAGATITGCGAACTAAACGTITMGCTGCFCTMAACA
AGACITITFCTGCTGCTGACATCGAAAAG
GTGAAAGAAAGTGCCAAGAAAAAACATGGCAAACCAGCTAAACGTATTITGAGAGCCAAAAACACCAATACAGCCGTAA
ATTGGGITGATMCGCTIITATCTMGAAA
AAACAGAGGITAACTTTAAAATTACTOTTAACTACAAACITCCAGACCAAAAGITGGGAAAATITGAACCAATMITGGG
ACGAAGATTITGGCTTATGACCAAAATCAA
ACCGCFCCTGATGCTFATGCGATTCTMAAATTMCGATGATACCGAACCITITGATTACAAGGGATATAAAATCAAATMI
TGTCTACTGGTGATTMGCITCAAAGTCAT
TGACCAAACAAACAGAAGITGATCAGCTAGCTFATAAGGGTGTGGACAAAACTACCAATITTFACAAAAAGTGGAAACA
CCAACGAAGGC GIVAAAACIVITAA
CATFCCAGATGCCCTAAAGACITITGAAAACATCAATAAAGAATATCITTATGGCTIVAACAATFCGTATCTGAAGITG
CTTAAACAAATTITACCGGGCAAATITGGACC
AATFCTMITGATATFCGACCAGAACTFATTGAAATGTGIVAGGGAATI'GGCTCTATCATGCGATI'GTCTAGTCTAAA
CCATGATAGITTGGACGCAATFCAATCTCTCAAAT
CCTMCITCACFCCTATTITGATCTCAAAGTAAAGGAAGAAATCAAAACAGAAGAATTGAGAGAAAAACCAGATAAAGAG
li 11 11 .1AACITGCTTCAACAAGTGATIVA
AAAACAAAAGAATAAACGCAAAGAAAAAGTFAATAGAACTGITGATGCCATTITGALTFRiGCGGCTGATGAGCAAGTA
CAAGTCATFGTAGGAGAGGGAGATCMGT
GTITCCACCAAAGGAACAAAAAAGAGACAAAACAACAGAACCATMATTGGTGTGCCAGAGCAGITGTGGAAAAACTAGA
AAAACCATCCAAACTACATGGGITGCAT
TTTAAGGAAATFCCACCACATTACACITCACATCAAGATMTTITGAACACAACAAGGATATTGAAAATCCAAAAGAAGT
CATGAAGTGTCGITIVAATACCACCGAAAA
TGTACCFCCITGGATGATCAAGAAAITCGCAAATTATCTFAAATGCGAAACAAAATATFATCITCAAGGAATGCAAGAT
TITCTAGAGCATTATGGTCTAGTAGAATACAAA
GATCACATCAAAAAGGGAAAAATCTCAATTGGGGATITTCAAAAACTTATCAAACTTGeltintiAGAAAGITGGAGAA
AAAGAGATTGTITITCCATGTAAAGGTGGTA
GAATCTATITGIVAACCTATI'GCTFAACAAATGAGTCTAAACCCATTli
____________________________ ITI
RAATGGCAGAAGATGCTATGITAATAATCCAGACCATCITGCTGCGATTAATGITGGCATF
TCFCTITMAATTITAATGCGAGAGCCAAGGTGGCGGAAAAAACCCCITGA
SEQ NO: 30>Wa2Cas12i coding sequence
ATGGCTAAGAAGGATITTATCGCTCGTCCCTACAATTCATFCCMCFCCCCAACGACAGAAAGCTMCITATCTGGAAGAA
ACTTGGACTGCCTACAAGTCAATCAAAAC
AGTACTGCACCGITFCCTCATCGCACCATACCGCGCTATFCCCITCCAGACCITTGCAAAAACCATCGAAAACACACAA
GAAGACGAATMCAATMGCATATGCCGITA
GAATCTIVAGACTAGTTCCAAAAGACITCTCCAAGAATGAAAACAACATACCCCCCGATATGCFCATTACCAACCITGC
TAGCTATACAAATATAAATCAATCACCAACCA
ATCFCTTGAGCTATGTAAACACCAACTACGATCCAGAAAAGTATAAGTGGATCGACTCACGCAACGAAGCCATCTCATI
'GTCCAAAGAAATCGCCATCAAACTCGATGAG
TTGGCAGACTACGCTACCACCATGCMGCGAGGACTGGCITCCACTFAACAAAGACACAGTCAACCGTMCGCCACCACTA
CCGGCCTATMCGCGCAGGaaaaaaaGAG
GATCGTACCCAAAAGGTACAAATGCTCAACGCATMCITTMCGGCTFAAAAACAACCMCCAAGGACTACAAACAGTATFC
GACCATCCITCTCAAGGCAMGATGC
CAAATCATGGGAAGAGGCTGITAAAAITTATAAAGGCGAATGCFCAGGTAGAACCAGTAGCTACCTGACAGAAAACCAT
GGAGACATITCCCCAGAAACTITGGAAAAA
CTAATTCAAAGTATIVAGAGAGATATMCTGACAAACAACACCCCATCAATCTACCTAAAAGAGAAGAAATTAAGGCATA
CTTGGAAAACCAGAGTGGTACTCCATACAA
TCTCAATCTCTGGTCACAAGCCCTACACAACGCTATCFCTFCTATCAAGAAGACAGATACTCGCAATITCAATACCACA
CTAGAAAAATATGAAAAAGAAATIVAACFCA
AGGAGTGCTFCCAAGATCGTGATGATGTAGAATTACTMGCAACAAATFCTITTCATCTCCATATCATAAGACCAACGAT
CFCITTGTCATTMCTCTGAGCATATCGCCAC
CAATCGCAAATACAATGTCGITGAGCAGATGTACCAACTCGCTACCGAACATGCCGATTITGAAACAGTG1TCACFCTC
CTCAAAGATGAATACGAAGAAAAAGGTATCA
AAACCCCAATCAAAAACATFCTTGAATACATITGGAACAACAAGAATGTGCCTGTAGGCACTFGGGGTAGAATTGCCAA
ATACAATCACCTGAAAGATAGATI'GGCTGGA
ATCAAAGCCAATCCTACCGITGAATCCAACCGTGGCATGACATTMGCAATFCTGCGATGGTMGCGAAGITATGCGATCC
AATCGCATITCGACCACCACGAAGAATAA
AGGCCAGATTITGGCCCAAATCCACAACGATAGGCCCGITGGGTCAAACAACATGATC1'GGCTGGAAATGACGCITIT
AAACAACCGGAAATGGCAAAAACACCACATC
CCGACCCACAATAATAAGTFCTITGAAGAAGTCCATGCTITCAATCCAGAACTGAACCAATCCGTGAATGTGCGAAATA
GAATGTATCGTFCTCAAAACTATFCGCAACTF
CCAACATCTCTGACCGATGGGCTGCAAGGCAACCCAAAAGCCAAGATITIVAACCGTCAATATCGTGCGCFCAATAACA
TGACCGCAAACGTGAITGATCCAAAGITGA
GMTATMTFAACAAAAAGGATGGCAGATFCGAAATTACCATCATTCACAATGITGAAGTGATCAGGGCCAGACGAGATCI
TCTGGTCGGGGATTACTTGGTCGCCATG
GATCAAAACCAGACTGCCACCAACACTTACGCTGTCATCCAGGTGGTTCACCCAAACACFCCTGACFCCCATGAATITC
GCAACCAATGGGTGAACTITATMAGAGTG
GCAAGATMAATCTTCTACFCTCAAITCTAGAGGCGAATACATMACCACITGAGTCATGATGGCGTGGATITGCAAGAAA
TCAAGGATFCTGAATGGATFCCAGCTGCTG
AGAAATFCTFAAACAAGTMCGAGCAATCAACAAGGACCGCACFCCAATCACCATCTCTAATACITCAAAGAGGGCTTAC
ACCTTCAACFCCATATATITCAAAATCTFAT
TGAATTATMCGTGCTAATGATCITGATCTGAAITTGGTGAGAGAGGAGATTCMCGTATI'GCCAACCGCAGMTITCGCC
CATGCGTCTGGGTAGTCTGTCGTGGACTA
CFCTFAAGATGITGGGCAACTITAGAAAITTGATTCATAGITATITCGATCACTGTGGTITCAAGGAAATGCCTGAAAG
GGAATCTAAAGACAAAACCATGTACGATCTGT
TGATCCATACCATCACAAAGCTGACAAACAACCGTGCCGAAAGAACGAGTAGGATMCTGOLRATMATGAATGTAGCCCA
TAAGTATAAAATTGGCACAACCGITGTG
CATGITGTCGITGAAGGCAGTCTAACCAAGACCGACAAATCCACCACCAAGGGTAATAACCGAAATACCACTGATI'GG
TGCFCAAGGGCTGTAGTCAAAAAGCTGGAA
GACATGTGCGIVTITTATGGGTFCAATITGAAACCAGTTFCGGCGCATFACACTAGTCACCAAGACCCATMGTFCATCG
GGCTGATFATGATGATCCCAAGCTTGCrritiC
GGTGTCGATATFCGTCGTATAGTCGGGCPGATTITGAAAAGTGGGG'PGAGAAGTCli
____________________ iTitiCTCCIUPGATFCGTPGGGCTACCGACAAAAAGAGCAATACTFGTTACAAG
GITGGGGCTGTGGAGTFCITTAAAAATTATAAAATCCCAGAGGACAAGATCACCAAGAAGCTGACCATAAAGGAATFCC
ITGAGATAATGTGTGCAGAGTCACACTATCC
GAATGAGTATGACGATATITMATFCCTCGCCGTGGAGGCAGGATITATCTGACAACGAAGAAGTMCMAGTGATFCGACC
CACCAAAGAGAAAGTGTGCATAGTCACA
CGGCMITGTCAAAATGAACCGGAAAGAGTATTATFCCTCAGATGCAGATGAGGTGGCTGCGATCAACATCTGCCTACAT
GACTGGGITGTCCCACTGAATMGACCAAT
94
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
CACTGCCTACCTGCTGGCTGGTGCTCTGACCACCTGAAAGAATGTGTGCAATGTCACACTCCAGACCCAGTACGAATAT
CCATGTAA
SEQ ID NO: 31>SiCas121 Codon optimized coding sequence
ATGAGTTCTGATGTGGTGCGGCCTTATAACACAAAGCTGCTCCCAGATAACAGAAAGCACAATATGTTCCTGCAGACCT
TCAAGCGGCTGAACAGCATCTCTCTGAACCA
CTTCGACCTGCTGATCTGCCTGTACGCTGCAATCACCAACAAGAAGGCCGAGGAATACAAGTCTGAAAAGGAAGCCCAC
