ENGINEERED CELLS FOR THERAPY

Abstract

Methods of culturing embryonic stem cells, induced pluripotent stem cells and/or differentiated cells in culture medium comprising activin are described. In one aspect, the disclosure features a pluripotent human stem cell, wherein the stem cell comprises: (i) a genomic edit that results in loss of function of Cytokine Inducible SH2 Containing Protein (CISH) and (ii) a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway, or a genomic edit that results in a loss of function of adenosine Ata receptor.

Claims

1. A pluripotent human stem cell, wherein the stem cell comprises: (i) a genomic edit that results in loss of function of Cytokine Inducible SH2 Containing Protein (CISH) and (ii) a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway, or a genomic edit that results in a loss of function of adenosine A2a receptor (ADORA2A).

2. The pluripotent human stem cell of claim 1, wherein the stem cell comprises a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway and a genomic edit that results in a loss of function of ADORA2A.

3. The pluripotent human stem cell of claim 1 or 2, wherein the stem cell comprises a genomic edit that results in a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor.

4. The pluripotent human stem cell of claim 3, wherein the TGF beta receptor is a TGF beta receptor II (TGFβRID.

5. The pluripotent human stem cell of any one of the preceding claims, wherein the stem cell expresses one or more pluripotency markers selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog.

6. A differentiated cell, wherein the differentiated cell is a daughter cell of the pluripotent human stem cell of any one of the preceding claims.

7. The differentiated cell of claim 6, wherein the differentiated cell is an immune cell.

8. The differentiated cell of claim 6, wherein the differentiated cell is a lymphocyte.

9. The differentiated daughter cell of claim 6, wherein the differentiated cell is a natural killer cell.

10. The differentiated cell of claim 6, wherein the stem cell is a human induced pluripotent stem cell (iPSC), and wherein the differentiated daughter cell is an iNK cell.

11. The differentiated cell of claim 6, wherein the cell: (a) does not express endogenous CD3, CD4, and/or CD8; and (b) expresses at least one endogenous gene encoding: (i) CD56 (NCAM), CD49, CD43, and/or CD45, or any combination thereof; (ii) NK cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII, cluster of differentiation 16 (CD16)); (iii) natural killer group-2 member D (NKG2D); (iv) CD69; (v) a natural cytotoxicity receptor; or any combination of two or more thereof.

12. The cell of any of the preceding claims, wherein the cell comprises one or more additional genomic edits.

13. The cell of claim 12, wherein the cell: (1) comprises at least one genomic edit characterized by an exogenous nucleic acid expression construct that comprises a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant of FcγRIII (CD16); (iii) interleukin 15 (IL-15); (iv) an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; (v) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vi) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vii) human leukocyte antigen G (HLA-G); (viii) human leukocyte antigen E (HLA-E); (ix) leukocyte surface antigen cluster of differentiation CD47 (CD47); or any combination of two or more thereof and/or (2) comprises at least one genomic edit that results in a loss of function of at least one of: (i) ADORA2A; (ii) T cell immunoreceptor with Ig and ITIM domains (TIGIT); (iii) β-2 microglobulin (B2M); (iv) programmed cell death protein 1 (PD-1); (v) class II, major histocompatibility complex, transactivator (CIITA); (vi) natural killer cell receptor NKG2A (natural killer group 2A); (vii) two or more HLA class II histocompatibility antigen alpha chain genes, and/or two or more HLA class II histocompatibility antigen beta chain genes; (viii) cluster of differentiation 32B (CD32B, FCGR2B); (ix) T cell receptor alpha constant (TRAC); or any combination of two or more thereof.

14. A human induced pluripotent stem cell (iPSC), wherein the iPSC comprises a genomic edit that results in a loss of function of adenosine A2a receptor (ADORA2A).

15. The human iPSC of claim 14, wherein the iPSC comprises a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway or a genomic edit that results in loss of function of Cytokine Inducible SH2 Containing Protein (CISH).

16. The human iPSC of claim 15, wherein the iPSC comprises a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway and a genomic edit that results in loss of function of CISH.

17. The human iPSC of claim 15 or 16, wherein the iPSC comprises a genomic edit that results in a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor.

18. The human iPSC of claim 17, wherein the TGF beta receptor is a TGF beta receptor II (TGFβRII).

19. The human iPSC of any one of claims 14-18, wherein the iPSC expresses one or more pluripotency markers selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog.

20. A differentiated cell, wherein the differentiated cell is a daughter cell of the human iPSC of any one of claims 14-19.

21. The differentiated cell of claim 20, wherein the differentiated cell is an immune cell.

22. The differentiated cell of claim 20, wherein the differentiated cell is a lymphocyte.

23. The differentiated daughter cell of claim 20, wherein the differentiated cell is a natural killer cell.

24. The differentiated cell of claim 20, wherein the differentiated daughter cell is an iNK cell.

25. The differentiated cell of claim 20, wherein the cell: (a) does not express endogenous CD3, CD4, and/or CD8; and (b) expresses at least one endogenous gene encoding: (i) CD56 (NCAM), CD49, CD43, and/or CD45, or any combination thereof; (ii) NK cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII, cluster of differentiation 16 (CD16)); (iii) natural killer group-2 member D (NKG2D); (iv) CD69; (v) a natural cytotoxicity receptor; or any combination of two or more thereof.

26. The cell of any of claims 14-25, wherein the cell comprises one or more additional genomic edits.

27. The cell of claim 26, wherein the cell: (1) comprises at least one genomic edit characterized by an exogenous nucleic acid expression construct that comprises a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant of FcγRIII (CD16); (iii) interleukin 15 (IL-15); (iv) an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; (v) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vi) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vii) human leukocyte antigen G (HLA-G); (viii) human leukocyte antigen E (HLA-E); (ix) leukocyte surface antigen cluster of differentiation CD47 (CD47); or any combination of two or more thereof and/or (2) comprises at least one genomic edit that results in a loss of function of at least one of: (i) cytokine inducible SH2 containing protein (CISH); (ii) T cell immunoreceptor with Ig and ITIM domains (TIGIT); (iii) β-2 microglobulin (B2M); (iv) programmed cell death protein 1 (PD-1); (v) class II, major histocompatibility complex, transactivator (CIITA); (vi) natural killer cell receptor NKG2A (natural killer group 2A); (vii) two or more HLA class II histocompatibility antigen alpha chain genes, and/or two or more HLA class II histocompatibility antigen beta chain genes; (viii) cluster of differentiation 32B (CD32B, FCGR2B); (ix) T cell receptor alpha constant (TRAC); or any combination of two or more thereof.

28. The cell of any one of claims 1-27, wherein: the genomic edit resulting in loss of function of CISH was produced using a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 258-364, 1155, and 1162; the genomic edit resulting in loss of function of TGFβRII was produced using a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 29-257, 1157, and 1161; and/or the genomic edit resulting in loss of function of ADORA2A was produced using a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 827-1143, 1159, and 1163.

29. The cell of any one of claims 1-28, wherein: the genomic edit resulting in loss of function of CISH was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease and (ii) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 258-364, 1155, and 1162; the genomic edit resulting in loss of function of TGFβRII was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease and (ii) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 29-257, 1157, and 1161; and/or the genomic edit resulting in loss of function of ADORA2A was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease and (ii) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 827-1143, 1159, and 1163.

30. A method of making the cell of any one of claims 1-29, the method comprising contacting the cell with one or more of: an RNA-guided nuclease and a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 258-364, 1155, and 1162; an RNA-guided nuclease and a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 29-257, 1157, and 1161; and/or an RNA-guided nuclease and a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 827-1143, 1159, and 1163.

31. A method of making the cell of any one of claims 1-30, the method comprising contacting the cell with one or more of: a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease and (ii) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 258-364, 1155, and 1162; a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease and (ii) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 29-257, 1157, and 1161; and/or a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease and (ii) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence according to any one of SEQ ID NO: 827-1143, 1159, and 1163.

32. The method of any one of claims 29-31, wherein the RNA-guided nuclease is a Cas12a variant.

33. The method of claim 32, wherein the Cas12a variant comprises one or more amino acid substitutions selected from M537R, F870L, and H800A.

34. The method of claim 32, wherein the Cas12a variant comprises amino acid substitutions M537R, F870L, and H800A.

35. The method of claim 32, wherein the Cas12a variant comprises the amino acid sequence of SEQ ID NO:1148.

36. The method of any one of claims 30-35, comprising contacting the cell with: (i) a guide RNA comprising a targeting domain sequence comprising the nucleotide sequence of SEQ ID NO: 1155 or 1162; a guide RNA comprises a targeting domain sequence comprising the nucleotide sequence of SEQ ID NO: 1157 or 1161; and a guide RNA comprises a targeting domain sequence comprising the nucleotide sequence of SEQ ID NO: 1159 or 1163; and (ii) an RNA-guided nuclease comprising the amino acid sequence of one of SEQ ID NO:1144-1151 (or a portion thereof).

37. A pluripotent human stem cell, wherein the stem cell comprises a disruption in the transforming growth factor beta (TGF beta) signaling pathway.

38. The pluripotent human stem cell of claim 34, wherein the stem cell comprises a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway.

39. The pluripotent human stem cell of claim 37 or 38, comprising a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor.

40. The pluripotent human stem cell of claim 39, wherein the TGF beta receptor is a TGF beta receptor II (TGFβRII).

41. The pluripotent human stem cell of any one of claims 37-40, further comprising a loss of function of an antagonist of interleukin signaling.

42. The pluripotent human stem cell of any one of claims 37-41, wherein the stem cell further comprises a genomic modification that results in the loss of function of an antagonist of interleukin signaling.

43. The pluripotent human stem cell of claim 41 or 42, wherein the antagonist of interleukin signaling is an antagonist of the IL-15 signaling pathway and/or of the IL-2 signaling pathway.

44. The pluripotent human stem cell of any one of claims 37-43, comprising a loss of function of Cytokine Inducible SH2 Containing Protein (CISH).

45. The pluripotent human stem cell of claim 44, wherein the stem cell comprises a genomic modification that results in the loss of function of CISH.

46. The pluripotent human stem cell of any one of claims 37-45, wherein the stem cell expresses one or more pluripotency markers selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog.

47. A differentiated cell, wherein the differentiated cell is a daughter cell of the pluripotent human stem cell of any one of claims 37-46.

48. The differentiated cell of claim 47, wherein the differentiated cell is an immune cell.

49. The differentiated cell of claim 47, wherein the differentiated cell is a lymphocyte.

50. The differentiated daughter cell of claim 47, wherein the differentiated cell is a natural killer cell.

51. The differentiated cell of claim 47, wherein the stem cell is a human induced pluripotent stem cell (iPSC), and wherein the differentiated daughter cell is an iNK cell.

52. The differentiated cell of claim 47, wherein the cell: (a) does not express endogenous CD3, CD4, and/or CD8; and (b) expresses at least one endogenous gene encoding: (i) CD56 (NCAM), CD49, CD43, and/or CD45, or any combination thereof; (ii) NK cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII, cluster of differentiation 16 (CD16)); (iii) natural killer group-2 member D (NKG2D); (iv) CD69; (v) a natural cytotoxicity receptor; or any combination of two or more thereof.

53. The cell of any of claims 37-52, wherein the cell comprises one or more additional genomic edits.

54. The cell of claim 53, wherein the cell: (1) comprises at least one genomic edit characterized by an exogenous nucleic acid expression construct that comprises a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant of FcγRIII (CD16); (iii) interleukin 15 (IL-15); (iv) an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; (v) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vi) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vii) human leukocyte antigen G (HLA-G); (viii) human leukocyte antigen E (HLA-E); (ix) leukocyte surface antigen cluster of differentiation CD47 (CD47); or any combination of two or more thereof; and/or (2) comprises at least one genomic edit that results in a loss of function of at least one of: (i) cytokine inducible SH2 containing protein (CISH); (ii) adenosine A2a receptor (ADORA2A); (iii) T cell immunoreceptor with Ig and ITIM domains (TIGIT); (iv) β-2 microglobulin (B2M); (v) programmed cell death protein 1 (PD-1); (vi) class II, major histocompatibility complex, transactivator (CIITA); (vii) natural killer cell receptor NKG2A (natural killer group 2A); (viii) two or more HLA class II histocompatibility antigen alpha chain genes, and/or two or more HLA class II histocompatibility antigen beta chain genes; (ix) cluster of differentiation 32B (CD32B, FCGR2B); (x) T cell receptor alpha constant (TRAC); or any combination of two or more thereof.

55. A method of culturing a pluripotent human stem cell, comprising culturing the stem cell in a medium comprising activin.

56. The method of claim 55, wherein the pluripotent human stem cell is an embryonic stem cell or an induced pluripotent stem cell.

57. The method of claim 55 or 56, wherein the pluripotent human stem cell does not express TGFβRII.

58. The method of any one of claims 55-57, wherein the pluripotent human stem cell is genetically engineered not to express TGFβRII.

59. The method of any one of claims 55-57, wherein the pluripotent human stem cell is genetically engineered to knock out a gene encoding TGFβRII.

60. The method of any one of claims 55-59, wherein the activin is activin A.

61. The method of any one of claims 55-60, wherein the medium does not comprise TGFβ.

62. The method of any one of claims 55-61, wherein the culturing is performed for a defined period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days, or more).

63. The method of any one of claims 55-62, wherein at one or more times during or following the culturing step, the pluripotent human stem cell maintains pluripotency (e.g., exhibits one or more pluripotency markers).

64. The method of claim 63, wherein at one or more times during or following the culturing step, the pluripotent human stem cell expresses a detectable level of one or more of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog.

65. The method of claim 63, wherein at a time during or following the culturing step, the pluripotent human stem cell is differentiated into cells of endoderm, mesoderm, and/or ectoderm lineage.

66. The method of claim 65, wherein the pluripotent human stem cell, or its progeny, is further differentiated into a natural killer (NK) cell.

67. The method of any one of claims 55-66, wherein the pluripotent human stem cell: (1) comprises at least one genomic edit characterized by an exogenous nucleic acid expression construct that comprises a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant of FcγRIII (CD16); (iii) interleukin 15 (IL-15); (iv) an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; (v) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vi) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vii) human leukocyte antigen G (HLA-G); (viii) human leukocyte antigen E (HLA-E); (ix) leukocyte surface antigen cluster of differentiation CD47 (CD47); or any combination of two or more thereof; and/or (2) comprises at least one genomic edit that results in a loss of function of at least one of: (i) cytokine inducible SH2 containing protein (CISH); (ii) adenosine A2a receptor (ADORA2A); (iii) T cell immunoreceptor with Ig and ITIM domains (TIGIT); (iv) β-2 microglobulin (B2M); (v) programmed cell death protein 1 (PD-1); (vi) class II, major histocompatibility complex, transactivator (CIITA); (vii) natural killer cell receptor NKG2A (natural killer group 2A); (viii) two or more HLA class II histocompatibility antigen alpha chain genes, and/or two or more HLA class II histocompatibility antigen beta chain genes; (ix) cluster of differentiation 32B (CD32B, FCGR2B); (x) T cell receptor alpha constant (TRAC); or any combination of two or more thereof.

68. A cell culture comprising (i) a pluripotent human stem cell and (ii) a cell culture medium comprising activin, wherein the pluripotent human stem cell comprises a disruption in the transforming growth factor beta (TGF beta) signaling pathway.

69. The cell culture of claim 68, wherein the stem cell comprises a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway.

70. The cell culture of claim 69, wherein the genomic edit is a genomic edit.

71. The cell culture of any one of claims 68-70, wherein the stem cell comprises a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor.

