IP Library › Patent Application 19099615

Patent Application

App. No. 19/099,615

CLASS II, TYPE V CRISPR SYSTEMS

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Patent No.: US None
App. No.: 19/099,615

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Abstract

Described herein are methods, compositions, and systems derived from uncultivated microorganisms useful for gene editing.

Claims (267)

1 . A method of disrupting a CD38 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

(b) an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a spacer sequence configured to hybridize to a region of said CD38 locus,

wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 4466-4503 and 5686; or

wherein said engineered guide RNA comprises a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS:

4428-4465 and 5685.

2 . The method of claim 1 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

3 . The method of claim 1 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

4 . The method of any one of claims 1-3 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, and 6033-6036.

5 . The method of any one of claims 1-4 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

6 . The method of any one of claims 1-5 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 4466, 4467, 4468, 4479, 4484, 4490, 4492, 4493, 4495, 4498.

7 . The method of any one of claims 1-6 , wherein said engineered guide RNA comprises a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 4428, 4429, 4430, 4436, 4441, 4446, 4452, 4454, 4455, 4460, or 4461.

8 . The method of any one of claims 1-7 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

9 . A method of disrupting a TIGIT locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

10 . The method of claim 9 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

11 . The method of claim 9 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

12 . The method of any one of claims 9-11 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, and 6033-6036.

13 . The method of any one of claims 9-12 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

14 . The method of any one of claims 9-13 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 4521, 4527, 4528, 4535, or 4536.

15 . The method of any one of claims 9-13 , wherein said engineered guide RNA comprises a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 4504, 4510, 4511, 4518, or 4519.

16 . The method of any one of claims 9-15 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

17 . A method of disrupting an AAVS1 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides complementary to a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 4569-4599; or

18 . The method of claim 17 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

19 . The method of claim 18 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

20 . The method of any one of claims 17-19 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, 6033-6036.

21 . The method of any one of claims 17-20 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

22 . The method of any one of claims 17-21 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 4574, 4577, 4578, 4579, 4582, 4584, 4585, 4586, 4587, 4589, 4590, 4591, 4592, 4593, 4595, 4596, or 4598.

23 . The method of any one of claims 17-21 , wherein said engineered guide RNA comprises a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 4543, 4546, 4547, 4548, 4551, 4553, 4554, 4555, 4556, 4558, 4559, 4560, 4561, 4562, 4565, or 4567.

24 . The method of any one of claims 17-23 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, hepatocyte, or precursor thereof.

25 . A method of disrupting a B2M locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

26 . The method of claim 25 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

27 . The method of claim 26 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

28 . The method of any one of claims 25-27 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857 and 6033-6036.

29 . The method of any one of claims 25-28 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

30 . The method of any one of claims 25-29 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 4676, 4678-4687, 4690, 4692, 4698-4707, 4720-4723, 4725-4726, 4732-4733, 4736-4737, 4741, or 4750-4751.

31 . The method of any one of claims 25-30 , wherein said engineered guide RNA comprises a nucleotide sequence having at least 80% identity to any one of SEQ ID NOS: 4600, 4602-4611, 4614, 4616, 4622-4631, 4644-4647, 4649-4650, 4656-4657, 4660-4661, 4665, or 4674-4675.

32 . The method of any one of claims 25-31 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

33 . A method of disrupting a CD2 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

34 . The method of claim 33 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

35 . The method of claim 34 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

36 . The method of any one of claims 33-35 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, and 6033-6036.

37 . The method of claim 36 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

38 . The method of any one of claims 33-37 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 4837, 4844, 4845, 4848, 4857-4858, 4883, 4887, 4892-4893, 4904-4909, 4914, 4916, or 4918.

39 . The method of claim 38 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 4752-4836 that target any one of SEQ ID NOs: 4837, 4844, 4845, 4848, 4857-4858, 4883, 4887, 4892-4893, 4904-4909, 4914, 4916, or 4918.

40 . The method of any one of claims 33-39 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

41 . A method of disrupting a CD5 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

42 . The method of claim 41 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

43 . The method of claim 42 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

44 . The method of any one of claims 41-43 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, and 6033-6036.