GTGACCGCCGATAGCCTGTGTGCCATCAAT
TGGTTCAGACCCATGAGCAAGAGATACAGCAAATACGCCACCACCACCTIVAACATGTTAGAACTGTTTAAGGAGTACA
GCGGCCACGAGCCTGATGCCTATTCCAAGA
ACTACCTGATGAGCAATATCGACAGCGACAGATTCGTGTGGGTGGATTGTAGGAAGTTCGCTAAGGACTTTGCCTATCA
GATGGAACTGGGTTTCCACGAGTTCACCGTG
TTGGCCGAAACCCTGCTGGCTAATTCTATCCTGGTGCTGAACGAGAGCACCAAGGCCAATTGGGCTTGGGGAACCGTGT
CTGCCCTGTACGGCGGCGGAGATAAGGAGG
ACAGCACACTGAAGAGCAAGATTCTGCTGGCCTTCGTGGACGCCCTGAACAACCACGAGCTGAAAACAAAGAGAGAAAT
CTTGAATCAAGTGTGTGAATCTCTGAAAT
ACCAGAGCTACCAGGACATGTACGTGGATTTTAGAAGCGTGGTTGACGAAAACGGCAACAAGAAGTCTCCTAACGGCTC
TATGCCTATCGTGACCAAGTTCGAGACAGA
CGACCTGATCAGCGACAACCAAAGAAAGGCCATGATCAGCAACTTCACTAAGAACGCCGCTGCCAAGGCAGCTAAGAAA
CCTATCCCTTACTTGGACCGCCTGAAGGA
GCACATGGTGTCCCTGTGCGACGAGTACAATGTGTATGCCTGGGCCGCGGCCATCACAAACAGCAACGCCGACGTGACC
GCCCGGAATACCAGAAACCTGACATTCATC
GGCGAACAGAACAGCAGACGAAAGGAACTGAGCGTGCTGCAGACAACAACCAACGAGAAGGCTAAGGACATCCTGAACA
AGATCAACGACAACCTGATTCAGGAGG
TGCGGTACACCCCTGCCCCTAAGCACCTGGGCAGAGATCTGGCCAACCTGTTTGATACACTGAAGGAAAAGGACATCAA
CAACATCGAGAACGAAGAAGAGAAACAGA
ACGTGATCAATGACTGTATCGAGCAGTACGTGGACGATTGCAGAAGCCTCAACCGGAACCCCATCGCAGCCCTCCTGAA
GCACATCTCTAGGTACTACGAGGATTTCAGC
GCCAAGAATTTCCTGGACGGCGCCAAGCTGAACGTGCTGACTGAGGTGGTGAACCGGCAGAAGGCCCACCCCACCATCT
GGAGCGAGAAGGCTTACACCTGGATCAGC
AAGTTCGACAAGAACCGGAGACAGGCCAACAGCAGCCTGGTCGGATGGGTTGTGCCCCCCGAGGAGGTGCACAAGGAGA
AAATCGCCGGACAGCAGAGCATGATGTG
GGTGACCCTCACCCTGCTGGACGACGGCAAGTGGGTCAAACATCACATCCCCTTCAGCGACAGCAGATACTACAGCGAA
GTGTACGCCTACAACCCTAATCTGCCTTATC
TGGACGGAGGCATCCCAAGACAGAGCAAGTTCGGCAACAAACCAACAACCAACCTGACAGCCGAGTCCCAGGCCCTCCT
GGCTAATTCTAAGTACAAGAAAGCCAAC
AAGAGCTTCCTGCGGGCTAAAGAGAATGCCACACACAACGTGCGGGTGTCCCCTAACACCTCTCTGTGCATTAGACTGC
TGAAGGACAGCGCCGGAAACCAGATGTTC
GACAAAATCGGCAACGTGCTCTTCGGCATGCAGATCAACCACAAGATCACCGTGGGAAAACCTAACTACAAGATCGAGG
TGGGCGACAGATTCCTGGGCTTCGATCAGA
ACCAGAGCGAGAACCACACCTACGCCGTGCTGCAGAGAGTGTCCGAGAGCAGTCACGACACCCACCACTTTAACGGCTG
GGACGTGAAGGTGCTGGAAAAGGGCAAA
GTGACCAGCGATGTGATCGTGCGGGACGAGGTCTACGACCAACTGTCTTACGAGGGCGTCCCCTACGATAGCAGCAAGT
TCGCCGAGTGGCGGGACAAGCGCAGAAGA
TTTGTGCTTGAGAACCTGAGCATCCAGCTGGAAGAGGGCAAGACCTTCCTGACAGAGTTCGACAAGCTGAATAAGGACA
GCCTGTACCGCTGGAACATGAACTACCTG
AAACTGCTGAGAAAGGCCATCCGGGCCGGAGGCAAAGAGTTCGCCAAGATCGCTAAGACAGAGATCTTCGAGCTGGCGG
TGGAAAGATTCGGCCCTATTAACCTGGGC
AGCCTGTCCCAGATCAGCCTTAAGATGATTGCCTCCTTTAAGGGCGTGGTCCAGTCCTACTTCTCCGTGAGCGGCTGCG
TGGATGATGCCTCCAAAAAGGCCCATGATTCT
ATGCTGTTCACATTTATGTGCGCCGCCGAAGAAAAGCGGACCAACAAGAGAGAAGAAAAGACCAACAGAGCCGCCAGCT
TTATCCTGCAAAAAGCCTACCTGCATGGC
TGCAAGATGATCGTGTGCGAGGACGACCTTCCTGTGGCCGACGGCAAGACAGGCAAAGCCCAGAATGCCGACCGGATGG
ACTGGTGCGCCAGAGCCCTGGCCAAGAA
GGTGAACGACGGCTGTGTTGCCATGAGCATCTGCTACAGAGCTATCCCTGCCTACATGAGCAGCCACCAGGACCCCTTT
GTGCACATGCAGGATAAGAAAACCAGCGTG
CTGCGGCCTAGATTCATGGAAGTTAATAAGGATAGCATCAGAGACTACCACGTGGCGGGCCTGAGAAGAATGCTGAACA
GCAAGAGTGACGCTGGCACCAGTGTTTATT
ACCGGCAAGCTGCCCTGCATTTCTGCGAAGCCCTGGGCGTGAGCCCTGAACTGGTGAAAAACAAGAAAACCCACGCCGC
CGAACTGGGCAAGCACATGGGCAGCGCT
ATGCTGATGCCCTGGAGAGGCGGTAGAGTGTACATCGCCAGCAAAAAGCTGACCTCCGATGCCAAATCAGTGAAGTACT
GCGGCGAGGATATGTGGCAGTACCACGCCG
ATGAGATCGCCGCTGTTAACATCGCCATGTATGAGGTGTGCTGCCAGACCGGCGCTTTCGGAAAGAAACAGAAAAAATC
GGACGAGCTGCCTGGA
SEQ ID NO: 32>S12Cas 121 Codon optimized coding sequence
ATGAGCTCTGACGTGGTGCGGCCTTACAATACCAAGCTGCTGCCAGACAACCGGAAGTACAACATGTTTCTGCAGACCT
TCAAGAGACTGAACCTGATCTCCAGCAACC
ACTTCGACCTGCTGGTGTGCCTGTACGCCGCTATCACCAACAAGAAAGCTGAGGAATACAAGAGCGAAAAAGAGGATCA
CGTTACAGCCGACAGCCTGTGTGCCATCA
ACTGGTTCCGGCCTATGTCTAAGCGGTACATCAAGTACGCTACAACCACCTTTAAGATGCTGGAACTGTTCAAGGAGTA
CAGCGGCCACGAGCCTGACACCTACAGCAA
GAACTACCTGATGTCTAATATCGTGAGCGATAGGTTCGTGTGGGTGGACTGCCGGAAATTCGCTAAGGACTTCGCCAAT
CAAATGGAACTGTCCTTCCACGAGTTCACCA
CCCTGAGTGAAACCCTGCTGGCTAACAGCATCCTGGTGCTAAATGAGTCTACAAAGGCCAACTGGGCCTGGGGCGCCGT
GAGTGCTCTGTACGGCGGCGGCGACAAAG
AGGACTCTACACTGAAAAGCAAGATCCTTCTGGCCTTTGTGGACGCCCTGAACAACCCTGAACTGAAAACACGTAGAGA
AATTCTGAACCACGTGTGCGAATCTCTGAA
GTATCAGAGCTACCAGGACATGTACGTCGATTTCAGAAGCGTGGTCGATGATAAGGGCAACAAGAAGAGCCCAAACGGC
AGCATGCCTATCGTGACCAAGTTCGAGAGC
GATGATCTGATCGGCGATAACCAGAGAAAGACAATGATCTCTAGCTTTACGAAGAACGCCGCCGCCAAGGCCAGCAAGA
AGCCCATCCCATACCTGGACATCCTCAAGG
ACCACATGATCAGCCTGTGTGAAGAGTACAACGTGTATGCCTGGGCCGCTGCCATCACCAACAGCAACGCCGACGTGAC
AGCCCGCAACACCAGAAACCTGACATTCAT
CGGAGAACAGAACACCCGGAGGAAGGAACTGAGCGTGCTGCAGACAAGCACCAACGAGAAGGCTAAAGACATCCTGAAC
AAAATCAACGACAACCTGATCCCTGAG
GTGCGGTACACACCTGCCCCTAAGCACCTGGGTCGGGACCTGGCCAATCTGTTCGAGATGTTCAAGGAAAAGGACATCA
ACCAGATCGGCAACGAGGAGGAGAAGCAG
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
AACGTGATCAACGACTGCATCGAACAGTACGTGGACGACTGTAGAAGCCTGAACAGAAACCCAGTGGCCGCCCTGCTAA
AGCACATCAGCGGATACTACGAGGATTTC
AGCGCCAAAAATTTCCTGGACGGCGCCAAGCTGAATGTGCTGACCGAAGTGGTCAACAGACAGAAGGCTCATCCTACAA
TCTGCAGCGAAAAGGCCTACACCTGGATT
AGCAAGATCGATAAGAACCGGCGGCAGGCCAATTCCTCCCTGGTCGGATGGGTGGTGCCCCCCGAGGAAGTGCACAAGG
AAAAGATTGCCGGCCAGCAGAGCATGATG
TGGGTGACACTGACACTGCTGGACGACGGCAAGTGGGTTAAGCACCACATCCCCTTCGCCGATTCTAGATACTACAGCG
AGGTGTATGCCTATAATCCTAACCTGCCTTAT
CTCGAGGGCGGCATCCCCAGACAGTCTAAGTTTGGCAACAAACCTACCACCAACCTGACCGCCGAATCTCAGGCCCTGT
TGGCCAACTCCAAGCACAAAAAAGCCAAC
AAGACCTTCCTGAGGGCCAAAGAGAACATCACCCACAACGTGAGAGTGTCTCCTAATACCAGCCTGTGCATCAGACCAC
TGAAGGACTCTGCTGGCAATCAAATGTTCG
ACAACATCGGCAACATGCTGTTCGGTATGCAGATCAACCATAGAATCACCGTAGGAAAACCCAACTACAAGATAGAGGT
GGGCGATAGATTTCTCGGATTCGACCAGAAT