72. The cell culture of claim 71, wherein the TGF beta receptor is a TGF beta receptor II (TGFβRII).

73. The cell culture of any one of claims 68-72, wherein the pluripotent human stem cell: (1) comprises at least one genomic edit characterized by an exogenous nucleic acid expression construct that comprises a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant of FcγRIII (CD16); (iii) interleukin 15 (IL-15); (iv) an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; (v) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vi) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vii) human leukocyte antigen G (HLA-G); (viii) human leukocyte antigen E (HLA-E); (ix) leukocyte surface antigen cluster of differentiation CD47 (CD47); or any combination of two or more thereof; and/or (2) comprises at least one genomic edit that results in a loss of function of at least one of: (i) cytokine inducible SH2 containing protein (CISH); (ii) adenosine A2a receptor (ADORA2A); (iii) T cell immunoreceptor with Ig and ITIM domains (TIGIT); (iv) β-2 microglobulin (B2M); (v) programmed cell death protein 1 (PD-1); (vi) class II, major histocompatibility complex, transactivator (CIITA); (vii) natural killer cell receptor NKG2A (natural killer group 2A); (viii) two or more HLA class II histocompatibility antigen alpha chain genes, and/or two or more HLA class II histocompatibility antigen beta chain genes; (ix) cluster of differentiation 32B (CD32B, FCGR2B); (x) T cell receptor alpha constant (TRAC); or any combination of two or more thereof.

74. A method of increasing a level of iNK cell activity comprising: (i) providing a pluripotent human stem cell comprising a disruption in the transforming growth factor beta (TGF beta) signaling pathway; and (ii) differentiating the pluripotent human stem cell into an iNK cell, wherein the iNK cell has a higher level of cell activity as compared to an iNK cell not comprising a disruption of the TGF beta signaling pathway.

75. The method of claim 74, wherein the iNK is differentiated from a pluripotent human stem cell cultured in a medium comprising activin.

76. The method of claim 74 or 75, further comprising culturing the pluripotent human stem cell in a medium comprising activin before and/or during the differentiating step.

77. The method of any one of claims 74-76, further comprising disrupting the transforming growth factor beta (TGF beta) signaling pathway in the pluripotent human stem cell.

78. The method of any one of claims 73-77, wherein the stem cell comprises a genomic edit that results in a loss of function of an agonist of the TGF beta signaling pathway.

79. The method of any one of claims 73-78, wherein the stem cell comprises a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor.

80. The method of claim 79, wherein the TGF beta receptor is a TGF beta receptor II (TGFβRII).

81. The method of any one of claims 73-80, wherein the pluripotent human stem cell: (1) comprises at least one genomic edit characterized by an exogenous nucleic acid expression construct that comprises a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant of FcγRIII (CD16); (iii) interleukin 15 (IL-15); (iv) an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; (v) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vi) an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; (vii) human leukocyte antigen G (HLA-G); (viii) human leukocyte antigen E (HLA-E); (ix) leukocyte surface antigen cluster of differentiation CD47 (CD47); or any combination of two or more thereof; and/or (2) comprises at least one genomic edit that results in a loss of function of at least one of: (i) cytokine inducible SH2 containing protein (CISH); (ii) adenosine A2a receptor (ADORA2A); (iii) T cell immunoreceptor with Ig and ITIM domains (TIGIT); (iv) β-2 microglobulin (B2M); (v) programmed cell death protein 1 (PD-1); (vi) class II, major histocompatibility complex, transactivator (CIITA); (vii) natural killer cell receptor NKG2A (natural killer group 2A); (viii) two or more HLA class II histocompatibility antigen alpha chain genes, and/or two or more HLA class II histocompatibility antigen beta chain genes; (ix) cluster of differentiation 32B (CD32B, FCGR2B); (x) T cell receptor alpha constant (TRAC); or any combination of two or more thereof.

82. A method of treating a subject having or at risk of cancer, the method comprising administering to the subject the cell of any one of claim 6-13, 20-29, or 47-54, thereby treating the cancer in the subject.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0067] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0068] FIG. 1 shows microscopy of cell morphology and flow cytometry of pluripotency markers of human induced pluripotent stem cells (hiPSCs) grown in various media in the absence or presence of Activin A (1 ng/ml or 4 ng/ml ActA).

[0069] FIG. 2 shows morphology of TGFβRII knockout hiPSCs (clone 7) or CISH/TGFβRII DKO hiPSCs (clone 7) cultured in media with or without Activin A (1 ng/mL, 2 ng/mL, 4 ng/mL, or 10 ng/mL).

[0070] FIG. 3 shows morphology of TGFβRII knockout hiPSCs (clone 9) cultured in media with our without Activin A (1 ng/mL, 2 ng/mL, 4 ng/mL, or 10 ng/mL).

[0071] FIG. 4A shows the bulk editing rates at the CISH and TGFβRII loci for single knockout and double knockout hiPSCs.

[0072] FIG. 4B shows expression of Oct4 and SSEA4 in TGFβRII knockout hiPSCs, CISH knockout hiPSCs, and double knockout hiPSCs cultured in Activin A.

[0073] FIG. 5 shows expression of Nanog and Tra-1-60 in TGFβRII knockout hiPSCs, CISH knockout hiPSCs, and double knockout hiPSCs cultured in Activin A.

[0074] FIG. 6 is a schematic of the procedure related to the STEMdiff™ Trilineage Differentiation Kit (STEMCELL Technologies Inc.).

[0075] FIG. 7A shows expression of differentiation markers of TGFβRII knockout hiPSCs, CISH knockout hiPSCs, and double knockout hiPSCs cultured in Activin A.

[0076] FIG. 7B shows karyotypes of TGFβRII/CISH double knockout hiPSCs cultured in Activin A.

[0077] FIG. 7C shows an expanded Activin A concentration curve performed on an unedited parental PSC line, an edited TGFβRII KO clone (C7), and an additional representative (unedited) cell line designated RUCDR. The minimum concentration of Activin A required to maintain each line varied slightly with the TGFβRII KO clone requiring a higher baseline amount of Activin A as compared to the parental control (0.5 ng/ml vs 0.1 ng/ml).

[0078] FIG. 7D shows the stemness marker expression in an unedited parental PSC line, an edited TGFβRII KO clone (C7), and an unedited RUCDR cell line, when cultured with the base medias alone (no supplemental Activin A). The TGFβRII KO iPSCs did not maintain stemness marker expression while the two unedited lines were able to maintain stemness marker expression in E8.

[0079] FIG. 8A is a schematic representation of an exemplary method for creating edited iPSC clones, followed by the differentiation to and characterization of enhanced CD56+ iNK cells.

[0080] FIG. 8B is a schematic of an iNK cell differentiation process utilizing STEMDiff APEL2 during the second stage of the differentiation process.

[0081] FIG. 8C is a schematic of an iNK cell differentiation process utilizing NK-MACS with 15% serum during the second stage of the differentiation process.

[0082] FIG. 8D shows the fold-expansion of unedited PCS-derived iNK cells and the percentage of iNK cells expressing CD45 and CD56 at day 39 of differentiation when differentiated using NK-MACS or Apel2 methods as depicted in FIG. 8C and FIG. 8B respectively.

[0083] FIG. 8E shows in the upper panel a heat map of the surface expression phenotypes (measured as a percentage of the population) of differentiated iNK cells derived from unedited PCS iPSCs when differentiated using NK-MACS or APEL2 methods as depicted in FIG. 8C and FIG. 8B respectively. The bottom panel displays representative histogram plots to illustrate the differences in the iNKs generated by the two methods.

[0084] FIG. 8F shows a heat map of the surface expression phenotypes (measured as a percentage of the population) of differentiated edited iNKs (TGFβRII knockout, CISH knockout, and double knockout (DKO)) and unedited parental iPSCs (WT) when differentiated using NK-MACS or APEL2 methods as depicted in FIG. 8C and FIG. 8B respectively.

[0085] FIG. 8G shows unedited iNK cell effector function when differentiated using NK-MACS or APEL2 methods as depicted in FIG. 8C and FIG. 8B respectively.

[0086] FIG. 9 shows differentiation phenotypes of edited clones (TGFβRII knockout, CISH knockout, and double knockout) as compared to parental wild type clones.

[0087] FIG. 10 shows surface expression phenotype of edited iNKs (TGFβRII knockout, CISH knockout, and double knockout) as compared to parental clone iNKs and wild type cells.

[0088] FIG. 11A shows surface expression phenotype of edited iNKs (TGFβRII knockout, CISH knockout, and double knockout) as compared to parental clone iNKs (“WT”) and peripheral blood-derived natural killer cells.

[0089] FIG. 11B is a flow cytometry histogram plot that shows the surface expression phenotype of edited iNK cells (TGFβRII/CISH double knockout) as compared to parental clone iNK cells (“unedited iNK cells”).

[0090] FIG. 11C shows surface expression phenotypes (measured as a percentage of the population) of edited iNK cells (TGFβRII/CISH double knockout) as compared to parental clone iNK cells (“unedited iNK cells”) at day 25, day 32, and day 39 post-hiPSC differentiation (average values from at least 5 separate differentiations).

[0091] FIG. 11D shows pSTAT3 expression phenotypes (measured as a percentage of the population) of edited CD56+ iNK cells (“CISH KO iNKs”) as compared to parental clone CD56+ iNK cells (“unedited iNKs”) at 10 minutes and 120 minutes following IL-15 induced activation. Briefly, the day 39 or day 40 iNKs are plated the day before in a cytokine starve condition. The next day the cells are stimulated with 10 ng/ml of IL15 for the length of time indicated. The cells are fixed immediately at the end of the time point, stained for CD56 followed by an intracellular stain. The cells were processed on a NovoCyte Quanteon and the data was analyzed in FlowJo. Data shown is a representative experiment of >3 experiments performed.

[0092] FIG. 11E shows pSMAD2/3 expression phenotypes (measured as a percentage of the population) of edited CD56+ iNK cells (TGFβRII/CISH double knockout, “DKO iNKs”) as compared to parental clone CD56+ iNK cells (“unedited iNK cells”) at 10 minutes and 120 minutes following IL-15 and TGF-β induced activation Briefly, the day 39 or day 40 iNKs were plated the day before in a cytokine starve condition. The next day the cells were stimulated with 10 ng/ml of IL-15 and 50 ng/ml of TGF-β for the length of time indicated. The cells were fixed immediately at the end of the time point, stained for CD56 followed by an intracellular stain. The cells were processed on a NovoCyte Quanteon and the data was analyzed in FlowJo. Data shown is a representative experiment of >3 experiments performed.

[0093] FIG. 11F shows IFN-γ expression phenotypes (measured as a percentage of the population) of edited CD56+ iNK cells (TGFβRII/CISH double knockout, “DKO IFNg”) as compared to parental clone CD56+ iNK cells (unedited iNKs, “WT IFNg”) with or without phorbol myristate acetate (PMA) and ionomycin (IMN) stimulation. The data is representative. It is generated from a single differentiation and each condition in the assay is run with 2 technical replicates. **p<0.05 vs unedited iNK cells (paired t test).

[0094] FIG. 11G shows TNF-α expression phenotypes (measured as a percentage of the population) of edited CD56+ iNK cells (TGFβRII/CISH double knockout, “DKO TNF a”) as compared to parental clone CD56+ iNK cells (unedited iNK cells, “WT TNFa”) with or without Phorbol myristate acetate (PMA) and Ionomycin (IMN) stimulation. The data is representative. It is generated from a single differentiation and each condition in the assay is run with 2 technical replicates. **p<0.05 vs unedited iNK cells (paired t test).

[0095] FIG. 12A is a schematic representation of an exemplary solid tumor cell killing assay, depicting the use of edited iNK cells (TGFβRII/CISH double knockout) to kill SK-OV-3 ovarian cells in the presence or absence of IL-15 and TGF-0.

[0096] FIG. 12B shows the results of a solid tumor killing assay as described in FIG. 12A. iNK cells function to reduce tumor cell spheroid size. Certain edited iNK cells (CISH single knockout, “CISH_2, 4, 5, and 8”) were not significantly different from the parental clone iNK cells (“WT_2”), while certain edited iNK cells (TGFβRII single knockout, “TGFβRII_7”, and TGFβRII/CISH double knockout “DKO”) functioned significantly better at effector-target (E:T) ratios of 1 or greater when measured in the presence of TGF-β as compared to parental clone iNK cells (“WT_2”). ****p<0.0001 vs unedited iNK cells (two-way ANOVA, Sidak's multiple comparisons test).

[0097] FIG. 12C shows edited iNK cell effector function as compared to unedited iNK cells.

[0098] FIG. 13 shows the results of an in-vitro serial killing assay, where iNK cells are serially challenged with hematological cancer cells (e.g., Nalm6 cells) in the presence of 10 ng/ml of IL-15 and 10 ng/ml of TGF-β; the X axis represents time, with tumor cells being added every 48 hours, while the Y axis represents killing efficacy as measured by normalized total red object area (e.g., presence of tumor cells). The data shows that edited iNK cells (TGFβRII/CISH double knockout) continue to kill hematological cancer cells while unedited iNK cells lose this function at equivalent time points.

[0099] FIG. 14 shows surface expression phenotypes (measured as a percentage of the population) of certain edited iNK clonal cells (CISH single knockout “CISH_C2, C4, C5, and C8”, TGFβRII single knockout “TGFβRII-C7”, and TGFβRII/CISH double knockout “DKO-C1”) as compared to parental clone iNK cells (“WT”) at day 25, day 32, and day 39 post-hiPSC differentiation when cultured in the presence of 1 ng/mL or 10 ng/mL IL-15.

[0100] FIG. 15A is a schematic of an in-vivo tumor killing assay. Mice were intraperitoneally inoculated with 1×10.sup.6 SKOV3-luc cells, mice are randomized, and 4 days later, 20×10.sup.6 iNK cells were introduced intraperitoneally. Mice were followed for up to 60 days post-tumor implantation. The X axis represents time since implantation, while the Y axis represents killing efficacy as measured by total bioluminescence (p/s).

[0101] FIG. 15B shows the results of an in-vivo tumor killing assay as described in FIG. 15A. An individual mouse is represented by each horizontal line. The data show that both unedited iNK cells (“unedited iNK”) and DKO edited iNK cells (TGFβRII/CISH double knockout) prevent tumor growth better than vehicle, while edited iNK cells kill tumor cells significantly better than vehicle in-vivo. Each experimental group had 9 animals each. ***p<0.001, ****p<0.0001 by a 2-way ANOVA analysis.

[0102] FIG. 15C shows the averaged results with standard error of the mean of the in-vivo tumor killing assay described in FIG. 15B. Populations of mice are represented by each horizontal line. The data show that DKO edited iNK cells (TGFβRII/CISH double knockout) prevent tumor growth and kill tumor cells significantly better than vehicle or unedited iNK cells in-vivo. ***p<0.001, ****p<0.0001 by a 2-way ANOVA analysis.

[0103] FIG. 16A shows surface expression phenotypes (measured as a percentage of the population) of bulk edited iNK cells (left panel—ADORA2A single knockout) or certain edited iNK clonal cells (right panel—ADORA2A single knockout) as compared to parental clone iNK cells (“PCS WT”) at day 25, day 32, and day 39 or at day 28, day 36, and day 39 post-hiPSC differentiation. Representative data from multiple differentiations.

[0104] FIG. 16B shows cyclic AMP (cAMP) concentration phenotypes following 5′-(N-Ethylcarboxamido)adenosine (“NECA”, adenosine agonist) activation for edited iNK clonal cells (ADORA2A single knockout) as compared to parental clone iNK cells (“unedited iNKs”). The Y axis represents average cAMP concentration in nM (a proxy for ADORA2A activation), while the X axis represents NECA concentration in nM.

[0105] FIG. 16C shows the results of an in-vitro serial killing assay, where iNK cells are serially challenged with hematological cancer cells (e.g., Nalm6 cells) in the presence of 100 μM NECA, and 10 ng/ml of IL-15; the X axis represents time, with tumor cells being added every 48 hours, while the Y axis represents killing efficacy as measured by total red object area (e.g., presence of tumor cells). The data shows that edited iNK cells (“ADORA2A KO iNK”) kill hematological cancer cells more effectively than unedited iNK cells (“Ctrl iNK”) under conditions that mimic adenosine suppression.

[0106] FIG. 17A shows surface expression phenotypes (measured as a percentage of the population) of certain edited iNK clonal cells (TGFβRII/CISH/ADORA2A triple knockout, “CRA_6” and “CR+A_8”) as compared to parental clone iNK cells (“WT_2”) at day 25, day 32, and day 39 post-hiPSC differentiation. Data is representative of multiple differentiations.

[0107] FIG. 17B shows cyclic AMP (cAMP) concentration phenotypes following NECA (adenosine agonist) activation for edited iNK clonal cells (TGFβRII/CISH/ADORA2A triple knockout, “TKO iNKs”) as compared to parental clone iNK cells (“unedited iNKs”). The Y axis represents average cAMP concentration in nM (a proxy for ADORA2A activation), while the X axis represents NECA concentration in nM.