45 . The method of claim 44 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

46 . The method of any one of claims 41-45 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 4946-4947, 4949, 4951, 4957-4960, 4963, 4967, or 4969.

47 . The method of any one of claims 41-45 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 4922-4945 that target any one of SEQ ID NOs: 4946-4947, 4949, 4951, 4957-4960, 4963, 4967, or 4969.

48 . The method of any one of claims 41-47 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

49 . A method of disrupting a mouse TRAC locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

50 . The method of claim 49 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

51 . The method of claim 50 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

52 . The method of any one of claims 49-51 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

53 . The method of claim 52 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

54 . The method of any one of claims 49-53 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5126-5130, 5133-5143, 5147-5150, 5172-5173, 5184-5189, or 5192-5194.

55 . The method of any one of claims 49-53 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 5056-5125 that target any one of SEQ ID NOs: 5126-5130, 5133-5143, 5147-5150, 5172-5173, 5184-5189, or 5192-5194.

56 . The method of any one of claims 49-55 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

57 . A method of disrupting a mouse TRBC1 or TRBC2 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

58 . The method of claim 57 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

59 . The method of claim 58 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

60 . The method of any one of claims 57-59 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3677-3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

61 . The method of any one of claims 57-60 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

62 . The method of any one of claims 57-61 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5211, 5213-5215, 5217, 5221, 5223, 5247, 5249-5250, 5252-5253, 5258-5259, or 5264.

63 . The method of any one of claims 57-61 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 5196-5210 or 5226-5246 that target any one of SEQ ID NOs: 5211, 5213-5215, 5217, 5221, 5223, 5247, 5249-5250, 5252-5253, 5258-5259, or 5264.

64 . The method of any one of claims 57-63 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

65 . A method of disrupting a human TRBC1 or TRBC2 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

66 . The method of claim 65 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

67 . The method of claim 66 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

68 . The method of any one of claims 65-67 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

69 . The method of any one of claims 65-68 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

70 . The method of any one of claims 65-69 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5661-5663, 5672-5675, or 5678.

71 . The method of any one of claims 65-69 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 5642-5660 that target any one of SEQ ID NOs: 5661-5663, 5672-5675, or 5678.

72 . The method of any one of claims 65-71 , wherein said cell is a eukaryotic cell, T-cell, hematopoietic stem cell, or precursor thereof.

73 . A method of disrupting an HPRT locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

74 . The method of claim 73 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

75 . The method of claim 74 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

76 . The method of any one of claims 73-75 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

77 . The method of any one of claims 73-76 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

78 . The method of any one of claims 73-77 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5562-5564 or 5568.

79 . The method of any one of claims 73-77 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 5482-5561 that target any one of SEQ ID NOs: 5562-5564 or 5568.

80 . The method of any one of claims 73 - 80 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

81 . A method of disrupting an APO-A1 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

82 . The method of claim 81 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

83 . The method of claim 82 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

84 . The method of any one of claims 81-83 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

85 . The method of any one of claims 81-84 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

86 . The method of any one of claims 81-85 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5861-5866 or 5868-5869.

87 . The method of any one of claims 81-85 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 5847-5860 that target any one of SEQ ID NOs: 5861-5866 or 5868-5869.

88 . The method of any one of claims 81-87 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

89 . A method of disrupting an ANGPTL3 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

90 . The method of claim 89 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

91 . The method of claim 90 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

92 . The method of any one of claims 89-91 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

93 . The method of any one of claims 89-92 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

94 . The method of any one of claims 89-93 , wherein said engineered guide RNA is configured to hybridize to a sequence having at least 20-22 consecutive nucleotides having at least 80% identity to any one of SEQ ID NOs: 5955-5963, 5968-5975, 5979-5987, 5989-5993, 5997, 5999, 6003-6010, 6014-6016, 6024-6025, or 6027-6030.

95 . The method of any one of claims 89-93 , wherein said engineered guide RNA has at least 80% sequence identity to any one of SEQ ID NOs: 5875-5952 that target any one of SEQ ID NOs: 5955-5963, 5968-5975, 5979-5987, 5989-5993, 5997, 5999, 6003-6010, 6014-6016, 6024-6025, or 6027-6030.