CAGAGCGAGAACCACACCTACGCAGTGCTGCAAAGAGTATCTGAGAGCAGCCACGGCACACACCACTTTAACGGCTGGG
ACGTGAAAGTGATCGAGAAGGGCAAGGT
GACCAGCGACGTGGTGGTGCGGGACGAGGTGTACGATCAGCTGTCCTACGAAGGCGTTCCTTACGACTCCCCTAAGTTT
ACCGAATGGCGGGAAAAACGGAGAAAGTT
CGTGCTGGAAAACATGAGCATCCAGATCGAGGAGGGCAAGACTTTTCTGACCGAGTTCGATAAGCTGAATAAAGACAGC
CTGTATAGATGGAACATGAACTACATGAAA
CTGCTGAGGAAGGCCATCAGAGCCGGCGGAAAAGAGTTCGCCAAGATCACCAAGGCCGAGATCTTCGAACTGGGCGTGA
TGAGATTCGGGCCTATGAACCTGGGCAGC
CTGAGCCAAGTGAGTCTCAAGATGATCGCCGCCTIVAAGGGAGTGATCCAGAGCTACTTCTCTGTGTCTGGCTGCATCG
ATGATGCTTCCAAGAAGGCCCACGACAGCA
TGCTGTTCGCCTTCCTGTGTAGCGCCGATGAAAAGCGGACCAACAAGCGGGAAGAAAAGACCAATCGGGCCGCCAGCTT
CATCCTIVAAAAGGCCTACTCCCACGGCT
GTAAAATGATTGTGTGCGAGGACGACCTTCCTATCGCCGATGGCAAAGTGGGAAAGGCCCAGAACGCCGACAGAATGGA
CTGGTGCGCCCGGAGCCTGGCTAAGAAAG
TGAACGATGGCTGCGTGGCCATGTCCATCTGCTACAGAGCCATCCCCGCCTACATGAGCTCCCACCAGGACCCCTTCAC
CCATATGCAGGATAAGAAAACCAGCGTGCTG
CGGCCTAGATTTATGGAAGTTGGCAAGGACAGCATCCGGGACCACCACGTGGCTGGCCTGAGACGGATGCTGAATAGCA
AGGGCAACACAGGCACCAGCGTGTACTAC
AGAGAGGCCGCACTGCGCTIVTGCGAGGCCCTGGGCGTGCTGCCTGAGCTGGTGAAGAATAAGAAAACACACGCCAGCG
AGCTGGGAAAGCATATGGGCAGCGCAAT
GCTGATGCCTTGGAGAGGCGGCAGAATCTACGTGGCCAGCAAGAAACTGACAAGCGACGCCAAATCTATCAAGTACTGC
GGCGAGGATATGTGGCAGTACCACGCCGA
CGAGATCGCTGCTATCAACATCGCCATGTACGAGGTC
SEQ ID NO: 33 >WiCas 12i Codon optimized coding sequence
ATGGGCATCTCTATCAGCAGACCTTACGGCACCAAACTGCGGCCTGATGCCAGAAAGAAAGAAATGCTGGATAAATTCT
TCACCACCCTGGCCAAAGGCCAGAGAGTGT
TCGCCGACCTGGGCCTGTGCATCTACGGCAGCCTGACACTGGAGATGGTGAAAAGACTGGAGCCTGAGAGCGACAGCGA
GCTGGTGTGCGCCATCGGCTGGTTCCGGC
TGGTGGATAAAGTGACCTGGAGCGAAAACGAGATCAAGCAGGAAAACCTGGTGCGGCAGTACGAAACCTACTCTGGCAA
GGAAGCCAGCGAGGTGATCAAGACCTAT
CTGAGCAGTCCCTCTTCTGATAAGTACGTGTGGATAGATTGCAGACAGAAGTTTCTGCGGTTCCAGCGGGACCTGGGCA
CAAGAAACCTGTCCGAGGATTTCGAGTGCA
TGCTGTTCGAGCAGTATCTGAGACTGACTAAGGGCGAGCTGGATGGACACACCGCCATGAGCAATATGTTCGGCACCAA
GACAAAGGAGGATAGAGCCACCAAGCTGC
GATACGCCGCCAGAATGAAGGAGTGGCTGGAAGCTAATGAGGAGATCACCTGGGAACAGTACCACCAGGCCCTGCAGGA
TAAGCTCGACGCGAACACTCTGGAGGAAG
CCGTGGATAACTACAAGGGCAAGGCTGGCGGAAGCAACCCTTTCTTTAGCTACACCCTGCTGAACCGAGGACAGATCGA
CAAGAAAACCCACGAGCAGCAGCTGAAGA
AGTTCAACAAGGTGCTGAAAACCAAGTCTAAGAACCTGAACTTCCCTAACAAAGAGAAGCTAAAGCAGTACCTCGAGAC
AGCGATCGGAATCCCCGTGGACGCTCAGG
TGTACGGCCAGATGTTTAACAACGGCGTGTCTGAGGTTCAACCTAAGACAACCAGAAACATGTCCTTTAGCATGGAAAA
GCTGGAGCTCCTGAACGAACTGAAGAGCCT
GAACAAGACCGACGGATTCGAGAGAGCCAACGAGGTGCTCAATGGCTTCTTCGACAGCGAACTGCACACAACAGAGGAC
AAATTCAATATCACAAGCAGATACCTGGG
CGGCGACAGAAACAACCGGCTCCCTAAGCTGTATGAGTTGTGGAAGAAGGAGGGCGTGGACAGAGAGGAGGGCATCCAG
CAATTTTCCCAAGGCATCCAGGACAAGAT
GGGCCAAATCCCTGTTAAGAACGTGCTCCGCTACATCTGGGAGTTCCGGGAAACCGTGAGCGCAGAAGATTTCGAGGCT
GCTGCCAAGGCCAACCAGCTGGAGGAAAA
GATCACCCGGACCAAAGCCCACCCCGTCGTGATCAGCAACAGATACTGGACCTTCGGGTCCAGCGCCCTGGTGGGCAAC
ATCATGCCTGCCGACAAGATGCACAAGGA
CCAGTACGCCGGCCAGAGCTTTAAGATGTGGCTGGAAGCTGAGCTGCACTACGACGGCAAgAAGGTGAAGCACCACCTG
CCCTIVTACAATGCCAGATTCTTCGAG GAG
GTGTACTGCTACCACCCATCAGTGGCCGAAGTGACCCCTTTTAAGACCAAGCAGTTCGGATATGCCATCGGCAAGGACA
TCCCAGCTGACGTGTCTGTGGTGCTGAAAGA
TAACCCCTACAAGAAGGCCACCAAGAGATTTCTGAGGGCCATCAGCAATCCAGTCGCCAACACTGTGGACGTGAACAAG
CCTACAGTGTGTAGCTTCATGATCAAGCGG
GAAAACGACGAGTACAAGCTGGTGATCAACAGAAAgATCGGAGTGGACAGACCCAAGAGAATCAAGGTGGGCAGAAAAG
TGATGGGCTACGACAGAAACCAGACCGC
CAGCGACACATATTGGATCGGCGAGCTGGTTCCTCATGGGACCACAGGCGCCTACAGAATCGGAGAATGGAGCGTGCAA
TACATTAAAAGCGGCCCTGTGCTTTCTTCTA
CACAGGGCGTGAACGATTCTACCACCGATCAGCTGATCTACAACGGAATGCCCAGCAGCAGCGAGCGGTTCAAGGCCTG
GAAGAAGTCCAGAATGAGCTTCATCCGGA
AGCTGATCAGACAGCTGAATGCCGAAGGCCTGGAAAGCAAAGGACAGGACTACGTGCCCGAGAACCCTAGCAGCTTCGA
CGTCAGAGGAGAAACACTGTACGTGTTTA
ACAGCAACTACATGAAAGCCCTGGTGTCCAAGCACAGGAAGGCCAAgAAGCCCGTGGAAGGCATCCTGGAAGAAATCGA
GGCTCTGACCTCCAAAGCCAAGGACAGC
TGCAGCCTGATGCGCCTGAGCTCTCTGAGCGACGCCGCCATGCAGGGCATCGCCAGCCTGAAGTCCCTGATCAACTCTT
ATTTCAACAAGAATGGCTGTAAAACCATCGA
GGACAAGGAAAAGTTCAACCCCGACCTGTACGTGAAGCTGGTCGAGGTCGAACAGAAAAGAACCAACAAGCGGAAGGAG
AAGGTGGGCCGGATCGCCGGCAGCCTG
GAACAGCTCGCCCTGCTGAATGGTGTTGACGTGGTGATCGGCGAGGCCGATCTGGGGGAAGTCAAGAAAGGCAAGTCTA
AgAAGCAGAATAGCAGAAACATGGACTGG
TGCGCCAAGCAGGTCGCTGAGCGCCTGGAATACAAACTGACCTTCCACTGTATCGGCTACTTCGGCGTGAACCCTATGT
ACACAAGCCACCAAGATCCTTTTGAACACCG
96
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
GAGAGTGGCCGACCACCTGGTGATGAGAGCTAGGTTCGAAGAGGTGAACGTTAGCAACGTAAGCGAATGGCACATGAGA
AACTTCAGCAATTACCTGCGGGCCGACAG
CGGCACAGGTCTGTACTACAAGCAAGCCACCCTGGACTTIVTGAAACATTACGACCTGGAGGAGCACGCCGACGACCTG
GAGAAACAGAATATCAAGTTCTACGATTTC
AGAAAGATCCTGGAGGACAAGCAGCTGACATCTGTTATAGTGCCTAAGCGGGGCGGCAGAATCTACATGGCCACAAACC
CCGTGACATCAGACAGCACCCCTGTGACCT
ACGCCGGCAAGACCTACAATAGATGCAACGCCGATGAGGTGGCTGCCGCTAATATCGCTATTTCTGTGCTGGCCCCTCA
CAGCAAGAAGGAAGAgAAAGAGGATAAGAT
CCCTATCATCAGCAAGAAGCCTAAGTCCAAGAACACCCCAAAGGCTAGAAAGAACCTGAAAACAAGCCAGCTGCCTCAG
AAG
SEQ ID NO: 34>Wi2Cas121 Codon optimized coding sequence
ATGGCCAGCAAACACGTGGTGCGGCCTTTTAACGGCAAAGTGACCGCTACCGGCAAGCGGCTGGCCTACCTGGAGGAAA
CCTTTCATTACCTGGAGAAGGCCGCCGGC
GGCGTGTCTACCCTGTTCGCCGCTCTGGGCAGCTACCTCGACGCCACAACCATCAGCAACCTGATCAACAAgAACCAGG
ACTTGGCTGTCGTGATCTTCCGGTACCACGT
GGTGCCTAAGGGCGAAGCCCACACACTGCCCGTGGGCACCGACATGGTGTCAAGGTTCGTGGCCGACTACGGCATGGAG
CCTAATGAGTTCCAAAGAGCCTACCTGGAT
AGCCCCATCGATCAGGAGAAGTACTGCTGGCAGGACAATCGGGACGTGGGATGTTGGCTGGGCGAACAGCTGGGTGTTT
CTGAGGCCGACATGCGGGCTATCGCCGTG
ACTTTTTACAACAACCAGATGCTGTACGACTGTGTGAAGGGAACTGGCAGCGGCAATGCCGTCTCTCTGCTGTTTGGCA