[0108] FIG. 17C shows the results of a solid tumor killing assay as described in FIG. 12A without IL-15. iNK cells function to reduce tumor cell spheroid size. The Y axis measures total integrated red object (e.g., presence of tumor cells), while the X axis represents the effector to target (E:T) cell ratio. The edited iNK cells (ADORA2A single knockout “ADORA2A”, TGFβRII/CISH double knockout “DKO”, or TGFβRII/CISH/ADORA2A triple knockout “TKO”) had lower EC50 rates when measured in the presence of TGF-β as compared to parental clone iNK cells (“Control”) (average values from at least 3 separate differentiations).

[0109] FIG. 18 shows the results of guide RNA selection assays for the loci TGFβRII, CISH, ADORA2A, TIGIT, and NKG2A utilizing in-vitro editing in iPSCs.

DETAILED DESCRIPTION

[0110] Some aspects of the disclosure are based, at least in part, on the recognition that, surprisingly, stem cells, e.g., embryonic stem cells or induced pluripotent stem cells, can be cultured in a culture medium that includes activin A, and that the presence of activin in the culture media abrogates a requirement for the presence of a TGF signaling agonist, e.g., of TGF beta, in the culture medium. Some aspects of the present disclosure relate to the recognition that, surprisingly, stem cells, including human stem cells, such as, for example, human embryonic stem cells or human induced pluripotent stem cells, retain their pluripotency when cultured in media comprising activin, e.g., activin A, even in the absence of a TGF beta signaling agonist, such as, for example, TGF beta, in the culture medium. Additionally, the disclosure is based, in part, on the recognition that, surprisingly, iPSCs lacking TGFβIIR (e.g., genetically knocked out, for example, via gene editing) can be cultured in a culture medium that includes activin, and that such cells not only grow but maintain their pluripotency. The present disclosure additionally encompasses cell cultures comprising embryonic stem cells and a culture medium comprising activin, as well as methods of culturing such stem cells and/or progeny thereof.

Definitions and Abbreviations

[0111] Unless otherwise specified, each of the following terms have the meaning set forth in this section.

[0112] The indefinite articles “a” and “an” refer to at least one of the associated noun, and are used interchangeably with the terms “at least one” and “one or more.” The conjunctions “or” and “and/or” are used interchangeably as non-exclusive disjunctions.

[0113] The term “cancer” (also used interchangeably with the terms, “hyperproliferative” and “neoplastic”), as used herein, refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Cancerous disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, e.g., malignant tumor growth, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state, e.g., cell proliferation associated with wound repair. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. In some embodiments, “cancer” includes malignancies of or affecting various organ systems, such as lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract. In some embodiments, “cancer” includes adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and/or cancer of the esophagus.

[0114] As used herein, the term “carcinoma” is refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The term carcinoma, as used herein, is well-recognized in the art. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. In some embodiments, carcinoma also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. In some embodiments, an “adenocarcinoma” is a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. In some embodiments, a “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0115] The term “differentiation” as used herein is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the features of a specialized cell such as, for example, a blood cell or a muscle cell. In some embodiments, a differentiated or differentiation-induced cell is one that has taken on a more specialized (“committed”) position within the lineage of a cell. For example, an iPSC can be differentiated into various more differentiated cell types, for example, a neural or a hematopoietic stem cell, a lymphocyte, a cardiomyocyte, and other cell types, upon treatment with suitable differentiation factors in the cell culture medium. In some embodiments, suitable methods, differentiation factors, and cell culture media for the differentiation of pluri- and multipotent cell types into more differentiated cell types are well known to those of skill in the art. In some embodiments, the term “committed”, is applied to the process of differentiation to refer to a cell that has proceeded through a differentiation pathway to a point where, under normal circumstances, it would or will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type (other than a specific cell type or subset of cell types) nor revert to a less differentiated cell type.

[0116] The terms “differentiation marker,” “differentiation marker gene,” or “differentiation gene,” as used herein refers to genes or proteins whose expression are indicative of cell differentiation occurring within a cell, such as a pluripotent cell. In some embodiments, differentiation marker genes include, but are not limited to, the following genes: CD34, CD4, CD8, CD3, CD56 (NCAM), CD49, CD45; NK cell receptor (cluster of differentiation 16 (CD16)), natural killer group-2 member D (NKG2D), CD69, NKp30, NKp44, NKp46, CD158b, FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NROB1, CXCR4, CYP2B6, GAT A3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), ZIC1, GATA1, GATA2, HDAC4, HDAC5, HDAC7, HDAC9, NOTCH1, NOTCH2, NOTCH4, PAX5, RBPJ, RUNX1, STAT1 and STAT3.

[0117] The terms “differentiation marker gene profile,” or “differentiation gene profile,” “differentiation gene expression profile,” “differentiation gene expression signature,” “differentiation gene expression panel,” “differentiation gene panel,” or “differentiation gene signature” as used herein refer to expression or levels of expression of a plurality of differentiation marker genes.

[0118] The term “edited iNK cell” as used herein refers to a natural killer cell which has been modified to change at least one expression product of at least one gene at some point in the development of the cell. In some embodiments, a modification can be introduced using, e.g., gene editing techniques such as CRISPR-Cas or, e.g., dominant-negative constructs. In some embodiments, an iNK cell is edited at a time point before it has differentiated into an iNK cell, e.g., at a precursor stage, at a stem cell stage, etc. In some embodiments, an edited iNK cell is compared to a non-edited iNK cell (an NK cell produced by differentiating an iPSC cell, which iPSC cell and/or iNK cell do not have modifications, e.g., genetic modifications).

[0119] The term “embryonic stem cell” as used herein refers to pluripotent stem cells derived from the inner cell mass of the embryonic blastocyst. In some embodiments, embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In some such embodiments, embryonic stem cells do not contribute to the extra-embryonic membranes or the placenta, i.e., are not totipotent.

[0120] The term “endogenous,” as used herein in the context of nucleic acids (e.g., genes, protein-encoding genomic regions, promoters), refers to a native nucleic acid or protein in its natural location, e.g., within the genome of a cell.

[0121] The term “exogenous,” as used herein in the context of nucleic acids, e.g., expression constructs, cDNAs, indels, and nucleic acid vectors, refers to nucleic acids that have artificially been introduced into the genome of a cell using, for example, gene-editing or genetic engineering techniques, e.g., CRISPR-based editing techniques.

[0122] The term “genome editing system” refers to any system having RNA-guided DNA editing activity.

[0123] The terms “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 or a Cpf1 (Cas12a) to a target sequence such as a genomic or episomal sequence in a cell.

[0124] The terms “hematopoietic stem cell,” or “definitive hematopoietic stem cell” as used herein, refer to CD34-positive stem cells. In some embodiments, CD34-positive stem cells are capable of giving rise to mature myeloid and/or lymphoid cell types. In some embodiments, the myeloid and/or lymphoid cell types include, for example, T cells, natural killer cells and/or B cells.

[0125] The terms “induced pluripotent stem cell” or “iPSC” as used herein to refer to a stem cell obtained from a differentiated somatic (e.g., adult, neonatal, or fetal) cell by a process referred to as reprogramming (e.g., dedifferentiation). In some embodiments, reprogrammed cells are capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. iPSCs are not found in nature.

[0126] The term “multipotent stem cell” as used herein refers to a cell that has the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm and endoderm), but not all three germ layers. Thus, in some embodiments, a multipotent cell may also be termed a “partially differentiated cell.” Multipotent cells are well-known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. In some embodiments, “multipotent” indicates that a cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent hematopoietic cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons. Accordingly, in some embodiments, “multipotency” refers to a state of a cell with a degree of developmental potential that is less than totipotent and pluripotent.

[0127] The term “pluripotent” as used herein refers to ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) or a given organism (e.g., human). For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Generally, pluripotency may be described as a continuum of developmental potencies ranging from an incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0128] The term “pluripotency” as used herein refers to a cell that has the developmental potential to differentiate into cells of all three germ layers (Ectoderm, mesoderm, and endoderm). In some embodiments, pluripotency can be determined, in part, by assessing pluripotency characteristics of the cells. In some embodiments, pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers including, but not limited to SSEA1 (mouse only), SSEA3/4, SSEA5, TRA1- 60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or CD50; (iv) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm); (v) teratoma formation consisting of the three somatic lineages; and (vi) formation of embryoid bodies consisting of cells from the three somatic lineages.

[0129] The term “pluripotent stem cell morphology” as used herein refers to the classical morphological features of an embryonic stem cell. In some embodiments, normal embryonic stem cell morphology is characterized as small and round in shape, with a high nucleus-to-cytoplasm ratio, the notable presence of nucleoli, and typical intercell spacing.

[0130] The term “polynucleotide” (including, but not limited to “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide”) as used herein refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides. In some embodiments, polynucleotides, nucleotide sequences, nucleic acids etc. can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. In some such embodiments, modifications can occur at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. In general, a nucleotide sequence typically carries genetic information, including, but not limited to, the information used by cellular machinery to make proteins and enzymes. In some embodiments, a nucleotide sequence and/or genetic information comprises double- or single-stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and/or sense and/or antisense polynucleotides. In some embodiments, nucleic acids containing modified bases.

[0131] Conventional IUPAC notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10; 13(9):3021-30, incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence may be encoded by either DNA or RNA, for example in gRNA targeting domains.

TABLE-US-00001 TABLE 1 IUPAC nucleic acid notation Character Base A Adenine T Thymine or Uracil G Guanine C Cytosine U Uracil K G or T/U M A or C R A or G Y C or T/U S C or G W A or T/U B C, G or T/U V A, C or G H A, C or T/U D A, G or T/U N A, C, G or T/U

[0132] The terms “potency” or “developmental potency” as used herein refers to the sum of all developmental options accessible to the cell (i.e., the developmental potency), particularly, for example in the context of cellular developmental potential, In some embodiments, the continuum of cell potency includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells.

[0133] The terms “prevent,” “preventing,” and “prevention” as used herein refer to the prevention of a disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.

[0134] The terms “protein,” “peptide” and “polypeptide” as used herein are used interchangeably to refer to a sequential chain of amino acids linked together via peptide bonds. The terms include individual proteins, groups or complexes of proteins that associate together, as well as fragments or portions, variants, derivatives and analogs of such proteins. Unless otherwise specified, peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus on the left, and proceeding to the carboxyl or C-terminus on the right. Standard one-letter or three-letter abbreviations can be used.

[0135] The terms “reprogramming” or “dedifferentiation” or “increasing cell potency” or “increasing developmental potency” as used herein refer to a method of increasing potency of a cell or dedifferentiating a cell to a less differentiated state. For example, in some embodiments, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. That is, in some embodiments, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state. In some embodiments, “reprogramming” refers to de-differentiating a somatic cell, or a multipotent stem cell, into a pluripotent stem cell, also referred to as an induced pluripotent stem cell, or iPSC. Suitable methods for the generation of iPSCs from somatic or multipotent stem cells are well known to those of skill in the art.

[0136] The terms “RNA-guided nuclease” and “RNA-guided nuclease molecule” are used interchangeably herein. In some embodiments, the RNA-guided nuclease is a RNA-guided DNA endonuclease enzyme. In some embodiments, the RNA-guided nuclease is a CRISPR nuclease. Non-limiting examples of RNA-guided nucleases are listed in Table 2 below, and the methods and compositions disclosed herein can use any combination of RNA-guided nucleases disclosed herein, or known to those of ordinary skill in the art. Those of ordinary skill in the art will be aware of additional nucleases and nuclease variants suitable for use in the context of the present disclosure, and it will be understood that the present disclosure is not limited in this respect.

TABLE-US-00002 TABLE 2 RNA-Guided Nucleases Length Nuclease (a.a.) PAM Reference SpCas9 1368 NGG Cong et al., Science. 2013; 339 (6121): 819-23 SaCas9 1053 NNGRRT Ran et al., Nature. 2015; 520 (7546): 186-91. (KKH) 1067 NNNRRT Kleinstiver et al., Nat Biotechnol. SaCas9 2015; 33 (12): 1293-1298 AsCpf1 1353 TTTV Zetsche et al., Nat Biotechnol. 2017; 35 (1): 31-34. (AsCas12a) LbCpf1 1274 TTTV Zetsche et al., Cell. 2015; 163 (3): 759-71. (LbCas12a) CasX 980 TTC Burstein et al., Nature. 2017; 542 (7640): 237-241. CasY 1200 TA Burstein et al., Nature. 2017; 542 (7640): 237-241. Cas12h1 870 RTR Yan et al., Science. 2019; 363 (6422): 88-91. Cas12i1 1093 TTN Yan et al., Science. 2019; 363 (6422): 88-91. Cas12c1 unknown TG Yan et al., Science. 2019; 363 (6422): 88-91. Cas12c2 unknown TN Yan et al., Science. 2019; 363 (6422): 88-91. eSpCas9 1423 NGG Chen et al., Nature. 2017; 550 (7676): 407-410. Cas9-HF1 1367 NGG Chen et al., Nature. 2017; 550 (7676): 407-410. HypaCas9 1404 NGG Chen et al., Nature. 2017; 550 (7676): 407-410. dCas9-Fok1 1623 NGG U.S. Pat. No. 9,322,037 Sniper-Cas9 1389 NGG Lee et al., Nat Commun. 2018; 9 (1): 3048. xCas9 1786 NGG, NG, Wang et al., Plant Biotechnol J. 2018; pbi. 13053. GAA, GAT AaCas12b 1129 TTN Teng et al. Cell Discov. 2018; 4: 63. evoCas9 1423 NGG Casini et al., Nat Biotechnol. 2018; 36 (3): 265-271. SpCas9-NG 1423 NG Nishimasu et al., Science. 2018; 361 (6408): 1259-1262. VRQR 1368 NGA Li et al., The CRISPR Journal, 2018; 01: 01 VRER 1372 NGCG Kleinstiver et al., Nature. 2016; 529 (7587): 490-5. NmeCas9 1082 NNNNGATT Amrani et al., Genome Biol. 2018; 19 (1): 214. CjCas9 984 NNNNRYAC Kim et al., Nat Commun. 2017; 8: 14500. BhCas12b 1108 ATTN Strecker et al., Nat Commun. 2019 Jan. 22; 10 (1): 212. BhCas12b 1108 ATTN Strecker et al., Nat Commun. 2019 Jan. 22; 10 (1): 212. V4 CasΦ 700-800 TBN Pausch et al., Science 2020; 369 (6501): 333-337. (where B is G, T, or C)

[0137] Additional suitable RNA-guided nucleases, e.g., Cas9 and Cas12 nucleases, will be apparent to the skilled artisan in view of the present disclosure, and the disclosure is not limited by the exemplary suitable nucleases provided herein. In some embodiments, a suitable nuclease is a Cas9 or Cpf1 (Cas12a) nuclease. In some embodiments, the disclosure also embraces nuclease variants, e.g., Cas9 or Cpf1 nuclease variants. In some embodiments, a nuclease is a nuclease variant, which refers to a nuclease comprising an amino acid sequence characterized by one or more amino acid substitutions, deletions, or additions as compared to the wild type amino acid sequence of the nuclease. In some embodiments, a suitable nuclease and/or nuclease variant may also include purification tags (e.g., polyhistidine tags) and/or signaling peptides, e.g., comprising or consisting of a nuclear localization signal sequence. Some non-limiting examples of suitable nucleases and nuclease variants are described in more detail elsewhere herein and also include those described in PCT application PCT/US2019/22374, filed Mar. 14, 2019, and entitled “Systems and Methods for the Treatment of Hemoglobinopathies,” the entire contents of which are incorporated herein by reference. In some embodiments, the RNA-guided nuclease is an Acidaminococcus sp. Cpf1 variant (AsCpf1 variant). In some embodiments, suitable Cpf1 nuclease variants, including suitable AsCpf1 variants will be known or apparent to those of ordinary skill in the art based on the present disclosure, and include, but are not limited to, the Cpf1 variants disclosed herein or otherwise known in the art. For example, in some embodiments, the RNA-guided nuclease is aAcidaminococcus sp. Cpf1 RR variant (AsCpf1-RR). In another embodiment, the RNA-guided nuclease is a Cpf1 RVR variant. For example, suitable Cpf1 variants include those having an M537R substitution, an H800A substitution, and/or an F870L substitution, or any combination thereof (numbering scheme according to AsCpf1 wild-type sequence).