96 . The method of any one of claims 89-95 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

97 . A method of disrupting a human Rosa26 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

98 . The method of claim 97 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

99 . The method of claim 98 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

100 . The method of any one of claims 97-99 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

101 . The method of any one of claims 97-100 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

102 . The method of any one of claims 97-101 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

103 . A method of disrupting a FAS locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

104 . The method of claim 103 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

105 . The method of claim 104 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

106 . The method of any one of claims 103-105 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

107 . The method of any one of claims 103-106 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

108 . The method of any one of claims 103-107 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

109 . A method of disrupting a PD-1 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

110 . The method of claim 109 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

111 . The method of claim 110 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

112 . The method of any one of claims 109-111 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

113 . The method of any one of claims 109-112 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

114 . The method of any one of claims 109-113 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

115 . An engineered nuclease system comprising:

(a) an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 215 or a variant thereof; and

wherein said system has reduced immunogenicity when administered to a human subject compared to an equivalent system comprising a Cas9 enzyme.

116 . The system of claim 115 , wherein said Cas9 enzyme is an SpCas9 enzyme.

117 . The system of claim 115-116 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

118 . The system of any one of claims 115-117 , wherein said immunogenicity is antibody immunogenicity.

119 . A method of disrupting a mouse HAO-1 locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

wherein said engineered guide RNA comprises the nucleotides of guide RNAs mH29-1_37, mH29-15_37, mH29-29_37 (SEQ ID NOs: 5779-5781) comprising the nucleotide modifications described in SEQ ID NOs: 5779-5781;

or wherein said engineered guide RNA comprises any one of SEQ ID NOs: 4184-4225.

120 . The method of claim 119 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

121 . The method of claim 120 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

122 . The method of any one of claims 119-121 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

123 . The method of any one of claims 119-122 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

124 . The method of any one of claims 119-123 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

125 . The method of any one of claims 119-124 , wherein said engineered guide RNA comprises the nucleotides of guide RNAs mH29-15_37 or mH29-29_37 (SEQ ID NOs: 5780-5781) comprising the nucleotide modifications described in SEQ ID NOs: 5780-5781.

126 . The method of any one of claims 119-125 , wherein said method further comprises disrupting expression of glycolate oxidase from said HAO-1 locus.

127 . A method of disrupting a human TRAC locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

wherein said engineered guide RNA comprises the nucleotides of MG29-1-TRAC-sgRNA-35 (SEQ ID NOs: 5681 or 5683) comprising the nucleotide modifications described in SEQ ID NOs: 5681 or 5683.

128 . The method of claim 127 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

129 . The method of claim 128 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

130 . The method of any one of claims 127-129 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

131 . The method of any one of claims 127-130 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

132 . The method of any one of claims 127-131 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

133 . A method of disrupting an albumin locus in a cell, comprising introducing to said cell:

(a) a class 2, type V Cas endonuclease; and

Wherein said engineered guide RNA comprises the nucleotides of mAlb298-37, mAlb2912-37, mAlb2918-37, or mAlb298-34 (SEQ ID NOs: 5756-5759) comprising the nucleotide modifications described in SEQ ID NOs: 5756-5759; or

wherein said engineered guide RNA comprises the nucleotides of mAlb29-8-44, mAlb29-8-50, mAlb29-8-50b, mAlb29-8-51b, mAlb29-8-52b, mAlb29-8-53b, or mAlb29-8-54b comprising the nucleotide modifications described in Table 5.

134 . The method of claim 133 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof.

135 . The method of claim 134 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

136 . The method of any one of claims 133-135 , wherein said engineered guide RNA comprises a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the non-degenerate nucleotides of any one of SEQ ID NOs: 3471, 3539, 3551-3559, 3608-3609, 3612, 3636-3637, 3640-3641, 3644-3645, 3648-3649, 3652-3653, 3656-3657, 3660-3661, 3664-3667, 3671-3672, 3678, 3695-3696, 3729-3730, 3734-3735, 3851-3857, or 6033-6036.