GCGGCAAgAAGTCCGACTACAGCATGAAGG
GAGTCATTGCCGGCAAGGCTGCCTCAGTGCTGGCTAAGTATAGACCTGCCACCTACCAGGATGCCAGAAAGATGATCCT
GGAAGCTAATGGCTIVACCAGCGTGAAAGA
TCTGGTCACATCTTACGGCATCACCGGCAGAAGCAGCGCCCTGCAAATCTTCATGGAAGGCATTGAAAGCGGACCTATC
TCCTCCAAAACATTGGACGCCAGAATCAAG
AAGTTCACGGAAGATAGTGAGCGGAACGGCCGCAAGAACCTGGTCCCCCACGCCGGCGCCATTAGAAATTGGCTGATCG
AGCAGGCCGGTTCTIVTGTGGAAAACTAC
CAAATGGCCTGGTGCGAGGTTTACGGCAACGTGAGCGCTGACTGGAACGCCAAGGTGGAAAGCAACTTCAACTTCGTGG
CCGAGAAGGTGAAAGCCCTGACCGAGCT
GAGCAATATCCAGAAGAGCACCCCTGATCTGGGCAAGGCTCTGAAACTGTTTGAGGAGTACCTGACCACATGCCAGGAC
GAGTTCGCCATCGCCCCATACCACTTCAGC
GTGATGGAAGAGGTGCGGATGGAAATGGCCACAGGCAGAGAGTTTAACGATGCATACGACGACGCTCTGAACAGCCTGG
ACATGGAAAGCAAGCAGCCTATCCAGCCT
CTGTGTAAATTCCTGATCGAGCGGGGCGGAAGCATCAGCTTCGACACCTTCAAGAGCGCCGCCAAATACCTGAAAACCC
AGAGCAAGATTGCCGGCAGATACCCTCATC
CATTCGTGAAGGGAAACCAGGGCTTCACATTCGGCTCCAAgAACATCTGGGCCGCCATAAACGACCCCATGATGGAGTA
CGCCGACGGCCGGATCGCCGGCGGCTCTGC
CATGATGTGGGTCACCGCTACCCTGCTGGACGGCAAGAAGTGGGTGAGACACCACATCCCCTTCGCCAACACAAGATAC
TTCGAGGAGGTTTACGCCAGCAAGAAGGG
CCTGCCTGTCCTGCCGTGCGCCAGAGATGGCAAGCACAGCTTTAAGCTGGGTAACAACCTGAGCGTGGAGAGAGTGGAA
AAGGTGAAGGAAGGCGGCAGAACAAAGG
CCACAAAGGCTCAGGAGAGAATCCTGAGCAACCTGACACACAACGTGCAGTTCGACAGCAGCACCACCTTCATCATCCG
GAGACAGGAGGAATCCTTTGTGATCTGCG
TGAACCACAGACACCCCGCCCCTCTGATGAAgAAGGAGATGGAAGTGGGCGACAAGATCATCGGCATCGACCAGAACGT
GACCGCCCCTACCACCTACGCCATCGTGGA
GAGGGTGGCCAGCGGAGGCATCGAGCGGAACGGCAAACAGTACAAGGTGACAGCCATGGGCGCCATCTCCTCTGTGCAG
AAAACCAGAGGCGGAGAGGTGGACGTGC
TGAGCTACATGGGTGTGGAGCTGTCCGACTCGAAGAACGGATTCCAGAGCCTGTGGAACAAGTGTCTGGACTTCGTGAC
CAAGCACGGCACAGAGAACGACGTGAAGT
ACTACAACAACACAGCCGTGTGGGCCAACAAGCTTTACGTGTGGCACAAGATGTACTIVAGACTGCTCAAGCAACTGAT
GAGAAGAGCCAAGGACCTGAAGCCTTTCA
GAGATCACCTGCAACACCTGCTGTTCCACCCTAACCTGTCTCCTCTGCAGCGGCATAGCCTGTCTCTTACAAGCCTGGA
GGCTACCAAGATCGTGCGCAATTGCATCCAC
AGCTATTTCAGCCTTCTCGGGCTGAAAACCCTGGATGAGAGAAAGGCAGCCGACATCAACCTGCTCGAGGTGCTGGAAA
AGCTGTATGCCGGCCTTGTGGAAAGAAGG
AAGGAGAGAACCAAGCTGACAGCCGGCCTGCTGGTCAGACTGTGCAACGAGCACGGAATTAGCTTTGCCGCCATCGAAG
GCGACCTGCCTGTGGTGGGCGAAGGCAA
GAGCAAGGCCGCTAACAACACCCAGCAGGACTGGACCGCCCGGGAACTGGAGAAGAGACTGAGCGAAATGGCTGAGGTG
GTGGGCATCAAGGTGATCGCTGTTCTAC
CACACTACACCAGCCACCAGGACCCTTTCGTTTACTCCAAGAATACCAAGAAAATGCGGTGCAGATGGAATTGGCGGAC
CACCAAGACCTTCACCGATAGAGATGCCCT
GAGCATCCGGAGAATCCTGAGCAAGCCCGAAACCGGAACCAACCTGTATTACCAGAAGGGACTGAAGGCCTTCGCCGAG
AAGCACGGCCTGGATCTGGCCGAAATGAA
GAAGCGGAAGGACGCCCAGTGGTACCTGGAAAGAATCCAGGATAAGAACTTCCTGGTGCCCATGAACGGCGGAAGAGTG
TACCTGAGCAGCGTGAAGCTGGCCGGCA
AAGAGACAATCGACATGGGCGGCGAGATTCTGTACCTGAACGACGCCGATCAGGTGGCCGCCCTCAACGTGCTGCTGGT
GAAGATC
SEQ ID NO: 35 >Wi3Cas121 Codon optimized coding sequence
ATGGCCAAAAAGGAACACATTATCAGACCTTTCAAGGGCACCCTGCCACTGCGGGGGGACAGACTGAGATACCTGCAGG
ACACCATGAAGTACATGAAGAAGGTTGAG
GACACCATCACCGAGCTGTGCGCCGCCGTGATCGCCTACGCCAAGCCTACAATCATCCAGCAGATTCTGGGAGAAGAAA
TCGAGACTACCTCCACCTTCTGCAGCTTCA
GACTGGTTGGGATTCATGAGAACTTCACTATGCCCCTGACAACCAATATGATCAAGCACTTCCAGAAAACCTTCAACAT
CAATCCTIVTGAGAAGCAGGCCATCTATCTG
AGCAGCGGATTTGATAGCGACAAATACAGATGGCAGGATACAAGCGAGGTGTCTAGAAATTTCGCTAATAAGTGCCGCC
TGACCAACCAGGAGTTCCAGGAGTTCGCCG
AGCAAGCTCTGTTAAACATGTGCTTTATCGGCTGTAGCGGATCTCCTGGCGCCACAAACGCCGTGTCCCAGATCTTCGG
CACCGGCGAAAAGTCTGATTACCAGCGGAA
GTCTCAGATCGCCAAGATCGCCGCTGATACCCTCGAGAACCACAAACCTAGCACATACGAGTCTGCTAGGCTGATGGTG
CTGAACACACTGGGACACAAGACGATCGAA
GATTGCGTGAACGACTACGGCGCTATTGGAGCCAAGTCCGCCTTCCGGCTGTTTATGGAAAGTAAAGAAATCGGCCCAA
TCACCAGCGAACAACTGACCACAAAAATCA
AGAAATTCAGAGAGGACCACAAGAAGAACAGCATCAAGAAGCAGCTGCCTCATGTGGAAAAGGTGCGGAACGCACTACT
GAGCCAGTTCAAGGAGCAGTACCTGCCA
AGCGCCTGGGCCGAGGCCTGGTGTAACATCATGGGAGAGTTCAATAGCAAGCTGTCCAACAACAACAATTTCATCGACC
AAAAAACCAAGATGGTCAACGACTGCGAC
97
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
AACATCAAAAAATCTAACCCCCAGCTGGATAAGGCCGTGAATATGCTGGACGAATGGAAGTACAAGAATTGGGACGACA
ATTCTGCCATCCACCCCTACCACATCGGCGA
TCTGAAAAAGCTGATGGCCATCTTCAACATCAACAATGAGGGCACCTTCGACGAGAGATTCAGCGCCAGCTGGGAGCAG
TTTTCTACCAGCCTGGAGTACGGCGAGAA
GCCCCCCGTGCGGGACCTGCTGGCCCACATCATCAAGAACATGAACGACCTGACTTACACCGACGTGATCAATGCCGCT
AAGTTCCTGAAGCTGCAAGATAATATCAGAA
ACAAGTATCCTCACCCTTTTGTGATGCCTAACAAGGGATGTACCTTCGGCAAGGATAACCTGTGGGGCGAGATCAATGA
TCCTACAGCTAAGATCAAGTCCACAGAGGAA
GTGGCCGGCCAGCGGCCTATGATGTGGCTGACCGCCAAGCTCCTGGACAACGGCAAATGGGTCGAGCACCATATCCCCT
TCGCCTCTAGCAGATACTTCGCCGAAGTGTA
CTACACCAACCCCGCCCTGCCTACCTTACCCATCGCCCGCGACGGCAAGCACAGCTACAAGCTGACCAAGACCATCGAC
GCCAACACCGCCAAAACCCTGGTGAACAA
CCCTAGAGACAAGGCCGCCAAGCTCATTGCCAGAACAAAGGCGAACACCACCCACAACGTGAAGTGGATCAAACCTACA
TACAGAATCCAGAAAGAGAACAACCAGT
TCGTGATCACCATCAATCACAGACACCCATGTATCACCCCTCCTAAGGAAATCATCTTGGGCGATAGAATCCTGTCATT
CGACCAAAACGAGACAGCCCCTACCGCCTTTA
GCATCCTGGAAAAGACCACCAAGGGCACAGAGTTCTGCGGCCACCACATCAAAGTGCTGAAAACCGGCATGCTGGAAGC
CAAGATCAAGACATCGAAGAAATCCATCG
ACGCCTTCACCTACATGGGCCCTATGGAGGACGACCACGCCAGCGGTTTCCCCACCCTGCTGAACATCTGTGAAAAGTT
CATCAGCGAGAACGGCGACGAGAAGGACA
AGAGCTTCAGCAGCAGAAAGCTGCCTTTTAAGAGAAGCCTGTATTTTTTCCACGGCAGCCACTTCGACCTGCTGAAGAA
GATGATCCGGAAGGCTAAAAATGACCCTAA
GAAACTGAAGCTGGTGAGAATCCACATCAACGAGATCCTATTCAACAGCAACCTGTCCCCTATCAAGCTGCACAGCCTG
AGCATCCACTCTATGGAGAACACAAAAAAG
GTGATCGCTGCCATCTCTTGCTACATGAACGTACACGAGTGGAAAACCATCGATGAGCAAAAAAACGCCGACATCACAC
TGTACAACGCCAAGGAAAAGCTGTACAACA
ACCTGGTTAATAGAAGAAAGGAAAGAGTGAAGGTGACCGCTGGCATGCTGATCCGGCTGGCCCGGGAAAACAACTGCAG
ATTCATGGTGGGCGAAGCCGAACTGCCAA