[0138] The term “subject” as used herein means a human or non-human animal. In some embodiments a human subject can be any age (e.g., a fetus, infant, child, young adult, or adult). In some embodiments a human subject may be at risk of or suffer from a disease, or may be in need of alteration of a gene or a combination of specific genes. Alternatively, in some embodiments, a subject may be a non-human animal, which may include, but is not limited to, a mammal. In some embodiments, a non-human animal is a non-human primate, a rodent (e.g., a mouse, rat, hamster, guinea pig, etc.), a rabbit, a dog, a cat, and so on. In certain embodiments of this disclosure, the non-human animal subject is livestock, e.g., avow, a horse, a sheep, a goat, etc. In certain embodiments, the non-human animal subject is poultry, e.g., a chicken, a turkey, a duck, etc.

[0139] The terms “treatment,” “treat,” and “treating,” as used herein refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress, ameliorate, reduce severity of, prevent or delay the recurrence of a disease, disorder, or condition or one or more symptoms thereof, and/or improve one or more symptoms of a disease, disorder, or condition as described herein. In some embodiments, a condition includes an injury. In some embodiments, an injury may be acute or chronic (e.g., tissue damage from an underlying disease or disorder that causes, e.g., secondary damage such as tissue injury). In some embodiments, treatment, e.g., in the form of a modified NK cell or a population of modified NK cells as described herein, may be administered to a subject after one or more symptoms have developed and/or after a disease has been diagnosed. Treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, in some embodiments, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of genetic or other susceptibility factors). In some embodiments, treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. In some embodiments, treatment results in improvement and/or resolution of one or more symptoms of a disease, disorder or condition.

[0140] The term “variant” as used herein refers to an entity such as a polypeptide, polynucleotide or small molecule that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity.

[0141] Stem Cells

[0142] Methods of the disclosure can be used to culture stem cells. Stem cells are typically cells that have the capacity to produce unaltered daughter cells (self-renewal; cell division produces at least one daughter cell that is identical to the parent cell) and to give rise to specialized cell types (potency). Stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic germ (EG) cells, germline stem (GS) cells, human mesenchymal stem cells (hMSCs), adipose tissue-derived stem cells (ADSCs), multipotent adult progenitor cells (MAPCs), multipotent adult germline stem cells (maGSCs) and unrestricted somatic stem cell (USSCs). Generally, stem cells can divide without limit. After division, the stem cell may remain as a stem cell, become a precursor cell, or proceed to terminal differentiation. A precursor cell is a cell that can generate a fully differentiated functional cell of at least one given cell type. Generally, precursor cells can divide. After division, a precursor cell can remain a precursor cell, or may proceed to terminal differentiation.

[0143] Pluripotent stem cells are generally known in the art. The present disclosure provides technologies (e.g., systems, compositions, methods, etc.) related to pluripotent stem cells. In some embodiments, pluripotent stem cells are stem cells that: (a) are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) are capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and/or (c) express one or more markers of embryonic stem cells (e.g., human embryonic stem cells express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). In some aspects, human pluripotent stem cells do not show expression of differentiation markers. In some embodiments, ES cells and/or iPSCs cultured using methods of the disclosure maintain their pluripotency (e.g., (a) are capable of inducing teratomas when transplanted in immunodeficient (SCID) mice; (b) are capable of differentiating to cell types of all three germ layers (e.g., can differentiate to ectodermal, mesodermal, and endodermal cell types); and/or (c) express one or more markers of embryonic stem cells).

[0144] In some embodiments, ES cells (e.g., human ES cells) can be derived from the inner cell mass of blastocysts or morulae. In some embodiments, ES cells can be isolated from one or more blastomeres of an embryo, e.g., without destroying the remainder of the embryo. In some embodiments, ES cells can be produced by somatic cell nuclear transfer. In some embodiments, ES cells can be derived from fertilization of an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or by means to generate ES cells, e.g., with homozygosity in the HLA region. In some embodiments, human ES cells can be produced or derived from a zygote, blastomeres, or blastocyst-staged mammalian embryo produced by the fusion of a sperm and egg cell, nuclear transfer, parthenogenesis, or the reprogramming of chromatin and subsequent incorporation of the reprogrammed chromatin into a plasma membrane to produce an embryonic cell. Exemplary human ES cells are known in the art and include, but are not limited to, MAO1, MAO9, ACT-4, No. 3, H1, H7, H9, H14 and ACT30 ES cells. In some embodiments, human ES cells, regardless of their source or the particular method used to produce them, can be identified based on, e.g., (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and/or (iii) ability to produce teratomas when transplanted into immunocompromised animals. In some embodiments, ES cells have been serially passaged as cell lines.

[0145] iPSCs

[0146] Induced pluripotent stem cells (iPSC) are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, such as an adult somatic cell (e.g., a fibroblast cell or other suitable somatic cell), by inducing expression of certain genes. iPSCs can be derived from any organism, such as a mammal. In some embodiments, iPSCs are produced from mice, rats, rabbits, guinea pigs, goats, pigs, cows, non-human primates or humans. iPSCs are similar to ES cells in many respects, such as the expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling time, embryoid body formation, teratoma formation, viable chimera formation, potency and/or differentiability. Various suitable methods for producing iPSCs are known in the art. In some embodiments, iPSCs can be derived by transfection of certain stem cell-associated genes (such asOct-3/4 (Pouf51) and Sox2) into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved through viral vectors, such as retroviruses, lentiviruses, or adenoviruses. Additional suitable reprogramming methods include the use of vectors that do not integrate into the genome of the host cell, e.g., episomal vectors, or the delivery of reprogramming factors directly via encoding RNA or as proteins has also been described. For example, cells can be transfected with Oct3/4, Sox2, Klf4, and/or c-Myc using a retroviral system or with OCT4, SOX2, NANOG, and/or LIN28 using a lentiviral system. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and can be isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. In one example, iPSCs from adult human cells are generated by the method described by Yu et al. (Science 318(5854):1224 (2007)) or Takahashi et al. (Cell 131:861-72 (2007)). In some embodiments, iPSCs are generated by a commercial source. In some embodiments, iPSCs are generated by a vendor. In some embodiments, iPSCs are generated by a contract research organization. Numerous suitable methods for reprogramming are known to those of skill in the art, and the present disclosure is not limited in this respect.

[0147] Genetically Engineered Stem Cells

[0148] In some embodiments, a stem cell (e.g., iPSC) described herein is genetically engineered to introduce a disruption in one or more targets described herein. For example, in some embodiments, a stem cell (e.g., iPSC) can be genetically engineered to knockout all or a portion of one or more target gene, introduce a frameshift in one or more target genes, and/or cause a truncation of an encoded gene product (e.g., by introducing a premature stop codon). In some embodiments, a stem cell (e.g., iPSC) can be genetically engineered to knockout all or a portion of a target gene using a gene-editing system, e.g., as described herein. In some such embodiments, a gene-editing system may be or comprise a CRISPR system, a zinc finger nuclease system, a TALEN, and/or a meganuclease.

[0149] TGF Signaling

[0150] In certain embodiments, the disclosure provides a genetically engineered stem cell, and/or progeny cell, comprising a disruption in TGF signaling, e.g., TGF beta signaling. This is useful, for example, in circumstances where it is desirable to generate a differentiated cell from pluripotent stem cell, wherein TGF signaling, e.g., TGF beta signaling is disrupted in the differentiated cell.

[0151] For example, TGF beta signaling inhibits or decreases the survival and/or activity of some differentiated cell types that are useful for therapeutic applications, e.g., TGF beta signaling is a negative regulator of natural killer cells, which can be used in immunotherapeutic applications. In some embodiments, it is desirable to generate a clinically effective number of natural killer cells comprising a genetic modification that disrupts TGF beta signaling, thus avoiding the negative effect of TGF beta on the clinical effectiveness of such cells. It is advantageous, in some embodiments, to source such NK cells from a pluripotent stem cell, instead, for example, from mature NK cells obtained from a donor. Modifying the stem cell instead of the differentiated cell has, among others, the advantage of allowing for clonal derivation, characterization, and/or expansion of a specific genotype, e.g., a specific stem cell clone harboring a specific genetic modification (e.g., a targeted disruption of TGFβRII in the absence of any undesired (e.g., off-target) modifications). In some embodiments, the stem cell, e.g., the human iPSC, is genetically engineered not to express one or more TGFβ receptor, e.g., TGFβRII, or to express a dominant negative variant of a TGFβ receptor, e.g., a dominant negative TGFβRII variant. Exemplary sequences of TGFβRII are set forth in KR710923.1, NM 001024847.2, and NM 003242.5. An exemplary dominant negative TGFβRII is disclosed in Immunity. 2000 February; 12(2):171-81.

[0152] Additional Loss-of-Function Modifications

[0153] In certain embodiments, the disclosure provides a genetically engineered stem cell, and/or progeny cell, that additionally or alternatively comprises a disruption in interleukin signaling, e.g., IL-15 signaling. IL-15 is a cytokine with structural similarity to Interleukin-2 (IL-2), which binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). Exemplary sequences of IL-15 are provided in NG 029605.2. Disruption of IL-15 signaling may be useful, for example, in circumstances where it is desirable to generate a differentiated cell from a pluripotent stem cell, but with certain signaling pathways (e.g., IL-15) disrupted in the differentiated cell. IL-15 signaling can inhibit or decrease survival and/or activity of some types of differentiated cells, such as cells that may be useful for therapeutic applications. For example, IL-15 signaling is a negative regulator of natural killer (NK) cells. CISH (encoded by the CISH gene) is downstream of the IL-15 receptor and can act as a negative regulator of IL-15 signaling in NK cells. As used herein, the term “CISH” refers to the Cytokine Inducible SH2 Containing Protein (see, e.g., Delconte et al., Nat Immunol. 2016 July; 17(7):816-24; exemplary sequences for CISH are set forth as NG 023194.1). In some embodiments, disruption of CISH regulation may increase activation of Jak/STAT pathways, leading to increased survival, proliferation and/or effector functions of NK cells. Thus, in some embodiments, genetically engineered NK cells (e.g., iNK cells, e.g., generated from genetically engineered hiPSCs comprising a disruption of CISH regulation) exhibit greater responsiveness to IL-15-mediated signaling than non-genetically engineered NK cells. In some such embodiments, genetically engineered NK cells exhibit greater effector function relative to non-genetically engineered NK cells.

[0154] In some embodiments, a genetically engineered stem cell and/or progeny cell, additionally or alternatively, comprises a disruption and/or loss of function in one or more of B2M, NKG2A, PD1, TIGIT, ADORA2a, CIITA, HLA class II histocompatibility antigen alpha chain genes, HLA class II histocompatibility antigen beta chain genes, CD32B, or TRAC.

[0155] As used herein, the term “B2M” ((32 microglobulin) refers to a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. Exemplary sequences for B2M are set forth as NG 012920.2.

[0156] As used herein, the term “NKG2A” (natural killer group 2A) refers to a protein belonging to the killer cell lectin-like receptor family, also called NKG2 family, which is a group of transmembrane proteins preferentially expressed in NK cells. This family of proteins is characterized by the type II membrane orientation and the presence of a C-type lectin domain. See, e.g., Kamiya-T et al., J Clin Invest 2019 https://doi.org/10.1172/JCI123955. Exemplary sequences for NKG2A are set forth as AF461812.1.

[0157] As used herein, the term “PD1” (Programmed cell death protein 1), also known CD279 (cluster of differentiation 279), refers to a protein found on the surface of cells that has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD1 is an immune checkpoint and guards against autoimmunity. Exemplary sequences for PD1 are set forth as NM_005018.3.

[0158] As used herein, the term “TIGIT” (T cell immunoreceptor with Ig and ITIM domains) refers to a member of the PVR (poliovirus receptor) family of immunoglobulin proteins. The product of this gene is expressed on several classes of T cells including follicular B helper T cells (TFH). Exemplary sequences for TIGIT are set forth in NM 173799.4.

[0159] As used herein, the term “ADORA2A” refers to the adenosine A2a receptor, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) superfamily, which is subdivided into classes and subtypes. This protein, an adenosine receptor of A2A subtype, uses adenosine as the preferred endogenous agonist and preferentially interacts with the G(s) and G(olf) family of G proteins to increase intracellular cAMP levels. Exemplary sequences of ADORA2a are provided in NG 052804.1.

[0160] As used herein, the term “CIITA” refers to the protein located in the nucleus that acts as a positive regulator of class II major histocompatibility complex gene transcription, and is referred to as the “master control factor” for the expression of these genes. The protein also binds GTP and uses GTP binding to facilitate its own transport into the nucleus. Mutations in this gene have been associated with bare lymphocyte syndrome type II (also known as hereditary MHC class II deficiency or HLA class II-deficient combined immunodeficiency), increased susceptibility to rheumatoid arthritis, multiple sclerosis, and possibly myocardial infarction. See, e.g., Chang et al., J Exp Med 180:1367-1374; and Chang et al., Immunity. 1996 February; 4(2):167-78, the entire contents of each of which are incorporated by reference herein. An exemplary sequence of CIITA is set forth as NG 009628.1.

[0161] In some embodiments, two or more HLA class II histocompatibility antigen alpha chain genes and/or two or more HLA class II histocompatibility antigen beta chain genes are disrupted, e.g., knocked out, e.g., by genomic editing. For example, in some embodiments, two or more HLA class II histocompatibility antigen alpha chain genes selected from HLA-DQA1, HLA-DRA, HLA-DPA1, HLA-DMA, HLA-DQA2, and HLA-DOA are disrupted, e.g., knocked out. For another example, in some embodiments, two or more HLA class II histocompatibility antigen beta chain genes selected from HLA-DMB, HLA-DOB, HLA-DPB1, HLA-DQB1, HLA-DQB3, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 are disrupted, e.g., knocked out. See, e.g., Crivello et al., J Immunol January 2019, ji1800257; DOI: https://doi.org/10.4049/jimmunol.1800257, the entire contents of which are incorporated herein by reference.

[0162] As used herein, the term “CD32B” (cluster of differentiation 32B) refers to a low affinity immunoglobulin gamma Fc region receptor II-b protein that, in humans, is encoded by the FCGR2B gene. See, e.g., Rankin-C T et al., Blood 2006 108(7):2384-91, the entire contents of which are incorporated herein by reference.

[0163] As used herein, the term “TRAC” refers to the T-cell receptor alpha subunit (constant), encoded by the TRAC locus.

[0164] Gain-of-Function Modifications

[0165] In some embodiments, a genetically engineered stem cell and/or progeny cell, additionally or alternatively, comprises a genetic modification that leads to expression of one or more of a CAR; a non-naturally occurring variant of FcγRIII (CD16); interleukin 15 (IL-15); an IL-15 receptor (IL-15R) agonist, or a constitutively active variant of an IL-15 receptor; interleukin 12 (IL-12); an IL-12 receptor (IL-12R) agonist, or a constitutively active variant of an IL-12 receptor; human leukocyte antigen G (HLA-G); human leukocyte antigen E (HLA-E); or leukocyte surface antigen cluster of differentiation CD47 (CD47).

[0166] As used herein, the term “chimeric antigen receptor” or “CAR” refers to a receptor protein that has been modified to give cells expressing the CAR the new ability to target a specific protein. Within the context of the disclosure, an cell modified to comprise a CAR may be used for immunotherapy to target and destroy cells associated with a disease or disorder, e.g., cancer cells.

[0167] CARs of interest include, but are not limited to, a CAR targeting mesothelin, EGFR, HER2 and/or MICA/B. To date, mesothelin-targeted CAR T-cell therapy has shown early evidence of efficacy in a phase I clinical trial of subjects having mesothelioma, non-small cell lung cancer, and breast cancer (NCT02414269). Similarly, CARs targeting EGFR, HER2 and MICA/B have shown promise in early studies (see, e.g., Li et al. (2018), Cell Death & Disease, 9(177); Han et al. (2018) Am. J. Cancer Res., 8(1):106-119; and Demoulin 2017) Future Oncology, 13(8); the entire contents of each of which are expressly incorporated herein by reference in their entireties).