137 . The method of any one of claims 133-136 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to the non-degenerate nucleotides of SEQ ID NO: 3609.

138 . The method of any one of claims 133-137 , wherein said cell is a eukaryotic cell, hepatocyte, T-cell, hematopoietic stem cell, or precursor thereof.

139 . The method of any one of claims 133-138 , wherein said engineered guide RNA comprises the nucleotides of mAlb298-37, mAlb2912-37, mAlb2918-37, or mAlb298-34 (SEQ ID NOs: 5756-5759) comprising the nucleotide modifications described in SEQ ID NOs: 5756-5759.

140 . An engineered guide RNA comprising:

a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a target sequence in a target DNA molecule; and

b) a protein-binding segment configured to bind to a class 2, type V Cas endonuclease, and

wherein said guide RNA comprises a nucleotide modification pattern depicted in any one of SEQ ID NOs: 5695-5701.

141 . The engineered guide RNA of claim 140 , wherein said guide RNA comprises mAlb29-8-44, mAlb29-8-50, mAlb29-8-37, or mAlb29-12-44.

142 . The engineered guide RNA of claim 140 , wherein said guide RNA comprises hH29-4_50, hH29-21_50, hH29-23_50, hH29-41_50, hH29-4_50b, hH29-21_50b, hH29-23_50b, or hH29-41_50b, mH29-1-50, mH29-15-50, mH29-29-50, mH29-1-50b, mH29-15-50b, or mH29-29-50b.

143 . The engineered guide RNA of claim 140 , wherein said DNA-targeting segment is configured to hybridize to an HAO-1 gene or an albumin gene.

144 . The engineered guide RNA of any one of claims 140-142 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

145 . The engineered guide RNA of any one of claims 140-144 , wherein said class 2, type V Cas endonuclease comprises an endonuclease having at least 75% sequence identity to SEQ ID NO: 215.

146 . An engineered nuclease system comprising:

(a) an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 1-3470 or a variant thereof, or a nucleotide sequence encoding said endonuclease; and

(b) a polynucleotide sequence encoding a CRISPR array, wherein said CRISPR array is configured to be processed by said endonuclease to an engineered guide RNA, wherein said engineered guide RNA is configured to form a complex with said endonuclease and said engineered guide RNA comprises a spacer sequence configured to hybridize to a target nucleic acid sequence,

wherein said spacer sequence is configured to hybridize to an albumin gene.

147 . The system of claim 146 , wherein said polynucleotide sequence comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 5712.

148 . The system of claim 146 or 147 , wherein said endonuclease comprises an endonuclease having at least 75% sequence identity to any one of SEQ ID NOs: 141, 215, 229, 261, or 1711-1722 or a variant thereof.

149 . The system of claim 148 , wherein said endonuclease comprises an endonuclease having at least 75% sequence identity to SEQ ID NO: 215.

150 . An engineered nuclease system comprising:

(a) an endonuclease having at least 75% sequence identity to SEQ ID NOs: 470 or a variant thereof; and

151 . The engineered nuclease system of claim 150 , wherein said engineered guide RNA comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 6031.

152 . The engineered nuclease system of claim 151 or 152 , wherein said endonuclease is configured to be selective for a 5′ PAM sequence comprising SEQ ID NO: 6032.

153 . An engineered nuclease system comprising:

(a) an endonuclease having at least at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 2824, 2841, or 2896, or a variant thereof; and

wherein said engineered guide RNA comprises a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOs: 6033, 6034, or 6035.

154 . The engineered nuclease system of claim 153 , wherein said endonuclease has at least 80% sequence identity to SEQ ID NO: 2824 and said engineered guide RNA has at least 80% sequence identity to SEQ ID NO: 6033.

155 . The engineered nuclease system of claim 153 , wherein said endonuclease has at least 80% sequence identity to SEQ ID NO: 2841 and said engineered guide RNA has at least 80% sequence identity to SEQ ID NO: 6034.