CACAGCAGCAGGGCAAGAGCAAGAAGAACAACAACAGCAAGCAGGACTGGTGCGCCAGAGACATCGCACAGAGATGCGA
GGATATGTGCGAGGTGGTGGGCATCAA
ATGGAACGGCGTGACACCTCACAACACCAGCCACCAGAATCCATTCATCTACAAGAACACCTCCGGCCAGCAGATGCGG
TGCAGATACAGCCTGGTCAAAAAGTCTGA
GATGACCGATAAGATGGCTGAGAAGATCCGGAACATTCTGCACGCCGAGCCTGTGGGCACAACCGCTTATTACAGAGAG
GGCATCCTGGAGTTTGCCAAGCACCACGGA
CTGGACCTGGGCATGATGAAGAAAAGAAGAGATGCCAAGTATTACGACAACCTGCCCGACGAATTTCTGCTGCCGACAA
GAGGCGGAAGAATATACCTGTCGGAAAAC
CAGCTGGGCGGCAACGAGACAATCGTGATCAACGGCAAGAAATACTTCGTGAATCAGGCCGACCAGGTGGCCGCCGTGA
ACATAGGGCTGCTGTACCTGCTGCCTAAG
AAGAACCAGAGC
SEQ ID NO: 36>SaCas 12i Codon optimized coding sequence
ATGAGCGAGAAGAAATTCCACATCAGACCCTACAGATGCAGCATCTCCCCTAACGCCCGGAAGGCCGACATGCTGAAGG
CTACCATCTCCTACCTGGACAGCCTGACCT
CTGTGTTCAGAAGCGGGTTTACCGCCCTGCTGGCTGGAATCGATCCTAGCACCGTGTCCAGGCTGGCTCCTAGCGGCGC
CGTGGGCAGCCCCGACCTGTGGAGCGCCGT
GAACTGGTTCAGAATCGTGCCCCTGGCCGAAGCCGGCGATGCCAGAGTCGGCCAGGCAAGCCTGAAAAACCTGTTTAGA
GGCTACGCCGGGCACGAACCTGACGAGG
AAGCCAGCATCTACATGGAAAGCAGAGTGGACGACAAACGGCACGCCTGGGTCGACTGCAGGGCCATGTTCAGAGCTAT
GGCCCTCGAGTGCGGCCTGGAGGAAGCCC
AGCTGGCTTCCGACGTGTTCGCCCTGGCCAGCAGAGAGGTGATCGTGTTCAAGGACGGCGAAATCAACGGCTGGGGCAT
CGCCAGTCTGCTGTTCGGCGAAGGAGAGA
AGGCTGATTCTCAGAAAAAGGTGGCCCTGCTGAGAAGCGTGAGACTGGCCCTCGAGGGCGATTACGCTACCTACGAGGA
GCTGTCTGGCCTGATGCTGGCCAAGACCG
GCGCCAGCTCTGGCTCCGATCTGCTGGACGAGTACAAACGGTCCGAAAAAGGTGGCTCTTCTGGAGGCAGACATCCTTT
CTTTGACGAGGTGTTTCGGAGAGGCGGCA
GAGTTAAACAGGAGGAAAGAGAGAGACTCCTGAAAAGCTGCGACACCGCAATCCAGAAGCAGGGACAGGCCCTGCCTCT
GTCTCACGTGGCCAGCTGGCGGCAGTGG
TTCCTGAGAAGAGTGACCCTGCTGAGGAATAGACGGCAGGAGAGCTTCGCTGTGTGCATCACAAACGCCCTGATGGACC
TGCAACCCAAGAACCTGAGAAATGTGCAC
TACGTGACCAACCCCAAGAGCGAGAAGGATAAGGGGGTTCTGGAACTGCGGGTGGACGTCAAAAACAACGAGGGCCCTG
ATGTGGCTGGCGCCCAAGCCGTGTTTGA
CGCCTACATGGCCAGACTTGCCCCAGATCTGAGATTCAGCGTGATGCCTAGACATCTGGGCTCACTGAAGGACCTGTAC
GCCTTGTGGGCCAAGCTGGGAAGAGATGAG
GCGATCGAGGAGTACCTGGAAGGCTATGAGGGCCCTTTCAGCAAAAGACCAATCGCCGGCATCCTGCAGATCATCCACG
CCCATCGGGGCAAGGTGGGGCACGACAGC
CTGTTGAGAGCCGCCAGACTTAACAGAGCTATGGATAGACTGGAGAGAAAAAGAGCCCACGCCTGTGCCGCCGGCAACA
AGGGATATGTGTACGGCAAGAGCAGCATG
GTGGGCCGGATCAACCCTCAGAGCCTTGAAGTGGGCGGACGGAAGTCTGGCCGGAGCCCCATGATGTGGGTGACACTGG
ACCTGGTCGACGGCGACAGATTCGCCCAG
CACCACCTGCCCTTTCAATCTGCCCGGTTCTTCAGCGAAGTGTACTGCCACGGAGACGGCCTGCCCGCCACCAGAGTGC
CAGGCATGGTCAGAAACCGGAGAAATGGC
CTGGCCATCGGAAATGGCCTGGGCGAGGGAGGACTGAGTGCTCTGAGAGCCGGAAGCGACCGGAGAAAGCGGGCTAACA
AGAGAACACTGAGAGCCCTGGAGAATAT
CACCCACAACGTGGAAATCGATCCTAGCACATCCTTCACACTGAGAGAGGACGGCATCATCATCAGCCACAGAATCGAG
AAGATCGAGCCTAAGCTGGTGGCTTTTGGA
GACAGAGCTCTGGGCTTCGACCTGAACCAGACCGGCGCCCACACCTTTGCCGTGCTGCAGAAGGTGGACAGCGGCGGGC
TGGATGTGGGTCACAGCCGGGTCAGCATT
GTGCTGACCGGCACCGTGCGGAGCATCTGCAAGGGCAATCAGGCCAGCGGGGGCCGGGACTACGACCTGCTGTCTTACG
ACGGCCCCGAGAGAGATGATGGCGCTTTT
ACCGCCTGGAGGTCTGACAGACAGGCCTTTCTGATGAGCGCCATTCGGGAACTGCCTACCCCTGCCGAGGGCGAGAAAG
ATTACAAGGCCGACCTGCTGTCCCAGATG
GCCAGCCTGGACCACTACCGGAGGCTGTACGCCTACAACAGAAAGTGCCTGGGCATCTACATCGGTGCCCTGCGGCGCG
CCACAAGACGGCAGGCCGTTGCCGCCTTC
AAGGACGAGATTCTGTCCATCGCCAACCACAGATGCGGCCCCCTGATGAGAGGCTCCCTGAGCGTCAACGGCATGGAAA
GCCTGGCCAACCTGAAGGGCCTGGCAACC
GCTTATCTGTCTAAGTTCAAGGACAGCAAGTCCGAGGACCTGCTGAGTAAGGACGAAGAAATGGCCGACCTGTACAGAG
CTTGCGCCAGACGCATGACCGGAAAAAGA
AAGGAACGGTACCGGCGTGCTGCCAGCGAAATCGTGAGACTGGCTAACGAGCACGGCTGTCTGTTCGTGTTCGGCGAGA
AGGAACTGCCTACAACCAGCAAGGGCAA
98
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
CAAGTCTAAACAGAACCAGCGGAACACCGACTGGTCGGCCCGGGCCATCGTGAAGGCCGTGAAGGAGGCCTGCGAGGGA
TGTGGCCTGGGCTTCAAGCCGGTGTGGA
AGGAATACTCTAGCTTGACCGACCCCTTCGAGAGGGACGGCGATGGCCGGCCTGCTCTGAGATGTAGATTCGCCAAGGT
GGCTGCTCCCGACAGCGAGCTCCCACCTAG
ACTGACAAAGGCCGTGGGAAGCTATGTGAAGAACGCCCTAAAGGCCGATAAGGCCGAGAAGAAACAAACATGTTACCAG
AGAGGAGCCATCGAGTTCTGCAGCAGGC
ACGGCATCGACGTCCGGAAAGCTACAGATAAGGCCATTCGGAAAGCTGTGCGGGGTAGCAGTGACCTATTAGTGCCTTT
CGATGGAGGCAGAACCTTCCTGCTATCAAC
AAGACTGAGCCCTGAGAGCAGAAAGGTGGAATGGGCCGGAAGAACACTGTACGAGTTCCCTTCTGATATGGTGGCCGCC
ATCAACATCGCCTGCCGGGGCCTGGAACC
TAGAAAGGCA
SEQ ID NO: 37>Sa2Cas12i Codon optimized coding sequence
ATGGACGAGCAGGCCGTGGTGAGCAGCGGCTCTGATAAGACCCTGAAGATCGTGAGGCCCTACAGAGCTAAGGTGACCG
CTACTGGAATCAGATTGGAAGGGATCAAA
AACACCCTGAATTACCTGAAGAGAACAGAGATTTGTCTGTCCAGACTGAACGCCGCTTGCGGCGCCTTTCTGACCCCTG
CCATCGTGGAGCAGATCTGTAAAGACGATC
CCGCCCTGGTGTGCGCCATAGCTAGATTCCAGCTGGTGCCTGTGGGCAGCGAAGCTACCCTGAGCGATAGCGGACTGAT
GCGGCACTTCAAGGCGGCGCTGGGCGAACT
GACCCCTCTGCAGGAAGCCTACCTGAACAGCAGTTATAACGATGAGCTGTACGCCTGGCAGGATACCCTGGTGCTGGCC
AGACAGATCATCGCGGAAACCGGCCTGACC
GAGGACCAGTTCCGGGCATTTGCCCACGCCTGCTTCAAGAACGGTAATATCATCGGTTGTGCCGGAGGCCCTGGCGCAA
GCAATGCCATTAGCGGCATCTTCGGCGAGG
GAATCAAGAGCGACTACAGCCTCCGCAGCGAGATGACAGCCGCTGTGGCTAAGGTGTTCGAGGAAAAGCGGCCCATCAC
ATACGAGGAAGCCAGAGCCCTGGCCCTCG
AAGCCACCGGCCACGCCTCTGTGCAGAGCTTTGTCGAGGCCTTTGGCAAACAGGGCAGAAAGGGCACCCTGATCCTGTT
CATGGAGGACACCAAAACAGGCGCCTTCC
CCTCCAACGAGTTCGACTATAAGCTGAAGAAGCTGAAGGAGGACGCAGAGCGGGTGGGCAGAAAGGGCATCATCCCACA
TCGGGACGTGATCGCCTCTTACCTCCGGA
ACCAGACCGGAGCCGACATCGAGTACAACAGCAAGGCCTGGTGCGAAAGCTACTGCTGCGCCGTTTCTGAATACAACAG
CAAGATGAGCAACAACGTGCGGTTCGCTA
CAGAGAAGAGCCTGGACCTGACTAAGCTGGACGAGACAATCAGGGAAACCCCAAAGATCAGCGAGGCCATGCTGGTGTT
CGAGAACTACATGGCCAGAATCGATGCCG
ACCTGAGGTTCATCGTGTCGAAGCACCACCTGGGAAACCTGGCCAAGTTCCGGCAAACAATGATGCACGTGTCCGCCAG
CGAGTTCGAGGAAGCCTTCAAGGCCATGT
GGGCCGATTACCTGGCTGGCTTGGAGTATGGCGAGAAACCTGCTATCTGCGAGCTGGTTAGATACGTGCTGACCCACGG
CAATGACCTGCCTGTGGAAGCCTTTTACGCC