[0168] CARs are well-known to those of ordinary skill in the art and include those described in, for example: WO13/063419 (mesothelin), WO15/164594 (EGFR), WO13/063419 (HER2), WO16/154585 (MICA and MICB), the entire contents of each of which are expressly incorporated herein by reference in their entireties. Any suitable CAR, NK-CAR, or other binder that targets a cell, e.g., an NK cell, to a target cell, e.g., a cell associated with a disease or disorder, may be expressed in the modified NK cells provided herein. Exemplary CARs, and binders, include, but are not limited to, CARs and binders that bind BCMA, CD19, CD22, CD20, CD33, CD123, androgen receptor, PSMA, PSCA, Mucl, HPV viral peptides (i.e., E7), EBV viral peptides, CD70, WT1, CEA, EGFRvIII, IL13Rα2, and GD2, CA125, CD7, EpCAM, Muc16, CD30. Additional suitable CARs and binders for use in the modified NK cells provided herein will be apparent to those of skill in the art based on the present disclosure and the general knowledge in the art. Such additional suitable CARs include those described in FIG. 3 of Davies and Maher, Adoptive T-cell Immunotherapy of Cancer Using Chimeric Antigen Receptor-Grafted T Cells, Archivum Immunologiae et Therapiae Experimentalis 58(3):165-78 (2010), the entire contents of which are incorporated herein by reference.

[0169] As used herein, the term “CD16” refers to a receptor (FcγRIII) for the Fc portion of immunoglobulin G, and it is involved in the removal of antigen-antibody complexes from the circulation, as well as other antibody-dependent responses.

[0170] As used herein, the term “IL-15/IL15RA” or “Interleukin-15” (IL-15) refers to a cytokine with structural similarity to Interleukin-2 (IL-2). Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is secreted by mononuclear phagocytes (and some other cells) following infection by virus(es). This cytokine induces cell proliferation of natural killer cells; cells of the innate immune system whose principal role is to kill virally infected cells. IL-15 Receptor alpha (IL15RA) specifically binds IL-15 with very high affinity, and is capable of binding IL-15 independently of other subunits. It is suggested that this property allows IL-15 to be produced by one cell, endocytosed by another cell, and then presented to a third party cell. IL15RA is reported to enhance cell proliferation and expression of apoptosis inhibitor BCL2L1/BCL2-XL and BCL2. Exemplary sequences of IL-15 are provided in NG 029605.2, and exemplary sequences of IL-15RA are provided in NM 002189.4. In some embodiments, the IL-15R variant is a constitutively active IL-15R variant. In some embodiments, the constitutively active IL-15R variant is a fusion between IL-15R and an IL-15R agonist, e.g., an IL-15 protein or IL-15R-binding fragment thereof. In some embodiments, the IL-15R agonist is IL-15, or an IL-15R-binding variant thereof. Exemplary suitable IL-15R variants include, without limitation, those described, e.g., in Mortier E et al, 2006; The Journal of Biological Chemistry 2006 281: 1612-1619; or in Bessard-A et al., Mol Cancer Ther. 2009 September; 8(9):2736-45, the entire contents of each of which are incorporated by reference herein.

[0171] As used herein, the term “IL-12” refers to interleukin-12, a cytokine that acts on T and natural killer cells. In some embodiments, a genetically engineered stem cell and/or progeny cell comprises a genetic modification that leads to expression of one or more of an interleukin 12 (IL12) pathway agonist, e.g., IL-12, interleukin 12 receptor (IL-12R) or a variant thereof (e.g., a constitutively active variant of IL-12R, e.g., an IL-12R fused to an IL-12R agonist (IL-12RA).

[0172] As used herein, the term “HLA-G” refers to the HLA non-classical class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. HLA-G is expressed on fetal derived placental cells. HLA-G is a ligand for NK cell inhibitory receptor KIR2DL4, and therefore expression of this HLA by the trophoblast defends it against NK cell-mediated death. See e.g., Favier et al., Tolerogenic Function of Dimeric Forms of HLA-G Recombinant Proteins: A Comparative Study In Vivo PLOS One 2011, the entire contents of which are incorporated herein by reference. An exemplary sequence of HLA-G is set forth as NG 029039.1.

[0173] As used herein, the term “HLA-E” refers to the HLA class I histocompatibility antigen, alpha chain E, also sometimes referred to as MHC class I antigen E. The HLA-E protein in humans is encoded by the HLA-E gene. The human HLA-E is a non-classical MHC class I molecule that is characterized by a limited polymorphism and a lower cell surface expression than its classical paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. HLA-E binds a restricted subset of peptides derived from the leader peptides of other class I molecules. HLA-E expressing cells escape allogeneic responses and lysis by NK cells. See e.g., Geornalusse-G et al., Nature Biotechnology 2017 35(8), the entire contents of which are incorporated herein by reference. Exemplary sequences of the HLA-E protein are provided in NM_005516.6.

[0174] As used herein, the term “CD47,” also sometimes referred to as “integrin associated protein” (IAP), refers to a transmembrane protein that in humans is encoded by the CD47 gene. CD47 belongs to the immunoglobulin superfamily, partners with membrane integrins, and also binds the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). CD47 acts as a signal to macrophages that allows CD47-expressing cells to escape macrophage attack. See, e.g., Deuse-T, et al., Nature Biotechnology 2019 37: 252-258, the entire contents of which are incorporated herein by reference.

[0175] Generation of iNK Cells

[0176] In some embodiments, the present disclosure provides methods of generating iNK cells (e.g., genetically modified iNK cells) that are derived from stem cells described herein.

[0177] In some embodiments, genetic modifications (e.g., genomic edits) present in an iNK cell of the present disclosure can be made at any stage during the reprogramming process from donor cell to iPSC, during the iPSC stage, and/or at any stage of the process of differentiating the iPSC to an iNK state, e.g., at an intermediary state, such as, for example, an iPSC-derived HSC state, or even up to or at the final iNK cell state.

[0178] For example, one or more genomic edits present in an edited iNK cell of the present disclosure may be made at one or more different cell stages (e.g., reprogramming from donor to iPSC, differentiation of iPSC to iNK). In some embodiments, one or more genomic edits present in modified genetically modified iNK cell provided herein is made before reprogramming a donor cell to an iPSC state. In some embodiments, all edits present in a genetically modified iNK cell provided herein are made at the same time, in close temporal proximity, and/or at the same cell stage of the reprogramming/differentiation process, e.g., at the donor cell stage, during the reprogramming process, at the iPSC stage, or during the differentiation process, e.g., from iPSC to iNK. In some embodiments, two or more edits present in a genetically modified iNK cell provided herein are made at different times and/or at different cell stages of the reprogramming/differentiation process from donor cell to iPSC to iNK. For example, in some embodiments, a first edit is made at the donor cell stage and a second (different) edit is made at the iPSC stage. In some embodiments, a first edit is made at the reprogramming stage (e.g., donor to iPSC) and a second (different) edit is made at the iPSC stage.

[0179] A variety of cell types can be used as a donor cell that can be subjected to reprogramming, differentiation, and/or genomic editing strategies described herein. For example, the donor cell can be a pluripotent stem cell or a differentiated cell, e.g., a somatic cell, such as, for example, a fibroblast or a T lymphocyte. In some embodiments, donor cells are manipulated (e.g., subjected to reprogramming, differentiation, and/or genomic editing) to generate iNK cells described herein.

[0180] A donor cell can be from any suitable organism. For example, in some embodiments, the donor cell is a mammalian cell, e.g., a human cell or a non-human primate cell. In some embodiments, the donor cell is a somatic cell. In some embodiments, the donor cell is a stem cell or progenitor cell. In certain embodiments, the donor cell is not or was not part of a human embryo and its derivation does not involve destruction of a human embryo.

[0181] In some embodiments, an edited iNK cell is derived from an iPSC, which in turn is derived from a somatic donor cell. Any suitable somatic cell can be used in the generation of iPSCs, and in turn, the generation of iNK cells. Suitable strategies for deriving iPSCs from various somatic donor cell types have been described and are known in the art. In some embodiments, a somatic donor cell is a fibroblast cell. In some embodiments, a somatic donor cell is a mature T cell.

[0182] For example, in some embodiments, a somatic donor cell, from which an iPSC, and subsequently an iNK cell is derived, is a developmentally mature T cell (a T cell that has undergone thymic selection). One hallmark of developmentally mature T cells is a rearranged T cell receptor locus. During T cell maturation, the TCR locus undergoes V(D)J rearrangements to generate complete V-domain exons. These rearrangements are retained throughout reprogramming of a T cells to an iPSC, and throughout differentiation of the resulting iPSC to a somatic cell.

[0183] In certain embodiments, a somatic donor cell is a CD8.sup.+ T cell, a CD8.sup.+naïve T cell, a CD4.sup.+ central memory T cell, a CD8.sup.+ central memory T cell, a CD4.sup.+ effector memory T cell, a CD4.sup.+ effector memory T cell, a CD4.sup.+ T cell, a CD4.sup.+ stem cell memory T cell, a CD8+ stem cell memory T cell, a CD4+ helper T cell, a regulatory T cell, a cytotoxic T cell, a natural killer T cell, a CD4+naïve T cell, a TH17 CD4.sup.+ T cell, a TH1 CD4.sup.+ T cell, a TH2 CD4.sup.+ T cell, a TH9 CD4.sup.+ T cell, a CD4.sup.+ Foxp3.sup.+ T cell, a CD4.sup.+ CD25.sup.+ CD127.sup.− T cell, or a CD4.sup.+ CD25.sup.+ CD127.sup.− Foxp3.sup.+ T cell.

[0184] T cells can be advantageous for the generation of iPSCs. For example, T cells can be edited with relative ease, e.g., by CRISPR-based methods or other gene-editing methods. Additionally, the rearranged TCR locus allows for genetic tracking of individual cells and their daughter cells. For example, if the reprogramming, expansion, culture, and/or differentiation strategies involved in the generation of NK cells a clonal expansion of a single cell, the rearranged TCR locus can be used as a genetic marker unambiguously identifying a cell and its daughter cells. This, in turn, allows for the characterization of a cell population as truly clonal, or for the identification of mixed populations, or contaminating cells in a clonal population. Another potential advantage of using T cells in generating iNK cells carrying multiple edits is that certain karyotypic aberrations associated with chromosomal translocations are selected against in T cell culture. Such aberrations can pose a concern when editing cells by CRISPR technology, and in particular when generating cells carrying multiple edits. Using T cell derived iPSCs as a starting point for the derivation of therapeutic lymphocytes can allow for the expression of a pre-screened TCR in the lymphocytes, e.g., via selecting the T cells for binding activity against a specific antigen, e.g., a tumor antigen, reprogramming the selected T cells to iPSCs, and then deriving lymphocytes from these iPSCs that express the TCR (e.g., T cells). This strategy can allow for activating the TCR in other cell types, e.g., by genetic or epigenetic strategies. Additionally, T cells retain at least part of their “epigenetic memory” throughout the reprogramming process, and thus subsequent differentiation of the same or a closely related cell type, such as iNK cells can be more efficient and/or result in higher quality cell populations as compared to approaches using non-related cells, such as fibroblasts, as a starting point for iNK derivation.

[0185] In some embodiments, a donor cell being manipulated, e.g., a cell being reprogrammed and/or undergoing genomic editing, is one or more of a long-term hematopoietic stem cell, a short term hematopoietic stem cell, a multipotent progenitor cell, a lineage restricted progenitor cell, a lymphoid progenitor cell, a myeloid progenitor cell, a common myeloid progenitor cell, an erythroid progenitor cell, a megakaryocyte erythroid progenitor cell, a retinal cell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmented epithelium cell, a trabecular meshwork cell, a cochlear hair cell, an outer hair cell, an inner hair cell, a pulmonary epithelial cell, a bronchial epithelial cell, an alveolar epithelial cell, a pulmonary epithelial progenitor cell, a striated muscle cell, a cardiac muscle cell, a muscle satellite cell, a neuron, a neuronal stem cell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell, an embryonic stem cell, a fibroblast, a monocyte-derived macrophage or dendritic cell, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g., a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell, a gastrointestinal epithelial cell, a biliary epithelial cell, a pancreatic ductal epithelial cell, an intestinal stem cell, a hepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, an osteoclast, an adipocyte, a preadipocyte, a pancreatic islet cell (e.g., a beta cell, an alpha cell, a delta cell), a pancreatic exocrine cell, a Schwann cell, or an oligodendrocyte.

[0186] In some embodiments, a donor cell is one or more of a circulating blood cell, e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem/progenitor cell (HSC), or endothelial cell (EC). In some embodiments, a donor cell is one or more of a bone marrow cell (e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), an MEP cell, myeloid progenitor cell (CMP/GMP), LP cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell). In some embodiments, a donor cell is one or more of a myeloid progenitor cell (e.g., a common myeloid progenitor (CMP) cell or granulocyte macrophage progenitor (GMP) cell). In some embodiments, a donor cell is one or more of a lymphoid progenitor cell, e.g., a common lymphoid progenitor (CLP) cell. In some embodiments, a donor cell is one or more of an erythroid progenitor cell (e.g., an MEP cell). In some embodiments, a donor cell is one or more of a hematopoietic stem/progenitor cell (e.g., a long term HSC (LT-HSC), short term HSC (ST-HSC), MPP cell, or lineage restricted progenitor (LRP) cell). In certain embodiments, the donor cell is a CD34.sup.+ cell, CD34+CD90.sup.+ cell, CD34.sup.+ CD38.sup.− cell, CD34.sup.+ CD90.sup.+ CD49f.sup.+ CD38.sup.− CD45RA.sup.− cell, CD105.sup.+ cell, CD31.sup.+, or CD133.sup.+ cell, or a CD34.sup.+ CD90.sup.+ CD133.sup.+ cell. In some embodiments, a donor cell is one or more of an umbilical cord blood CD34.sup.+ HSPC, umbilical cord venous endothelial cell, umbilical cord arterial endothelial cell, amniotic fluid CD34.sup.+ cell, amniotic fluid endothelial cell, placental endothelial cell, or placental hematopoietic CD34.sup.+ cell. In some embodiments, a donor cell is one or more of a mobilized peripheral blood hematopoietic CD34.sup.+ cell (after the patient is treated with a mobilization agent, e.g., G-CSF or Plerixafor). In some embodiments, a donor cell is a peripheral blood endothelial cell. In some embodiments, a donor cell is a peripheral blood natural killer cell.

[0187] In some embodiments, a donor cell is a dividing cell. In some embodiments, a donor cell is a non-dividing cell.

[0188] In some embodiments, a genetically modified (e.g., edited) iNK cell resulting from one or more methods and/or strategies described herein, are administered to a subject in need thereof, e.g., in the context of an immuno-oncology therapeutic approach. In some embodiments, donor cells, or any cells of any stage of the reprogramming, differentiating, and/or editing strategies provided herein, can be maintained in culture or stored (e.g., frozen in liquid nitrogen) using any suitable method known in the art, e.g., for subsequent characterization or administration to a subject in need thereof.

[0189] Genome Editing Systems

[0190] Genome editing systems of the present disclosure may be used, for example, to edit stem cells. In some embodiments, genome editing systems of the present disclosure include at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA-guided nuclease. These two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single-strand break (an SSB or nick), a double-strand break (a DSB) and/or a point mutation.

[0191] Naturally occurring CRISPR systems are organized evolutionarily into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 June; 9(6): 467-477 (“Makarova”)), and while genome editing systems of the present disclosure may adapt components of any type or class of naturally occurring CRISPR system, the embodiments presented herein are generally adapted from Class 2, and type II or V CRISPR systems. Class 2 systems, which encompass types II and V, are characterized by relatively large, multidomain RNA-guided nuclease proteins (e.g., Cas9 or Cpf1) and one or more guide RNAs (e.g., a crRNA and, optionally, a tracrRNA) that form ribonucleoprotein (RNP) complexes that associate with (i.e., target) and cleave specific loci complementary to a targeting (or spacer) sequence of the crRNA. Genome editing systems according to the present disclosure similarly target and edit cellular DNA sequences, but differ significantly from CRISPR systems occurring in nature. For example, the unimolecular guide RNAs described herein do not occur in nature, and both guide RNAs and RNA-guided nucleases according to this disclosure may incorporate any number of non-naturally occurring modifications.