156 . The engineered nuclease system of claim 153 , wherein said endonuclease has at least 80% sequence identity to SEQ ID NO: 2896 and said engineered guide RNA has at least 80% sequence identity to SEQ ID NO: 6035.

157 . The engineered nuclease system of any one of claims 153-156 , wherein said endonuclease is configured to be selective for a 5′ PAM sequence comprising any one of SEQ ID NOs: 6037-6039.

158 . A lipid nanoparticle comprising:

(a) any of the endonucleases described herein;

(b) any of the engineered guide RNAs described herein:

(d) a sterol;

(e) a neutral lipid; and

(f) a PEG-modified lipid.

159 . The lipid nanoparticle of claim 158 , wherein said cationic lipid comprises C12-200, said sterol comprises cholesterol, said neutral lipid comprises DOPE, or said PEG-modified lipid comprises DMG-PEG2000.

160 . The lipid nanoparticle of claim 158 , wherein said cationic lipid comprises 98N12-5 (TETA5-LAP), DLin DMA, DLin-K-DMA (2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA, or C12-200.

161 . An engineered nuclease system comprising:

(a) an endonuclease comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6274-6281 or 6340-6551, or a variant thereof, wherein said endonuclease is a class 2, type V endonuclease, or a nucleotide sequence encoding said endonuclease; and

162 . The engineered nuclease system of claim 161 , wherein said endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6274-6281.

163 . The engineered nuclease system of claim 161 or 162 , wherein said endonuclease is configured to bind to a protospacer adjacent motif (PAM) sequence having at least 80% sequence identity to any one of SEQ ID NOs: 6332-6339.

164 . An engineered nuclease system comprising:

(a) an endonuclease comprising a PI (PAM interacting) domain having at least 80% sequence identity to a PI domain of any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, or 6281, or a variant thereof, or a nucleotide sequence encoding said endonuclease, wherein said endonuclease is a class 2, type V endonuclease and said endonuclease is configured to be selective for a 5′ PAM of any one of SEQ ID NOs: 6326-6339; and

165 . The engineered nuclease system of claim 164 , wherein said endonuclease comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2811, 2819, 2878, 2916, 2963, 3009, 6274, 6275, 6276, 6279, 6280, or 6281, or a variant thereof.

166 . The engineered nuclease system of any one of claims 161-165 , wherein said guide RNA comprises a sequence with at least 80% sequence identity to non-degenerate nucleotides of any one of SEQ ID NOs: 6284-6325.

167 . The engineered nuclease system of any one of claims 161-166 , wherein said guide RNA spacer sequence comprises a sequence complementary to a eukaryotic, fungal, plant, mammalian, or human genomic polynucleotide sequence.

168 . The engineered nuclease system of any one of claims 161-167 , wherein said endonuclease comprises at least one of a S168R, E172R, N577R, or Y170R mutation when a sequence of said endonuclease is optimally aligned to SEQ ID NO: 215.

169 . The engineered nuclease system of any one of claims 161-168 , further comprising a single- or double-stranded DNA repair template comprising from 5′ to 3′: a first homology arm comprising a sequence of at least 20 nucleotides 5′ to said target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides 3′ to said target sequence.

170 . The engineered nuclease system of claim 169 , wherein said first or second homology arm comprises a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides.

171 . The engineered nuclease system of claim 169 or 170 , wherein said first and second homology arms are homologous to a genomic sequence of a prokaryote, bacteria, fungus, or eukaryote.

Assignments (2)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 20, 2026

From: THOMAS, BRIAN C.; ALEXANDER, LISA; BROOKS, ALAN; BROWN, CHRISTOPHER; BUTTERFIELD, CRISTINA NOEL; CASTELLE, CINDY; DEVOTO, AUDRA; GOLTSMAN, DANIELA S.A.; MATHEUS CARNEVALI, PAULA B.; NOCEDAL, ISABEL; TEMOCHE-DIAZ, MORAYMA

To: METAGENOMI, INC.

Reel/Frame 074138/0210 →

CHANGE OF NAME Recorded Feb 19, 2026

From: METAGENOMI, INC.

To: METAGENOMI THERAPEUTICS, INC.

Reel/Frame 074765/0807 →