GCCTGCAAGTTTCTGTCCCTGGACGACAAGATCAAGAACAGATACCCTCATCCTTTCGTGCCCGGCAACAAGGGCTATA
CATTCGGCGCAAAGAACCTCTGGGCCGAGA
TCAACGACCCTTTCAAGCCTATCAGACAGGGCAATCCTGAGGTAGCCGGCCAAAGACCCATGATGTGGGCCACAGCTGA
TCTGCTGGACAACAACAAGTGGGTGCTGC
ACCATATTCCTTTTGCCTCGAGCAGATACTTTGAGGAAGTGTACTACACAGACCCATCTCTCCCAACCGCCCAGAAGGC
CAGAGACGGCAAGCACGGCTACAGACTGGG
AAAGGTGCTGGATGAGGCCGCCAGAGAAAGACTGAAGGCCAACAACAGACAAAGAAAGGCCGCCAAGGCCATCGAGCGG
ATCAAGGCCAATTGCGAGCACAATGTG
GCCTGGGACCCTACCACCACCTTCATGCTGCAACTGGACAGCGAGGGCAACGTGAAGATGACCATCAACCACAGACACA
TCGCCTACCGGGCTCCTAAGGAAATCGGC
GTGGGCGACCGGGTTATCGGCATCGACCAGAACGAAACCGCCCCTACAACATACGCCATCTTGGAAAGAACGGAAAACC
CCCGGGACCTGGAATATAACGGCAAGTACT
ACAGAGTGGTGAAGATGGGCAGCGTGACCTCTCCTAACGTGTCCAAATACAGAACCGTGGACGCCCTGACTTACGACGG
CGTGTCTCTGAGCGACGACGCCAGCGGAG
CCGTGAACTTCGTCGTGCTGTGCAGAGAGTTCTTCGCCGCTCATGGCGACGACGAGGGCCGGAAATACCTGGAGAGAAC
CCTGGGCTGGAGCTCCAGCCTGTATAGCTT
CCACGGCAACTACTTCAAGTGCCTGACCCAGATGATGCGGAGAAGCGCCCGCTCTGGCGGCGATCTGACCGTGTACCGC
GCTCACCTGCAGCAGATCCTGTTTCAGCAC
AACCTGTCCCCTCTGAGAATGCACAGCCTGAGCCTGCGGAGCATGGAATCTACCATGAAGGTGATCAGCTGCATGAAGT
CTTACATGAGCCTGTGCGGCTGGAAAACCG
ATGCTGACAGAATCGCCAACGACCGGAGCCTGTTCGAAGCCGCCAGAAAGCTGTACACATCTCTGGTCAATCGGCGGAC
CGAAAGAGTGCGGGTGACAGCAGGCATCC
TTATGAGACTGTGTCTGGAGCACAATGTGCGGTTTATCCACATGGAGGACGAGCTGCCTGTGGCTGAAACCGGCAAAAG
CAAAAAAAGCAACGGCGCCAAGATGCACT
GGTGTGCCCGGGAGCTGGCAGTTAGACTGTCTCAGATGGCCGAAGTGACCAGCGTTAAGTTCACCGGAGTGAGCCCCCA
CTACACTAGTCACCAGGACCCCTTCGTGCA
CTCTAAAACCAGCAAAGTGATGCGCGCCAGATGGTCCTGGCGGAACCGGGCCGACTTCACAGATAAGGACGCCGAGAGA
ATCCGGACTATCCTGGGCGGCGATGACGC
CGGGACCAAAGCTTACTACAGAAGCGCCCTGGCCGAGTTCGCCAGCAGATACGGCCTGGATATGGAGCAAATGAGAAAG
AGACGGGATGCCCAGTGGTACCAGGAGAG
ACTGCCTGAAACCTTCATCATCCCCCAGAGAGGCGGGAGAGTGTACCTGAGCTCCCACGACCTGGGCAGCGGCCAGAAA
GTGGACGGCATCTACGGCGGAAGGGCCTT
CGTGAATCACGCTGATGAGGTGGCCGCCCTTAACGTGGCTCTGGTCCGCCTC
SEQ ID NO: 38>Sa3Cas12i Codon optimized coding sequence
ATGAAAACAGAGACACTGATCCGCCCTTACCCCGGCAAGCTGAACCTGCAGCCTCGGCGGGCCCAATTCCTGGAGGATT
CAATCCAGTACCACCAGAAAATGACCGAGT
TCTTCTACCAGTTCCTGCAGGCCGTAGGCGGCGCGACCACACATCAGAACATCAGCGATTTCATTGACAACAAGGCCAC
TGATGAGCACCAGGCCACCCTTCTCTTCCA
GGTCGTGTCCAAGGACAGCACCACCCCTGAGTGCCCTGCCGAGGAACTGCTGGCCAGATTCGCCCAGTACACCGGCAAA
CAGCCCAACGAGGCCGTGACCCACTACCT
GACCAGCAGAATCAACACCGACAAGTACAGATGGCAGGACAATAGACTACTGGCCCAGAACATCGCCAGCCAACTTAAC
ATCTCCGAGACACAATTCCAGGAAATCGC
GCACGCTATCCTCAGCAACAACCTGTACATCGGACAGACCGCCAGCAACGCTGCCGCCAACTTCATCTCTCAGGTGACC
GGCACCGGCCAGAAAGCCCCAAAGGCTGC
CAGACTGGACGTGCTGTTCCAGACGAACCAAGCCCTGGCCAAAACCCAGCCTACAACCTTTGGCCAGCTCCAGCAGATT
ATCGTGCAGGCTTGTGGAGAAAGCACCAC
CGACGCCGTGCTGGCCAAGTTCGGCAACAAAGGTGCCGCCACCTCGCTGCAGCTGGCTCTGAAAACCGACCCCAACACC
ACCCTGGATCAGAAAAAGTATGAGGCCCT
99
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
GCAAAAGAAATTCGCCGAGGACGAAACAAAGTACCGGAACAAGGTTGACATTCCCCACAAAACGCAGCTGAGAAATCTG
ATCCTGAACACAAGCAATCAATTTTGCAA
CTGGCACACAAAGCCTGCCATCGAGGCTTTTAAGTGCGCCATCGCCGACATCCAGAGCAAGGTGTCCAACAACCTGAGG
ATCATGCAGGAGAAGGCCAAGCTGTACGA
GGCCTTCAGAAACGTGGACCCCCAGGTGCAGATCGCTGTCCAAGCCCTGGAGAATCACATGAACACCCTCGAAGAACCC
TACGCCCCTTACGCCCACAGCTTCGGCAG
CGTGAAGGACTTCTATGAGGACCTGAACAACGGCAGCAATCTGGACGAGGCAATTCAGACCATCGTGCACGATTCTGAT
AACTTCAACCGGAAGCCTGATCCTAACTGG
CTGAGAATCATCGCCCCACTGCACTCTAGCCACAGCGCCTCTCAGATCATGGAAGCTGTGAAATACCTGAGCAGCAAGC
AGGACTACGAACTGAGGAAGCCCTTCCCAT
TCGTGGCCACCAACCTGCCTGCCACATACGGCAAGTTCAATATCCCCGGCACCCTGAACCCTCCTACAGACTCTCTGCA
CGGCAGACTGAACGGCTCTCACAGCAACAT
GTGGCTGACAGCCCTGCTGCTGGACGGCAGAGACTGGAAGAACCACCACCTGTGCTTCGCCAGCAGCAGATACTTCGAA
GAAGTCTACTTCACCAACCCTAGCCTGCC
CACCACCGATAAAGTGCGGTCCCCAAAGTGCGGCTTTACCCTGAAGAGCGTGCTGGACAGCGAGGCTAAGGATAGAATC
CGTAATGCCCCTAAGAGCAGAACCAAGGC
CGTGAAGGCCATCGAGAGAATTAAGGCTAATTCTACCCACAACGTGGCCTGGAACCCCGAGACAAGCTTCCAGATGCAG
AAGAGAAACGACGAGTTCTACATCACAATC
AACCACAGGATCGAGATGGAAAAGATCCCCGGCCAAAAGAAAACAGACGACGGCTTCACCATCCACCCCAAGGGCCTGT
TTGCTATCCTGAAGGAAGGAGATAGAATC
CTGAGCCAGGATCTGAATCAGACAGCCGCTACACACTGCGCCGTGTACGAGGTGGCCAAGCCTGACCAGAACACCTTCA
ACCACCATGGCATCCACCTGAAGCTGATCG
CCACCGAAGAACTGAAGATGCCTCTGAAAACCAAGAAGTCTACCATCCCAGATGCCCTGTCATACCAGGGCATCCACGC
CCACGACCGGGAAAACGGCCTGCAGCAGC
TGAAGGACGCTTGCGGAGCCTTCATCTCACCTAGACTGGACCCCAAGCAGAAGGCCACCTGGGACAACAGCGTGTCCAA
GAAAGAAAACCTGTACCCTTTCATCACCG
CCTACATGAAGCTGCTGAAGAAGGTGATGAAGGCGGGCCGGCAGGAGCTGAAGCTGTTTCGGACTCATCTGGATCACAT
CCTGTTCAAACACAATCTCAGCCCTCTGAA
ACTGCACGGCGTGAGCATGATCGGCCTGGAGAGCAGCAGAGCTACAAAAAGCGTGATCAACAGCTTCTTCAACCTGCAG
AACGCTAAGACTGAGCAGCAGCAGATCGC
CTTAGACAGACCCCTGTTCGAGGCCGGCAAGACACTGATCAATAATCAGACCAGAAGAAGGCAGGAAAGAGTGCGGCTG
GAAACATCTCTGACCATGAGACTGGCCCA
TAAGTATAACGCTAAAGCCATCATCATTGAGGGAGAGCTGCCTCACAGCTCCACCGGCACATCTCAGTACCAGAACAAC
GTGCGGCTGGATTGGAGTGCCAAGAAGAGC
GCCAAGCTGAAAACCGAAAGCGCCAACTGCGCTGGAATCGCCATCTGCCAGATCGACCCTTGTCACACCTCCCACCAGA
ACCCTTTTCGGCACACCCCTACAAACCCTG
ACCTGCGGCCACGGTTCGCCCAGGTGAAGAAAGGCAAGATGTTCCAGTACCAGCTTAATGGCCTCCAGCGGCTGCTGAA
TCCTAGATCAAAGTCTAGCACAGCAATCTA
CTACCGGCAGGCCGTGCAAAGCT Ill
GTGCCCACCACAACCTGACCGAGAGAGACATCACCTCTGCCAAATTTCCCAGCGACCTGGAAAAGAAGATCAAGGACGA
CAC
CTACCTGATCCCTCAGAGAGGCGGCCGGATCTACATCAGTAGCTTCCCTGTTACAAGCTGCGCCAGACCTTGCACAAGC
AACCATTATTTCGGCGGAGGCCAGTTCGAGT
GTAATGCTGATGCCGTGGCCGCCGTGAACATCATGCTGAAGGTCCACCCT
SEQ ID NO: 39>WaCas12i Codon optimized coding sequence
ATGCCTATCCGGGGCTATAAGTGCACCGTGGTGCCTAATGTGCGGAAAAAGAAACTGCTGGAGAAAACATACAGCTACC
TGCAGGAGGGCAGCGACGTGTTTTTCGATC