[0192] Genome editing systems can be implemented (e.g. administered or delivered to a cell or a subject) in a variety of ways, and different implementations may be suitable for distinct applications. For instance, a genome editing system is implemented, in certain embodiments, as a protein/RNA complex (a ribonucleoprotein, or RNP), which can be included in a pharmaceutical composition that optionally includes a pharmaceutically acceptable carrier and/or an encapsulating agent, such as a lipid or polymer micro- or nanoparticle, micelle, liposome, etc. In certain embodiments, a genome editing system is implemented as one or more nucleic acids encoding the RNA-guided nuclease and guide RNA components described above (optionally with one or more additional components); in certain embodiments, the genome editing system is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus; and in certain embodiments, the genome editing system is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.

[0193] It should be noted that the genome editing systems of the present disclosure can be targeted to a single specific nucleotide sequence, or may be targeted to—and capable of editing in parallel—two or more specific nucleotide sequences through the use of two or more guide RNAs. The use of multiple gRNAs is referred to as “multiplexing” throughout this disclosure, and can be employed to target multiple, unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain and, in some cases, to generate specific edits within such target domain. For example, International Patent Publication No. WO 2015/138510 by Maeder et al. (“Maeder”) describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene that results in the creation of a cryptic splice site, which in turn reduces or eliminates the function of the gene. The genome editing system of Maeder utilizes two guide RNAs targeted to sequences on either side of (i.e., flanking) the point mutation, and forms DSBs that flank the mutation. This, in turn, promotes deletion of the intervening sequence, including the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.

[0194] As another example, WO 2016/073990 by Cotta-Ramusino, et al. (“Cotta-Ramusino”) describes a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (a Cas9 that makes a single strand nick such as S. pyogenes D10A), an arrangement termed a “dual-nickase system.” The dual-nickase system of Cotta-Ramusino is configured to make two nicks on opposite strands of a sequence of interest that are offset by one or more nucleotides, which nicks combine to create a double strand break having an overhang (5′ in the case of Cotta-Ramusino, though 3′ overhangs are also possible). The overhang, in turn, can facilitate homology directed repair events in some circumstances. And, as another example, WO 2015/070083 by Palestrant et al. (“Palestrant”) describes a gRNA targeted to a nucleotide sequence encoding Cas9 (referred to as a “governing RNA”), which can be included in a genome editing system comprising one or more additional gRNAs to permit transient expression of a Cas9 that might otherwise be constitutively expressed, for example in some virally transduced cells. These multiplexing applications are intended to be exemplary, rather than limiting, and the skilled artisan will appreciate that other applications of multiplexing are generally compatible with the genome editing systems described here.

[0195] Genome editing systems can, in some instances, form double strand breaks that are repaired by cellular DNA double-strand break mechanisms such as NHEJ or HDR. These mechanisms are described throughout the literature, for example by Davis & Maizels, PNAS, 111(10):E924-932, Mar. 11, 2014 (“Davis”) (describing Alt-HDR); Frit et al. DNA Repair 17(2014) 81-97 (“Frit”) (describing Alt-NHEJ); and Iyama and Wilson III, DNA Repair (Amst.) 2013-August; 12(8): 620-636 (“Iyama”) (describing canonical HDR and NHEJ pathways generally).

[0196] Where genome editing systems operate by forming DSBs, such systems optionally include one or more components that promote or facilitate a particular mode of double-strand break repair or a particular repair outcome. For instance, Cotta-Ramusino also describes genome editing systems in which a single stranded oligonucleotide “donor template” is added; the donor template is incorporated into a target region of cellular DNA that is cleaved by the genome editing system, and can result in a change in the target sequence.

[0197] In certain embodiments, genome editing systems modify a target sequence, or modify expression of a target gene in or near the target sequence, without causing single- or double-strand breaks. For example, a genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA, thereby modifying the target sequence or its expression. As one example, an RNA-guided nuclease can be connected to (e.g., fused to) a cytidine deaminase functional domain, and may operate by generating targeted C-to-A substitutions. Exemplary nuclease/deaminase fusions are described in Komor et al. Nature 533,420-424 (19 May 2016) (“Komor”). Alternatively, a genome editing system may utilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a dead Cas9 (dCas9), and may operate by forming stable complexes on one or more targeted regions of cellular DNA, thereby interfering with functions involving the targeted region(s) including, without limitation, mRNA transcription, chromatin remodeling, etc.

[0198] Guide RNA (gRNA) Molecules

[0199] Guide RNAs (gRNAs) of the present disclosure may be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing). gRNAs and their component parts are described throughout the literature, for instance in Briner et al. (Molecular Cell 56(2), 333-339, Oct. 23, 2014 (“Briner”)), and in Cotta-Ramusino.

[0200] In bacteria and archaea, type II CRISPR systems generally comprise an RNA-guided nuclease protein such as Cas9, a CRISPR RNA (crRNA) that includes a 5′ region that is complementary to a foreign sequence, and a trans-activating crRNA (tracrRNA) that includes a 5′ region that is complementary to, and forms a duplex with, a 3′ region of the crRNA. While not intending to be bound by any theory, it is thought that this duplex facilitates the formation of—and is necessary for the activity of—the Cas9/gRNA complex. As type II CRISPR systems were adapted for use in gene editing, it was discovered that the crRNA and tracrRNA could be joined into a single unimolecular or chimeric guide RNA, in one non-limiting example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3′ end) and the tracrRNA (at its 5′ end). (Mali et al. Science. 2013 Feb. 15; 339(6121): 823-826 (“Mali”); Jiang et al. Nat Biotechnol. 2013 March; 31(3): 233-239 (“Jiang”); and Jinek et al., 2012 Science August 17; 337(6096): 816-821 (“Jinek 2012”)).

[0201] Guide RNAs, whether unimolecular or modular, include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including without limitation “guide sequences” (Hsu et al., Nat Biotechnol. 2013 September; 31(9): 827-832, (“Hsu”)), “complementarity regions” (Cotta-Ramusino), “spacers” (Briner) and generically as “crRNAs” (Jiang). Irrespective of the names they are given, targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5′ terminus of in the case of a Cas9 gRNA, and at or near the 3′ terminus in the case of a Cpf1 gRNA.

[0202] In addition to the targeting domains, gRNAs typically (but not necessarily, as discussed below) include a plurality of domains that may influence the formation or activity of gRNA/Cas9 complexes. For instance, as mentioned above, the duplexed structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeat:anti-repeat duplex) interacts with the recognition (REC) lobe of Cas9 and can mediate the formation of Cas9/gRNA complexes. (Nishimasu et al., Cell 156, 935-949, Feb. 27, 2014 (“Nishimasu 2014”) and Nishimasu et al., Cell 162, 1113-1126, Aug. 27, 2015 (“Nishimasu 2015”)). It should be noted that the first and/or second complementarity domains may contain one or more poly-A tracts, which can be recognized by RNA polymerases as a termination signal. The sequence of the first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for instance through the use of A-G swaps as described in Briner, or A-U swaps. These and other similar modifications to the first and second complementarity domains are within the scope of the present disclosure.

[0203] Along with the first and second complementarity domains, Cas9 gRNAs typically include two or more additional duplexed regions that are involved in nuclease activity in vivo but not necessarily in vitro. (Nishimasu 2015). A first stem-loop one near the 3′ portion of the second complementarity domain is referred to variously as the “proximal domain,” (Cotta-Ramusino) “stem loop 1” (Nishimasu 2014 and 2015) and the “nexus” (Briner). One or more additional stem loop structures are generally present near the 3′ end of the gRNA, with the number varying by species: s. pyogenes gRNAs typically include two 3′ stem loops (for a total of four stem loop structures including the repeat:anti-repeat duplex), while S. aureus and other species have only one (for a total of three stem loop structures). A description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner.

[0204] While the foregoing description has focused on gRNAs for use with Cas9, it should be appreciated that other RNA-guided nucleases have been (or may in the future be) discovered or invented which utilize gRNAs that differ in some ways from those described to this point. For instance, Cpf1 (“CRISPR from Prevotella and Franciscella 1”) is a recently discovered RNA-guided nuclease that does not require a tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771 Oct. 22, 2015 (“Zetsche I”)). A gRNA for use in a Cpf1 genome editing system generally includes a targeting domain and a complementarily domain (alternately referred to as a “handle”). It should also be noted that, in gRNAs for use with Cpf1, the targeting domain is usually present at or near the 3′ end, rather than the 5′ end as described above in connection with Cas9 gRNAs (the handle is at or near the 5′ end of a Cpf1 gRNA).

[0205] Those of skill in the art will appreciate, however, that although structural differences may exist between gRNAs from different prokaryotic species, or between Cpf1 and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that includes one or more chemical modifications and/or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.

[0206] More generally, skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, and not only those gRNAs that are compatible with a particular species of Cas9 or Cpf1. By way of illustration, the term gRNA can, in certain embodiments, include a gRNA for use with any RNA-guided nuclease occurring in a Class 2 CRISPR system, such as a type II or type V or CRISPR system, or an RNA-guided nuclease derived or adapted therefrom.

[0207] gRNA Design

[0208] Methods for selection and validation of target sequences as well as off-target analyses have been described previously, e.g., in Mali; Hsu; Fu et al., 2014 Nat Biotechnol 32(3): 279-84, Heigwer et al., 2014 Nat methods 11(2):122-3; Bae et al. (2014) Bioinformatics 30(10): 1473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182. As a non-limiting example, gRNA design may involve the use of a software tool to optimize the choice of potential target sequences corresponding to a user's target sequence, e.g., to minimize total off-target activity across the genome. While off-target activity is not limited to cleavage, the cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.

[0209] For example, methods for selection and validation of target sequences as well as off-target analyses can be performed using cas-offinder (Bae S, Park J, Kim J-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics. 2014; 30:1473-5). Cas-offinder is a tool that can quickly identify all sequences in a genome that have up to a specified number of mismatches to a guide sequence.

[0210] As another example, methods for scoring how likely a given sequence is to be an off-target (e.g., once candidate target sequences are identified) can be performed. An exemplary score includes a Cutting Frequency Determination (CFD) score, as described by Doench J G, Fusi N, Sullender M, Hegde M, Vaimberg E W, Donovan K F, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34:184-91.

[0211] gRNA Modifications

[0212] In certain embodiments, gRNAs as used herein may be modified or unmodified gRNAs. In certain embodiments, a gRNA may include one or more modifications. In certain embodiments, the one or more modifications may include a phosphorothioate linkage modification, a phosphorodithioate (PS2) linkage modification, a 2′-O-methyl modification, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5′ end of the gRNA, at the 3′ end of the gRNA, or combinations thereof.

[0213] In certain embodiments, a gRNA modification may comprise one or more phosphorodithioate (PS2) linkage modifications.

[0214] In some embodiments, a gRNA used herein includes one or more or a stretch of deoxyribonucleic acid (DNA) bases, also referred to herein as a “DNA extension.” In some embodiments, a gRNA used herein includes a DNA extension at the 5′ end of the gRNA, the 3′ end of the gRNA, or a combination thereof. In certain embodiments, the DNA extension may be 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 DNA bases long. For example, in certain embodiments, the DNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 DNA bases long. In certain embodiments, the DNA extension may include one or more DNA bases selected from adenine (A), guanine (G), cytosine (C), or thymine (T). In certain embodiments, the DNA extension includes the same DNA bases. For example, the DNA extension may include a stretch of adenine (A) bases. In certain embodiments, the DNA extension may include a stretch of thymine (T) bases. In certain embodiments, the DNA extension includes a combination of different DNA bases. In certain embodiments, a DNA extension may comprise a sequence set forth in Table 3.

[0215] Exemplary suitable 5′ extensions for Cpf1 guide RNAs are provided in Table 3 below:

TABLE-US-00003 TABLE 3 Exemplary Cpf1 gRNA 5′ Extensions SEQ ID NO: 5′ extension sequence 5′ modification  1 rCrUrUrUrU +5 RNA  2 rArArGrArCrCrUrUrUrU +10 RNA  3 rArUrGrUrGrUrUrUrUrUrGrUrCrArArArArGrArCrCrUrUrUrU +25 RNA  4 rArGrGrCrCrArGrCrUrUrGrCrCrGrGrUrUrUrUrUrUrArGrUrCrG +60 RNA rUrGrCrUrGrCrUrUrCrArUrGrUrGrUrUrUrUrUrGrUrCrArArArA rGrArCrCrUrUrUrU  5 CTTTT +5 DNA  6 AAGACCTTTT +10 DNA  7 ATGTGTTTTTGTCAAAAGACCTTTT +25 DNA  8 AGGCCAGCTTGCCGGTTTTTTAGTCGTGCTGCTTCATGTGTTTTTGTCAAAA +60 DNA GACCTTTT  9 TTTTTGTCAAAAGACCTTTT +20 DNA 10 GCTTCATGTGTTTTTGTCAAAAGACCTTTT +30 DNA 11 GCCGGTTTTTTAGTCGTGCTGCTTCATGTGTTTTTGTCAAAAGACCTTTT +50 DNA 12 TAGTCGTGCTGCTTCATGTGTTTTTGTCAAAAGACCTTTT +40 DNA 13 C*C*GAAGTTTTCTTCGGTTTT +20 DNA + 2xPS 14 T*T*TTTCCGAAGTTTTCTTCGGTTTT +25 DNA + 2xPS 15 A*A*CGCTTTTTCCGAAGTTTTCTTCGGTTTT +30 DNA + 2xPS 16 G*C*GTTGTTTTCAACGCTTTTTCCGAAGTTTTCTTCGGTTTT +41 DNA + 2xPS 17 G*G*CTTCTTTTGAAGCCTTTTTGCGTTGTTTTCAACGCTTTTTCCGAAGTT +62 DNA + 2xPS TTCTTCGGTTTT 18 A*T*GTGTTTTTGTCAAAAGACCTTTT +25 DNA + 2xPS 19 AAAAAAAAAAAAAAAAAAAAAAAAA +25 A 20 TTTTTTTTTTTTTTTTTTTTTTTTT +25 T 21 mA*mU*rGrUrGrUrUrUrUrUrGrUrCrArArArArGrArCrCrUrUrUrU +25 RNA + 2xPS 22 mA*mA*rArArArArArArArArArArArArArArArArArArArArArArA Poly A RNA + 2xPS 23 mU*mU*rUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrUrU PolyU RNA + 2xPS All bases are in upper case Lowercase “r” represents RNA, 2′-hydroxy; bases not modified by an “r” are DNA All bases are linked via standard phosphodiester bonds except as noted: “*” represents phosphorothioate modification “PS” represents phosphorothioate modification

[0216] In certain embodiments, a gRNA used herein includes a DNA extension as well as a chemical modification, e.g., one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2′-O-methyl modifications, or one or more additional suitable chemical gRNA modification disclosed herein, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5′ end of the gRNA, at the 3′ end of the gRNA, or combinations thereof.

[0217] Without wishing to be bound by theory, it is contemplated that any DNA extension may be used with any gRNA disclosed herein, so long as it does not hybridize to the target nucleic acid being targeted by the gRNA and it also exhibits an increase in editing at the target nucleic acid site relative to a gRNA which does not include such a DNA extension.

[0218] In some embodiments, a gRNA used herein includes one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an “RNA extension.” In some embodiments, a gRNA used herein includes an RNA extension at the 5′ end of the gRNA, the 3′ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension may be 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 RNA bases long. For example, in certain embodiments, the RNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 RNA bases long. In certain embodiments, the RNA extension may include one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC), or uracil (rU), in which the “r” represents RNA, 2′-hydroxy. In certain embodiments, the RNA extension includes the same RNA bases. For example, the RNA extension may include a stretch of adenine (rA) bases. In certain embodiments, the RNA extension includes a combination of different RNA bases, In certain embodiments, a gRNA used herein includes an RNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2′-O-methyl modifications, one or more additional suitable gRNA modification, e.g., chemical modification, disclosed herein, or combinations thereof. In certain embodiments, the one or more modifications may be at the 5′ end of the gRNA, at the 3′ end of the gRNA, or combinations thereof. In certain embodiments, a gRNA including a RNA extension may comprise a sequence set forth herein.

[0219] It is contemplated that gRNAs used herein may also include an RNA extension and a DNA extension. In certain embodiments, the RNA extension and DNA extension may both be at the 5′ end of the gRNA, the 3′ end of the gRNA, or a combination thereof. In certain embodiments, the RNA extension is at the 5′ end of the gRNA and the DNA extension is at the 3′ end of the gRNA. In certain embodiments, the RNA extension is at the 3′ end of the gRNA and the DNA extension is at the 5′ end of the gRNA.

[0220] In some embodiments, a gRNA which includes a modification, e.g., a DNA extension at the 5′ end and/or a chemical modification as disclosed herein, is complexed with a RNA-guided nuclease, e.g., an AsCpf1 nuclease, to form an RNP, which is then employed to edit a target cell, e.g., a pluripotent stem cell or a daughter cell thereof.

[0221] Additional suitable gRNA modifications will be apparent to those of ordinary skill in the art based on the present disclosure. Suitable gRNA modifications include, for example, those described in PCT application PCT/US2018/054027, filed on Oct. 2, 2018, and entitled “MODIFIED CPF1 GUIDE RNA;” in PCT application PCT/US2015/000143, filed on Dec. 3, 2015, and entitled “GUIDE RNA WITH CHEMICAL MODIFICATIONS;” in PCT application PCT/US2016/026028, filed Apr. 5, 2016, and entitled “CHEMICALLY MODIFIED GUIDE RNAS FOR CRISPR/CAS-MEDIATED GENE REGULATION;” and in PCT application PCT/US2016/053344, filed on Sep. 23, 2016, and entitled “NUCLEASE-MEDIATED GENOME EDITING OF PRIMARY CELLS AND ENRICHMENT THEREOF;” the entire contents of each of which are incorporated herein by reference.

[0222] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5′ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5′ end) and/or at or near the 3′ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3′ end). In some cases, modifications are positioned within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, a stem loop structure of a Cas9 or Cpf1 gRNA, and/or a targeting domain of a gRNA.

[0223] As one example, the 5′ end of a gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., a G(5′)ppp(5′)G cap analog, a m7G(5′)ppp(5′)G cap analog, or a 3′-O-Me-m7G(5′)ppp(5′)G anti reverse cap analog (ARCA)), as shown below:

##STR00001##

[0224] The cap or cap analog can be included during either chemical or enzymatic synthesis of the gRNA.

[0225] Along similar lines, the 5′ end of the gRNA can lack a 5′ triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5′ triphosphate group.

[0226] Another common modification involves the addition, at the 3′ end of a gRNA, of a plurality (e.g., 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a polyA tract. The polyA tract can be added to a gRNA during chemical or enzymatic synthesis, using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase).

[0227] Guide RNAs can be modified at a 3′ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:

##STR00002##

wherein “U” can be an unmodified or modified uridine.

[0228] The 3′ terminal U ribose can be modified with a 2′3′ cyclic phosphate as shown below:

##STR00003##

wherein “U” can be an unmodified or modified uridine.

[0229] Guide RNAs can contain 3′ nucleotides that can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.

[0230] In certain embodiments, sugar-modified ribonucleotides can be incorporated into a gRNA, e.g., wherein the 2′ OH-group is replaced by a group selected from H, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH.sub.2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In certain embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group. In certain embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2′-O-methyl, 2′-O-methoxyethyl, or 2′-Fluoro modified including, e.g., 2′-F or 2′-O-methyl, adenosine (A), 2′-F or 2′-O-methyl, cytidine (C), 2′-F or 2′-O-methyl, uridine (U), 2′-F or 2′-O-methyl, thymidine (T), 2′-F or 2′-O-methyl, guanosine (G), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

[0231] Guide RNAs can also include “locked” nucleic acids (LNA) in which the 2′ OH-group can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, including without limitation methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH.sub.2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH.sub.2).sub.n-amino (wherein amino can be, e.g., NH.sub.2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0232] In certain embodiments, a gRNA can include a modified nucleotide which is multicyclic (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with α-L-threofuranosyl-(3′.fwdarw.2′)).

[0233] Generally, gRNAs include the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified gRNAs can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2′ position, other sites are amenable to modification, including the 4′ position. In certain embodiments, a gRNA comprises a 4′-S, 4′-Se or a 4′-C-aminomethyl-2′-O-Me modification.

[0234] In certain embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into a gRNA. In certain embodiments, 0- and N-alkylated nucleotides, e.g., N6-methyl adenosine, can be incorporated into a gRNA. In certain embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.

[0235] Guide RNAs can also include one or more cross-links between complementary regions of the crRNA (at its 3′ end) and the tracrRNA (at its 5′ end) (e.g., within a “tetraloop” structure and/or positioned in any stem loop structure occurring within a gRNA). A variety of linkers are suitable for use. For example, guide RNAs can include common linking moieties including, without limitation, polyvinylether, polyethylene, polypropylene, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), and copolymers thereof.

[0236] In some embodiments, a bifunctional cross-linker is used to link a 5′ end of a first gRNA fragment and a 3′ end of a second gRNA fragment, and the 3′ or 5′ ends of the gRNA fragments to be linked are modified with functional groups that react with the reactive groups of the cross-linker. In general, these modifications comprise one or more of amine, sulfhydryl, carboxyl, hydroxyl, alkene (e.g., a terminal alkene), azide and/or another suitable functional group. Multifunctional (e.g. bifunctional) cross-linkers are also generally known in the art, and may be either heterofunctional or homofunctional, and may include any suitable functional group, including without limitation isothiocyanate, isocyanate, acyl azide, an NHS ester, sulfonyl chloride, tosyl ester, tresyl ester, aldehyde, amine, epoxide, carbonate (e.g., Bis(p-nitrophenyl) carbonate), aryl halide, alkyl halide, imido ester, carboxylate, alkyl phosphate, anhydride, fluorophenyl ester, HOBt ester, hydroxymethyl phosphine, O-methylisourea, DSC, NHS carbamate, glutaraldehyde, activated double bond, cyclic hemiacetal, NHS carbonate, imidazole carbamate, acyl imidazole, methylpyridinium ether, azlactone, cyanate ester, cyclic imidocarbonate, chlorotriazine, dehydroazepine, 6-sulfo-cytosine derivatives, maleimide, aziridine, TNB thiol, Ellman's reagent, peroxide, vinylsulfone, phenylthioester, diazoalkanes, diazoacetyl, epoxide, diazonium, benzophenone, anthraquinone, diazo derivatives, diazirine derivatives, psoralen derivatives, alkene, phenyl boronic acid, etc. In some embodiments, a first gRNA fragment comprises a first reactive group and the second gRNA fragment comprises a second reactive group. For example, the first and second reactive groups can each comprise an amine moiety, which are crosslinked with a carbonate-containing bifunctional crosslinking reagent to form a urea linkage. In other instances, (a) the first reactive group comprises a bromoacetyl moiety and the second reactive group comprises a sulfhydryl moiety, or (b) the first reactive group comprises a sulfhydryl moiety and the second reactive group comprises a bromoacetyl moiety, which are crosslinked by reacting the bromoacetyl moiety with the sulfhydryl moiety to form a bromoacetyl-thiol linkage. These and other cross-linking chemistries are known in the art, and are summarized in the literature, including by Greg T. Hermanson, Bioconjugate Techniques, 3rd Ed. 2013, published by Academic Press.

[0237] Additional suitable gRNA modifications will be apparent to those of ordinary skill in the art based on the present disclosure. Suitable gRNA modifications include, for example, those described in PCT application PCT/US2018/054027, filed on Oct. 2, 2018, and entitled “MODIFIED CPF1 GUIDE RNA;” in PCT application PCT/US2015/000143, filed on Dec. 3, 2015, and entitled “GUIDE RNA WITH CHEMICAL MODIFICATIONS;” in PCT application PCT/US2016/026028, filed Apr. 5, 2016, and entitled “CHEMICALLY MODIFIED GUIDE RNAS FOR CRISPR/CAS-MEDIATED GENE REGULATION;” and in PCT application PCT/US2016/053344, filed on Sep. 23, 2016, and entitled “NUCLEASE-MEDIATED GENOME EDITING OF PRIMARY CELLS AND ENRICHMENT THEREOF;” the entire contents of each of which are incorporated herein by reference.

[0238] Exemplary gRNAs

[0239] Non-limiting examples of guide RNAs suitable for certain embodiments embraced by the present disclosure are provided herein, for example, in the Tables below. Those of ordinary skill in the art will be able to envision suitable guide RNA sequences for a specific nuclease, e.g., a Cas9 or Cpf-1 nuclease, from the disclosure of the targeting domain sequence, either as a DNA or RNA sequence. For example, a guide RNA comprising a targeting sequence consisting of RNA nucleotides would include the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence, and this contain uracil instead of thymidine nucleotides. For example, a guide RNA comprising a targeting domain sequence consisting of RNA nucleotides, and described by the DNA sequence TCTGCAGAAATGTTCCCCGT (SEQ ID NO: 24) would have a targeting domain of the corresponding RNA sequence UCUGCAGAAAUGUUCCCCGU (SEQ ID NO: 25). As will be apparent to the skilled artisan, such a targeting sequence would be linked to a suitable guide RNA scaffold, e.g., a crRNA scaffold sequence or a chimeric crRNA/tracrRNA scaffold sequence. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For AsCpf1, for example, a suitable scaffold sequence comprises the sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 26) added to the 5′-terminus of the targeting domain. In the example above, this would result in a Cpf1 guide RNA of the sequence UAAUUUCUACUCUUGUAGAUUCUGCAGAAAUGUUCCCCGU (SEQ ID NO: 27). Those of skill in the art would further understand how to modify such a guide RNA, e.g., by adding a DNA extension (e.g., in the example above, adding a 25-mer DNA extension as described herein would result, for example, in a guide RNA of the sequence ATGTGTTTTTGTCAAAAGACCTTTTrUrArArUrUrUrCrUrArCrUrCrUrUrGrUrArGrArU rUrCrUrGrCrArGrArArArUrGrUrUrCrCrCrCrGrU) (SEQ ID NO: 28). It will be understood that the exemplary targeting sequences provided herein are not limiting, and additional suitable sequences, e.g., variants of the specific sequences disclosed herein, will be apparent to the skilled artisan based on the present disclosure in view of the general knowledge in the art.

[0240] In some embodiments the gRNA for use in the disclosure is a gRNA targeting TGFβRII (TGFβRII gRNA). In some embodiments, the gRNA targeting TGFβRII is one or more of the gRNAs described in Table 4.