TGTTCCTGTCACTGTATGGCGGCATCGCCCCTAAGATGATCCCTCAGGATCTGGGCATCAACGAGCAAGTGATCTGTGC
CGCAAACTGGTTCAAGATCGTGGAAAAGACC
AAGGACTGCATCGCCGACGACGCCCTGCTGAACCAGTTTGCCCAGTACTACGGCGAGAAGCCTAACGAGAAGGTTGTGC
AGTTTCTGACAGCTTCTTATAACAAAGATA
AGTACGTGTGGGTCGACTGCCGTCAAAAGTTCTACACCCTGCAGAAAGACCTGGGAGTGCAGAACCTCGAGAACGACCT
GGAGTGCCTGATCCGCGAGGACCTGCTGC
CTGTGGGATCTGATAAGGAAGTGAATGGATGGCACAGCATCAGCAAACTCTTCGGCTGCGGCGAGAAGGAGGACAGAAC
CATCAAGGCCAAGATTCTGAACGGCCTGT
GGGAGCGGATCGAGAAGGAAGATATTCTGACCGAGGAGGACGCCAGAAACGAGCTGCTGCATAGCGCTGGCGTGCTGAC
CCCTAAGGAGTTCAGAAAGGTGTACAAG
GGCGCCGCCGGCGGACGGGACTGCTACCACACCCTGCTGGTTGACGGCAGAAACTTCACCTTCAACCTGAAAACCCTGA
TCAAGCAGACCAAGGACAAGCTCAAGGA
AAAGTCCGTGGATGTGGAAATCCCCAACAAGGAGGCCCTGAGGCTGTACCTGGAAAAGCGAATCGGAAGATCTTTCGAG
CAGAAGCCTTGGTCCGAGATGTACAAAAC
CGCCCTGAGCGCTGTTATGCCCAAGAACACCCTGAATTACTGCTTTGCCATCGATAGACACGCCCAGTACACGAAGATC
CAGACCCTGAAGCAACCTTACGACTCTGCCA
TCACCGCCCTGAACGGCTTCTTCGAGAGCGAATGCTTCACCGGGAGCGACGTGTTCGTGATCAGCCCTAGCCACCTGGG
AAAAACCCTGAAGAAGCTGTACAACTACA
AGGACGTTGAGAGCGGAATCAGCGAGATCGTCGAGGACGAGGATAATAGCCTGCGGAGCGGCGTGAACGTGAATCTGCT
TCGGTACATCTTCACACTGAAGGATATGTT
CAGCGCCGAGGACTTCATCAAGGCCGCCGAGTACAACGTAGTGTTTGAGAGATACAATAGACAGAAAGTCCACCCTACA
GTGAAGGGCAATCAAAGCTTCACATTTGGC
AACAGCGCTCTGTCTGGCAAGGTGATCCCTCCATCTAAGTGTCTGAGCAACCTGCCTGGACAGATGTGGCTGGCCATCA
ATCTGCTGGACCAGGGCGAGTGGAAGGAGC
ACCACATTCCCTTCCACAGCGCCAGATTCTACGAGGAAATCTACGCTACATCTGATAACCAGAACAACCCCGTGGACCT
GCGGACCAAGAGATTCGGCTGTTCTCTGAAC
AAGACCTTCAGCGCCGCTGACATCGAGAAGGTGAAGGAGTCTGCCAAGAAAAAGCACGGAAAGGCCGCTAAGAGAATCC
TGCGTGCCAAGAACACAAACACCGCCGT
GAACTGGGTGGATTGCGGCTTCATGCTGGAAAAGACCGAAGTGAACTTCAAAATCACCGTCAATTACAAACTGCCCGAT
CAGAAGCTGGGCAAGTTCGAGCCTATCGTG
GGCACAAAAATCCTGGCTTATGACCAGAATCAGACCGCCCCAGATGCCTACGCCATCCTGGAAATTTGCGACGATTCTG
AAGCCTTCGACTACAAGGGCTACAAAATCAA
ATGTCTGAGCACCGGGGACCTGGCCAGCAAGTCCCTGACAAAGCAGACAGAAGTGGACCAGCTGGCATATAAGGGCGTA
GACAAAACCAGCAACTTCTACAAGAAGT
GGAAGCAGCAGCGGAGACTTTTTGTGAAGAGCCTGAATATCCCAGACGCCCTGAAATCTTTTGAAAACATCAACAAGGA
GTACCTGTACGGCTTTAACAATAGTTACCT
GAAGCTACTGAAGCAAATTCTGAGAGGCAAATTCGGACCTATCCTGGTGGACATCAGACCTGAGCTGATCGAGATGTGC
CAGGGCATCGGCAGCATCATGCGGCTGTCC
AGCTTGAACCACGACAGCCTGGACGCCATTCAGTCCCTGAAGAGCCTGCTGCACTCTTACTTCGACCTGAAGGTGAAGG
AAGAAATCAAGACCGAAGAGCTGAGAGA
GAAGGCCGATAAGGAAGTGTTTAAGCTGCTGCAACAGGTGATCCAGAAGCAGAAGAATAAGAGAAAGGAAAAGGTGAAC
AGAACAGTGGATGCTATCCTGACACTGG
100
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
CCGCCGACGAGCAAGTGCAGGIVATCGTGGGCGAAGGCGACCTGTGCGTGTCCACCAAGGGCACCAAAAAGAGACAGAA
CAACCGGACAATCGACTGGIVCGCGAG
AGCCGTGGIVGAGAAACTGGAAAAAGCCTGCAAGCTGCACGGCCTGCACITCAAGGAAATCCCCCCCCACTACACCAGC
CACCAGGACTGTITCGAGCACAACAAGG
ACATCGAGAATCCTAAGGAAGIVATGAAGIVTAGATIVAACAGCAGCGAGAACGTGGCCCCTIVGATGATTAAGAAGTF
CGCCAACTACCTFAAATGCGAGACAAAATA
CTACGTGCAGGGCATGCAGGACITCCIVGAACATTACGGCCTGGTGGAATACAAGGACCATATCAAGAAGGGAAAGATC
AGTATCGGCGATTITCAGAAACTGATCAAG
CTGGCCCTGGAAAAAGTAGGCGAGAAGGAAATCGIVTITCCTIVCAAAGGCGGCAGAATCTACCTGAGCACCTACTGTC
TGACCAACGAGIVCAAACCCATCGTGTIVA
ACGGCAGACGGTGCTATGTGAACAACGCCGACCACGTGGCCGCTATCAACGTGGGCATCTGCCTGITGAATFTCAACGC
CAGAGCTAAGGTGGCTGAAAAGACACCA
SEQ ID NO: 40>Wa2Cas12i Codon optimized coding sequence
ATGGCCAAGAAGGACITCATCGCCAGACCITACAACACCITIVTGCTGCCTAACGACAGAAAGCTGGCTFACCTGGAAG
AAACATGGACCGCCTACAAGAGCATCAAG
ACCGIVCIVCACAGATITCTGATCGCGGCCTATGGCGCCATCCCCITCCAGACATFCGCCAAAACCATIVAAAACACCC
AAGAGGACGAGCTGCAACTGGCCTATGCCGT
GCGGATGTIVAGACTGGTGCCCAAGGACITCAGCAAGAACGAGAACAACATIVCACCTGACATGCTGATCAGCAAGCTG
GCCAGCTACACCAATATCAACCAGTCCCCA
ACAAACGTFCTCAGCTACGTGAATAGCAACTACGACCCAGAGAAATACAAGIVGATCGAITCTAGAAACGAGGCCATCA
GCCTGAGCAAGGAGATCGGCATCAAGCTGG
ACGAGCTCGCTGATTACGCCACCACCATGCTGTGGGAGGATTGGCTGCCCCTGAACAAGGACACAGTGAACGGCTGGGG
AACCACCTCTGGCCTGITCGGCGCCGGCA
AAAAAGAGGATAGGACCCAAAAGGTGCAGATGCTGAACGCCCTGCTGCTGGGCCTGAAAAACAACCCCCCCAAGGATTA
CAAGCAGTACAGCACCATCCTACTGAAGG
CATTTGATGCCAAGAGCTGGGAAGAGGCCGTGAAGATTTACAAAGGCGAGTGTFCTGGCCGAACAAGTAGITACCTGAC
TGAGAAGCACGGTGACATCAGCCCTGAGA
CACTGGAAAAGCTGATCCAGAGCATCCAGCGGGACATCGCCGACAAACAGCACCCAATCAACCTGCCAAAGAGAGAAGA
AATCAAAGCCTACCTGGAGAAACAGTCT
GGCACCCCATACAACCTGAACCTGTGGAGCCAGGCCCTGCACAACGCCATGAGCTCTATCAAGAAAACCGACACCAGAA
ATITCAACTCTACCCTGGAGAAGTACGAG
AAGGAAATCCAGCTGAAGGAGTGCCITCAAGATGGCGACGATGIVGAGCTGCTGGGGAACAAGTTITTCTCTIVTCCTF
ACCACAAGACAAATGATGIVTICGTGATCT
GCTCTGAACACATCGGAACAAATAGAAAGTACAACGTGGIVGAGCAGATGTATCAGCTGGCCAGCGAGCACGCCGACIT
CGAGACAGTITIVACCCTGCTGAAGGACG
AGTATGAGGAAAAGGGCATCAAGACACCCATCAAAAACATCCTGGAGTACATCTGGAACAACAAGAACGTCCCIVTGGG
CACATGGGGCCGGATCGCTAAATACAACC
AGCTGAAGGACAGATFACCAGGGATCAAGGCCAATCCCACAGTGGAATGCAACAGAGGCATGACATITGGCAACAGCGC
CATGGTGGGCGAAGTGATGCGCFCCAACC
GGATCAGCACCAGCACCAAGAACAAGGGCCAGATCTIGGCCCAGATGCACAACGACCGGCCTGIVGGCAGCAACAACAT
GATITGGCTGGAAATGACCCTCCIVAACA
ACGGCAAGIVGCAGAAGCACCACATCCCCACACACAACAACAAATITITCGAGGAAGTGCACGCCTIVAACCCTGAACT
GAAGCAGAGCGTGAACGTGAGAAACAGA
ATGTACAGAAGCCAGAACTACTCACAGCTGCCTACCAGCCTGACCGACGGCCTGCAGGGAAATCCTAAGGCCAAGATCT
FCAAGAGACAGTACAGAGCCCTGAACAAC
ATGACCGCTAATGTGATCGACCCTAAGCTGTCCTFCATCGTGAACAAGAAAGATGGAAGAITCGAGATCAGCATCATCC
ACAACGTGGAAGTGATCCGAGCCAGACGGG
ACGTGCTGGIVGGCGACTACCTGGTGGGCATGGACCAAAACCAGACGGCITCTAATACCTACGCCGTCATGCAGGTGGI
VCAGCCTAACACCCCCGACAGCCATGAGIT
CAGAAACCAGTGGGIVAAGTIVATCGAGAGCGGCAAGATCGAGAGCFCAACACTGAACIVCCGGGGTGAGTACATCGAC
CAGCTGAGCCACGATGGCGTCGACCTGCA
GGAGATFAAGGATTCTGAGTGGATFCCTGCCGCCGAAAAATI'CCTGAACAAGCTAGGAGCTATCAACAAAGACGGCAC
CCCCATCAGCATCTCCAACACCAGCAAACGG
GCCTACACATIVAATAGCATCTATITCAAAATCCTGCTGAATFATCTGAGAGCCAACGACGTGGACCTGAATCTGGTGC
GGGAAGAGATCCTGCGGATCGCCAACGGCAG
ATIVAGCCCTATGCGGCTGGGATCTCTGIVCTGGACCACACTAAAAATGCTGGGCAATITCCGGAACCTAATI'CACAG
CTACITCGACCACTGTGGCTITAAGGAAATGC
CTGAGAGAGAAAGCAAGGACAAGACCATGTACGATCTGCTGATGCACACCATCACCAAGCTGACCAACAAGCGGGCCGA
GCGCACCAGCAGAATCGCTGGAAGCCTG
ATGAACGTGGCFCACAAGTACAAGATCGGCACAAGCGTGGIVCACGTGGTGGTGGAAGGCFCTCTGAGCAAAACCGACA
AGAGCAGCFCCAAGGGCAACAATCGGAA
TACCACAGACTGGTGCAGCCGGGCCGTGGIVAAGAAGCITGAAGATATGTGCGTGITCTACGGCITCAACCTGAAAGCC
GTGAGCGCCCACTACACCAGCCACCAGGA
CCCTCTGGTIVATAGAGCCGATFACGATGATCCTAAGITGGCCCTGAGATGCAGATACFCTIVITACAGCAGAGCTGAT
ITIVAGAAGTGGGGCGAAAAATCTITCGCCGC
CGTGATCAGATGGGCCACAGACAAGAAGAGCAACACCTGCTACAAGGTGGGAGCCGTAGAGTFCTIVAAGAACTACAAA
ATCCCTGAGGACAAGATCACCAAAAAGCT
GACCATCAAAGAGTFCCTGGAAATFATGTGCGCTGAGAGCCACTACCCTAATGAGTACGACGACATICI'GATCCCTAG
AAGGGGCGGCAGAATCTACCTCACAACTAAGA
AGCTGCTGIVCGATAGCACCCACCAGAGAGAGTCTGTGCATAGCCATACCGCCGTGGTGAAGATGAACGGCAAGGAATA
CTATAGCAGCGACGCCGATGAGGIVGCTGC
TATCAATATCTGCCTGCACGACIVGGIVGIVCCCCTGAATIVGACAAATCACTGCCTGCCTGCCGGATGGIVTAGCGAC
CACCTGAAGGAATGCGTGCAATGTCACACCC
CTGATCCTGIVAGAATCAGCATG
SpCas9 protein, SEQ ID NO: 47
MDKKYSIGLDIGTNSVGWAVITDEYIOTPSKKFICVLGNTDRHSIICKNIJGALLFDSGETAEATRLKRTARRRYTRRI
NIUCYLQEIFSNEMAIOTDDSFFHRLEESFLVEEDKKHER
HPIFGNIVDEVAYEEKYPTIYHLRICKLVDSTDKADLRLIYLALAHMIKFRGEFLIEGDLNPDNSDVDELFIQLVQTYN
QLFEENPINASGVDAKAILSARISKSERLENLIAQLPG
EICKNGLEGNLIALSLGLTPNEKSNFDLAEDAKLQLSICDITDDDLDNLLAQIGDQYADLFLAAKNISDAILISDILIW
NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPE
KYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEICMDGTEELLVKLNREDLLEKQRTEDNGSIPHQIELGELHAIL
REQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF
101
CA 03237337 2024-05-02
WO 2023/078314 PCT/CN2022/129376
AWMTRKSEETITPWNFEEVVDKGASAQSFIERMINFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS
GEQKKAIVDLLFKINRKVTVKQLKEDYFKKIEC
FDSVEISGVEDRFNASLGTYHDLLKIIKDKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKR
RRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA
NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVEN
TQLQNEKLYLYYLQNGRDMYVDQELDINIRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKN
YWRQLLNAKLITQRKEDNLTKAERGGLSELDKA
GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDERKDFQFYKVREINNYHHAHDAYLNAVV
GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ
EIGKATAKYFFYSNIVINFEKTEITLANGEIRKRPLIEINGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGE
SKESILPKRNSDKLIARKKDWDPKKYGGEDSPTVAYS
VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ
KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ
LEVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKR
YTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
SpCas9 scaffold sequence, SEQ ID NO: 48
CC ATTA C A GTAGG A GC
ATACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG
C
LbCas12a protein, SEQ ID NO: 49
MAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLE
INLRKEIAKAFKGNEGYKSLEKKDIIETILPEFLDDK
DEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDY
DVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIK
GLNEYINLYNQKTKQKLPKEXPLYKQVLSDRESLSFYGEGYTSDEEVLEVERNTLNKNSEIFSSIKKLEKLEKNEDEYS
SAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDI
HLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADEVLEKSLKK
NDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRD
ESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKY
AKCLQKIDKDDVNGNYEKINYKLLPGINKMLP
KVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREV
EEQGYKVSFESASKKEVDKLVEEGKLYMFQIYN
KDFSDKSHGTINLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKFTPDNPKKTTTLSYDVY
KDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLK
HDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNENGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELK
AGYISQVVHKICELVEKYDAVIALEDLNSGEKNSRVK
VEKQVYQKFEKMLIDKLNYMVDKKSFIPCATGGALKGYQITNKFESEKSMSTQNGFIFYIPAWLTSKIDPSTGEVNLLK
TKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNE
SRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALM
SLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSR
NYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
LbCas12a DR sequence, SEQ ID NO: 50
TAATTTCTACTAAGTGTAGATCC ATTA C A GTA GG A GC ATAC
SEQ ID NO: 51>SiCas12i-crRNA
CTAGCAATGACTCAGAAATGTGTC CCCAGTTGACACCC ATTA C A GTA GG A GC ATA C
SEQ ID NO: 52 >S12Cas 12i-crRNA
ATCGCAACATCTTAGAAATCCGTCCTTAGTTGACGGCC ATTAC A GTA GG A GC ATAC
SEQ ID NO: 53 >WiCas12i-crRNA
TCTCAACGATAGTCAGACATGTGTCCCCAGTGACACCC ATTAC A GTA GG A GC ATAC
SEQ ID NO: 54 >Wi2Cas12i-crRNA
CTCAAAGTGTCAAAAGAATGTCCCTGCTAATGGGACCC ATTA C A GTA GG A GC ATAC
SEQ ID NO: 55 >Wi3Cas12i-crRNA
TCCCAAAGTGGCAAAAGAATCTCCCTGTTAATGGGAGCC ATTAC A GTA GG A GC ATAC
SEQ ID NO: 56>SaCa512i-crRNA
GTCTAACTGCCATAGAATCGTGCCTGCAATTGGCACCC ATTA C A GTA GG A GC ATA C
102
CA 03237337 2024-05-02
WO 2023/078314
PCT/CN2022/129376
SEQ ID NO: 57 >Sa2Cas12i-crRNA
TCGGGGCACCAAAATAATCTCCTTGGTAATGGGAGCCATTACAGTAGGAGCATAC
SEQ ID NO: 58>Sa3Cas12i-crRNA
CCACAACAACCAAAAGAATGTCCCTGAAAGTGGGACCCATTACAGTAGGAGCATAC
SEQ ID NO: 59 >WaCas12i-crRNA
GTAACAGTGGCTAAGTAATGTGTCTTCCAATGACACCCATTACAGTAGGAGCATAC
SEQ ID NO: 60 >Wa2Cas12i-crRNA
GAGAGAATGTGTGCAAAGTCACACCCATTACAGTAGGAGCATAC
103