TABLE-US-00004 TABLE 4 Exemplary TGFβRII gRNAs gRNA Targeting Domain Sequence Name Sequence  (DNA) Length Enzyme SEQ ID NO: TGFBR24326 CAGGACGATGTGCAGCGGCC 20 AsCpf1 RR   29 TGFBR24327 ACCGCACGTTCAGAAGTCGG 20 AsCpf1 RR   30 TGFBR24328 ACAACTGTGTAAATTTTGTG 20 AsCpf1 RR   31 TGFBR24329 CAACTGTGTAAATTTTGTGA 20 AsCpf1 RR   32 TGFBR24330 ACCTGTGACAACCAGAAATC 20 AsCpf1 RR   33 TGFBR24331 CCTGTGACAACCAGAAATCC 20 AsCpf1 RR   34 TGFBR24332 TGTGGCTTCTCACAGATGGA 20 AsCpf1 RR   35 TGFBR24333 TCTGTGAGAAGCCACAGGAA 20 AsCpf1 RR   36 TGFBR24334 AAGCTCCCCTACCATGACTT 20 AsCpf1 RR   37 TGFBR24335 GAATAAAGTCATGGTAGGGG 20 AsCpf1 RR   38 TGFBR24336 AGAATAAAGTCATGGTAGGG 20 AsCpf1 RR   39 TGFBR24337 CTACCATGACTTTATTCTGG 20 AsCpf1 RR   40 TGFBR24338 TACCATGACTTTATTCTGGA 20 AsCpf1 RR   41 TGFBR24339 TAATGCACTTTGGAGAAGCA 20 AsCpf1 RR   42 TGFBR24340 TTCATAATGCACTTTGGAGA 20 AsCpf1 RR   43 TGFBR24341 AAGTGCATTATGAAGGAAAA 20 AsCpf1 RR   44 TGFBR24342 TGTGTTCCTGTAGCTCTGAT 20 AsCpf1 RR   45 TGFBR24343 TGTAGCTCTGATGAGTGCAA 20 AsCpf1 RR   46 TGFBR24344 AGTGACAGGCATCAGCCTCC 20 AsCpf1 RR   47 TGFBR24345 AGTGGTGGCAGGAGGCTGAT 20 AsCpf1 RR   48 TGFBR24346 AGGTTGAACTCAGCTTCTGC 20 AsCpf1 RR   49 TGFBR24347 CAGGTTGAACTCAGCTTCTG 20 AsCpf1 RR   50 TGFBR24348 ACCTGGGAAACCGGCAAGAC 20 AsCpf1 RR   51 TGFBR24349 CGTCTTGCCGGTTTCCCAGG 20 AsCpf1 RR   52 TGFBR24350 GCGTCTTGCCGGTTTCCCAG 20 AsCpf1 RR   53 TGFBR24351 TGAGCTTCCGCGTCTTGCCG 20 AsCpf1 RR   54 TGFBR24352 GCGAGCACTGTGCCATCATC 20 AsCpf1 RR   55 TGFBR24353 GGATGATGGCACAGTGCTCG 20 AsCpf1 RR   56 TGFBR24354 AGGATGATGGCACAGTGCTC 20 AsCpf1 RR   57 TGFBR24355 CGTGTGCCAACAACATCAAC 20 AsCpf1 RR   58 TGFBR24356 GCTCAATGGGCAGCAGCTCT 20 AsCpf1 RR   59 TGFBR24357 ACCAGGGTGTCCAGCTCAAT 20 AsCpf1 RR   60 TGFBR24358 CACCAGGGTGTCCAGCTCAA 20 AsCpf1 RR   61 TGFBR24359 CCACCAGGGTGTCCAGCTCA 20 AsCpf1 RR   62 TGFBR24360 GCTTGGCCTTATAGACCTCA 20 AsCpf1 RR   63 TGFBR24361 GAGCAGTTTGAGACAGTGGC 20 AsCpf1 RR   64 TGFBR24362 AGAGGCATACTCCTCATAGG 20 AsCpf1 RR   65 TGFBR24363 CTATGAGGAGTATGCCTCTT 20 AsCpf1 RR   66 TGFBR24364 AAGAGGCATACTCCTCATAG 20 AsCpf1 RR   67 TGFBR24365 TATGAGGAGTATGCCTCTTG 20 AsCpf1 RR   68 TGFBR24366 GATTGATGTCTGAGAAGATG 20 AsCpf1 RR   69 TGFBR24367 CTCCTCAGCCGTCAGGAACT 20 AsCpf1 RR   70 TGFBR24368 GTTCCTGACGGCTGAGGAGC 20 AsCpf1 RR   71 TGFBR24369 GCTCCTCAGCCGTCAGGAAC 20 AsCpf1 RR   72 TGFBR24370 TGACGGCTGAGGAGCGGAAG 20 AsCpf1 RR   73 TGFBR24371 TCTTCCGCTCCTCAGCCGTC 20 AsCpf1 RR   74 TGFBR24372 AACTCCGTCTTCCGCTCCTC 20 AsCpf1 RR   75 TGFBR24373 CAACTCCGTCTTCCGCTCCT 20 AsCpf1 RR   76 TGFBR24374 CCAACTCCGTCTTCCGCTCC 20 AsCpf1 RR   77 TGFBR24375 ACGCCAAGGGCAACCTACAG 20 AsCpf1 RR   78 TGFBR24376 CGCCAAGGGCAACCTACAGG 20 AsCpf1 RR   79 TGFBR24377 AGCTGATGACATGCCGCGTC 20 AsCpf1 RR   80 TGFBR24378 GGGCGAGGGAGCTGCCCAGC 20 AsCpf1 RR   81 TGFBR24379 CGGGCGAGGGAGCTGCCCAG 20 AsCpf1 RR   82 TGFBR24380 CCGGGCGAGGGAGCTGCCCA 20 AsCpf1 RR   83 TGFBR24381 TCGCCCGGGGGATTGCTCAC 20 AsCpf1 RR   84 TGFBR24382 ACATGGAGTGTGATCACTGT 20 AsCpf1 RR   85 TGFBR24383 CAGTGATCACACTCCATGTG 20 AsCpf1 RR   86 TGFBR24384 TGTGGGAGGCCCAAGATGCC 20 AsCpf1 RR   87 TGFBR24385 TGTGCACGATGGGCATCTTG 20 AsCpf1 RR   88 TGFBR24386 CGAGGATATTGGAGCTCTTG 20 AsCpf1 RR   89 TGFBR24387 ATATCCTCGTGAAGAACGAC 20 AsCpf1 RR   90 TGFBR24388 GACGCAGGGAAAGCCCAAAG 20 AsCpf1 RR   91 TGFBR24389 CTGCGTCTGGACCCTACTCT 20 AsCpf1 RR   92 TGFBR24390 TGCGTCTGGACCCTACTCTG 20 AsCpf1 RR   93 TGFBR24391 CAGACAGAGTAGGGTCCAGA 20 AsCpf1 RR   94 TGFBR24392 GCCAGCACGATCCCACCGCA 20 AsCpf1 RVR   95 TGFBR24393 AAGGAAAAAAAAAAGCCTGG 20 AsCpf1 RVR   96 TGFBR24394 ACACCAGCAATCCTGACTTG 20 AsCpf1 RVR   97 TGFBR24395 ACTAGCAACAAGTCAGGATT 20 AsCpf1 RVR   98 TGFBR24396 GCAACTCCCAGTGGTGGCAG 20 AsCpf1 RVR   99 TGFBR24397 TGTCATCATCATCTTCTACT 20 AsCpf1 RVR  100 TGFBR24398 GACCTCAGCAAAGCGACCTT 20 AsCpf1 RVR  101 TGFBR24399 AGGCCAAGCTGAAGCAGAAC 20 AsCpf1 RVR  102 TGFBR24400 AGGAGTATGCCTCTTGGAAG 20 AsCpf1 RVR  103 TGFBR24401 CCTCTTGGAAGACAGAGAAG 20 AsCpf1 RVR  104 TGFBR24402 TTCTCATGCTTCAGATTGAT 20 AsCpf1 RVR  105 TGFBR24403 CTCGTGAAGAACGACCTAAC 20 AsCpf1 RVR  106 TGFbR2036 GGCCGCTGCACATCGTCCTG 20 SpyCas9  107 TGFbR2037 GCGGGGTCTGCCATGGGTCG 20 SpyCas9  108 TGFbR2038 AGTTGCTCATGCAGGATTTC 20 SpyCas9  109 TGFbR2039 CCAGAATAAAGTCATGGTAG 20 SpyCas9  110 TGFbR2040 CCCCTACCATGACTTTATTC 20 SpyCas9  111 TGFbR2041 AAGTCATGGTAGGGGAGCTT 20 SpyCas9  112 TGFbR2042 AGTCATGGTAGGGGAGCTTG 20 SpyCas9  113 TGFbR2043 ATTGCACTCATCAGAGCTAC 20 SpyCas9  114 TGFbR2044 CCTAGAGTGAAGAGATTCAT 20 SpyCas9  115 TGFbR2045 CCAATGAATCTCTTCACTCT 20 SpyCas9  116 TGFbR2046 AAAGTCATGGTAGGGGAGCT 20 SpyCas9  117 TGFbR2047 GTGAGCAATCCCCCGGGCGA 20 SpyCas9  118 TGFbR2048 GTCGTTCTTCACGAGGATAT 20 SpyCas9  119 TGFbR2049 GCCGCGTCAGGTACTCCTGT 20 SpyCas9  120 TGFbR2050 GACGCGGCATGTCATCAGCT 20 SpyCas9  121 TGFbR2051 GCTTCTGCTGCCGGTTAACG 20 SpyCas9  122 TGFbR2052 GTGGATGACCTGGCTAACAG 20 SpyCas9  123 TGFbR2053 GTGATCACACTCCATGTGGG 20 SpyCas9  124 TGFbR2054 GCCCATTGAGCTGGACACCC 20 SpyCas9  125 TGFbR2055 GCGGTCATCTTCCAGGATGA 20 SpyCas9  126 TGFbR2056 GGGAGCTGCCCAGCTTGCGC 20 SpyCas9  127 TGFbR2057 GTTGATGTTGTTGGCACACG 20 SpyCas9  128 TGFbR2058 GGCATCTTGGGCCTCCCACA 20 SpyCas9  129 TGFbR2059 GCGGCATGTCATCAGCTGGG 20 SpyCas9  130 TGFbR2060 GCTCCTCAGCCGTCAGGAAC 20 SpyCas9  131 TGFbR2061 GCTGGTGTTATATTCTGATG 20 SpyCas9  132 TGFbR2062 CCGACTTCTGAACGTGCGGT 20 SpyCas9  133 TGFbR2063 TGCTGGCGATACGCGTCCAC 20 SpyCas9  134 TGFbR2064 CCCGACTTCTGAACGTGCGG 20 SpyCas9  135 TGFbR2065 CCACCGCACGTTCAGAAGTC 20 SpyCas9  136 TGFbR2066 TCACCCGACTTCTGAACGTG 20 SpyCas9  137 TGFbR2067 CCCACCGCACGTTCAGAAGT 20 SpyCas9  138 TGFbR2068 CGAGCAGCGGGGTCTGCCAT 20 SpyCas9  139 TGFbR2069 ACGAGCAGCGGGGTCTGCCA 20 SpyCas9  140 TGFbR2070 AGCGGGGTCTGCCATGGGTC 20 SpyCas9  141 TGFbR2071 CCTGAGCAGCCCCCGACCCA 20 SpyCas9  142 TGFbR2072 CCATGGGTCGGGGGCTGCTC 20 SpyCas9  143 TGFbR2073 AACGTGCGGTGGGATCGTGC 20 SpyCas9  144 TGFbR2074 GGACGATGTGCAGCGGCCAC 20 SpyCas9  145 TGFbR2075 GTCCACAGGACGATGTGCAG 20 SpyCas9  146 TGFbR2076 CATGGGTCGGGGGCTGCTCA 20 SpyCas9  147 TGFbR2077 CAGCGGGGTCTGCCATGGGT 20 SpyCas9  148 TGFbR2078 ATGGGTCGGGGGCTGCTCAG 20 SpyCas9  149 TGFbR2079 CGGGGTCTGCCATGGGTCGG 20 SpyCas9  150 TGFbR2080 AGGAAGTCTGTGTGGCTGTA 20 SpyCas9  151 TGFbR2081 CTCCATCTGTGAGAAGCCAC 20 SpyCas9  152 TGFbR2082 ATGATAGTCACTGACAACAA 20 SpyCas9  153 TGFbR2083 GATGCTGCAGTTGCTCATGC 20 SpyCas9  154 TGFbR2084 ACAGCCACACAGACTTCCTG 20 SpyCas9  155 TGFbR2085 GAAGCCACAGGAAGTCTGTG 20 SpyCas9  156 TGFbR2086 TTCCTGTGGCTTCTCACAGA 20 SpyCas9  157 TGFbR2087 CTGTGGCTTCTCACAGATGG 20 SpyCas9  158 TGFbR2088 TCACAAAATTTACACAGTTG 20 SpyCas9  159 TGFbR2089 GACAACATCATCTTCTCAGA 20 SpyCas9  160 TGFbR2090 TCCAGAATAAAGTCATGGTA 20 SpyCas9  161 TGFbR2091 GGTAGGGGAGCTTGGGGTCA 20 SpyCas9  162 TGFbR2092 TTCTCCAAAGTGCATTATGA 20 SpyCas9  163 TGFbR2093 CATCTTCCAGAATAAAGTCA 20 SpyCas9  164 TGFbR2094 CACATGAAGAAAGTCTCACC 20 SpyCas9  165 TGFbR2095 TTCCAGAATAAAGTCATGGT 20 SpyCas9  166 TGFbR2096 TTTTCCTTCATAATGCACTT 20 SpyCas9  167 TGFBR24024 CACAGTTGTGGAAACTTGAC 20 AsCpf1  168 TGFBR24039 CCCAACTCCGTCTTCCGCTC 20 AsCpf1  169 TGFBR24040 GGCTTTCCCTGCGTCTGGAC 20 AsCpf1  170 TGFBR24036 CTGAGGTCTATAAGGCCAAG 20 AsCpf1  171 TGFBR24026 TGATGTGAGATTTTCCACCT 20 AsCpf1  172 TGFBR24038 CCTATGAGGAGTATGCCTCT 20 AsCpf1  173 TGFBR24033 AAGTGACAGGCATCAGCCTC 20 AsCpf1  174 TGFBR24028 CCATGACCCCAAGCTCCCCT 20 AsCpf1  175 TGFBR24031 CTTCATAATGCACTTTGGAG 20 AsCpf1  176 TGFBR24032 TTCATGTGTTCCTGTAGCTC 20 AsCpf1  177 TGFBR24029 TTCTGGAAGATGCTGCTTCT 20 AsCpf1  178 TGFBR24035 CCCACCAGGGTGTCCAGCTC 20 AsCpf1  179 TGFBR24037 AGACAGTGGCAGTCAAGATC 20 AsCpf1  180 TGFBR24041 CCTGCGTCTGGACCCTACTC 20 AsCpf1  181 TGFBR24025 CACAACTGTGTAAATTTTGT 20 AsCpf1  182 TGFBR24030 GAGAAGCAGCATCTTCCAGA 20 AsCpf1  183 TGFBR24027 TGGTTGTCACAGGTGGAAAA 20 AsCpf1  184 TGFBR24034 CCAGGTTGAACTCAGCTTCT 20 AsCpf1  185 TGFBR24043 ATCACAAAATTTACACAGTTG 21 SauCas9  186 TGFBR24065 GGCATCAGCCTCCTGCCACCA 21 SauCas9  187 TGFBR24110 GTTAGCCAGGTCATCCACAGA 21 SauCas9  188 TGFBR24099 GCTGGGCAGCTCCCTCGCCCG 21 SauCas9  189 TGFBR24064 CAGGAGGCTGATGCCTGTCAC 21 SauCas9  190 TGFBR24094 GAGGAGCGGAAGACGGAGTTG 21 SauCas9  191 TGFBR24108 CGTCTGGACCCTACTCTGTCT 21 SauCas9  192 TGFBR24058 TTTTTCCTTCATAATGCACTT 21 SauCas9  193 TGFBR24075 CCATTGAGCTGGACACCCTGG 21 SauCas9  194 TGFBR24057 CTTCTCCAAAGTGCATTATGA 21 SauCas9  195 TGFBR24103 GCCCAAGATGCCCATCGTGCA 21 SauCas9  196 TGFBR24060 TCATGTGTTCCTGTAGCTCTG 21 SauCas9  197 TGFBR24048 GTGATGCTGCAGTTGCTCATG 21 SauCas9  198 TGFBR24087 TCTCATGCTTCAGATTGATGT 21 SauCas9  199 TGFBR24081 TCCCTATGAGGAGTATGCCTC 21 SauCas9  200 TGFBR24044 CATCACAAAATTTACACAGTT 21 SauCas9  201 TGFBR24077 ATTGAGCTGGACACCCTGGTG 21 SauCas9  202 TGFBR24080 CAGTCAAGATCTTTCCCTATG 21 SauCas9  203 TGFBR24046 AGGATTTCTGGTTGTCACAGG 21 SauCas9  204 TGFBR24101 TCCACAGTGATCACACTCCAT 21 SauCas9  205 TGFBR24079 AGCAGAACACTTCAGAGCAGT 21 SauCas9  206 TGFBR24072 CCGGCAAGACGCGGAAGCTCA 21 SauCas9  207 TGFBR24074 GATGTCAGAGCGGTCATCTTC 21 SauCas9  208 TGFBR24062 TCATTGCACTCATCAGAGCTA 21 SauCas9  209 TGFBR24054 CTTCCAGAATAAAGTCATGGT 21 SauCas9  210 TGFBR24045 AGATTTTCCACCTGTGACAAC 21 SauCas9  211 TGFBR24049 ACTGCAGCATCACCTCCATCT 21 SauCas9  212 TGFBR24098 AGCTGGGCAGCTCCCTCGCCC 21 SauCas9  213 TGFBR24090 TGACGGCTGAGGAGCGGAAGA 21 SauCas9  214 TGFBR24076 CATTGAGCTGGACACCCTGGT 21 SauCas9  215 TGFBR24078 AGCAAAGCGACCTTTCCCCAC 21 SauCas9  216 TGFBR24067 CGCGTTAACCGGCAGCAGAAG 21 SauCas9  217 TGFBR24063 GAAATATGACTAGCAACAAGT 21 SauCas9  218 TGFBR24107 AGACAGAGTAGGGTCCAGACG 21 SauCas9  219 TGFBR24047 CAGGATTTCTGGTTGTCACAG 21 SauCas9  220 TGFBR24096 CTCCTGTAGGTTGCCCTTGGC 21 SauCas9  221 TGFBR24105 ACAGAGTAGGGTCCAGACGCA 21 SauCas9  222 TGFBR24056 GCTTCTCCAAAGTGCATTATG 21 SauCas9  223 TGFBR24068 GCAGCAGAAGCTGAGTTCAAC 21 SauCas9  224 TGFBR24093 TGAGGAGCGGAAGACGGAGTT 21 SauCas9  225 TGFBR24055 CTTTGGAGAAGCAGCATCTTC 21 SauCas9  226 TGFBR24053 CTCCCCTACCATGACTTTATT 21 SauCas9  227 TGFBR24106 GACAGAGTAGGGTCCAGACGC 21 SauCas9  228 TGFBR24092 CTGAGGAGCGGAAGACGGAGT 21 SauCas9  229 TGFBR24102 GGGCATCTTGGGCCTCCCACA 21 SauCas9  230 TGFBR24082 CCAAGAGGCATACTCCTCATA 21 SauCas9  231 TGFBR24051 AGAATGACGAGAACATAACAC 21 SauCas9  232 TGFBR24097 CCTGACGCGGCATGTCATCAG 21 SauCas9  233 TGFBR24073 AGCGAGCACTGTGCCATCATC 21 SauCas9  234 TGFBR24104 GCAGGTTAGGTCGTTCTTCAC 21 SauCas9  235 TGFBR24050 ACCTCCATCTGTGAGAAGCCA 21 SauCas9  236 TGFBR24052 TAAAGTCATGGTAGGGGAGCT 21 SauCas9  237 TGFBR24061 TCAGAGCTACAGGAACACATG 21 SauCas9  238 TGFBR24086 TCTCAGACATCAATCTGAAGC 21 SauCas9  239 TGFBR24066 CATCAGCCTCCTGCCACCACT 21 SauCas9  240 TGFBR24089 CGCTCCTCAGCCGTCAGGAAC 21 SauCas9  241 TGFBR24071 AACCTGGGAAACCGGCAAGAC 21 SauCas9  242 TGFBR24095 TCCACGCCAAGGGCAACCTAC 21 SauCas9  243 TGFBR24100 GAGGTGAGCAATCCCCCGGGC 21 SauCas9  244 TGFBR24069 CAGCAGAAGCTGAGTTCAACC 21 SauCas9  245 TGFBR24083 TCCAAGAGGCATACTCCTCAT 21 SauCas9  246 TGFBR24070 AGCAGAAGCTGAGTTCAACCT 21 SauCas9  247 TGFBR24088 CCAGTTCCTGACGGCTGAGGA 21 SauCas9  248 TGFBR24085 AGGAGTATGCCTCTTGGAAGA 21 SauCas9  249 TGFBR24084 TTCCAAGAGGCATACTCCTCA 21 SauCas9  250 TGFBR24042 CAACTGTGTAAATTTTGTGAT 21 SauCas9  251 TGFBR24059 TGAAGGAAAAAAAAAAGCCTG 21 SauCas9  252 TGFBR24091 CGTCTTCCGCTCCTCAGCCGT 21 SauCas9  253 TGFBR24109 CCAGGTCATCCACAGACAGAG 21 SauCas9  254 TGFBR2736 GCCTAGAGTGAAGAGATTCAT 21 SpyCas9  255 TGFBR2737 GTTCTCCAAAGTGCATTATGA 21 SpyCas9  256 TGFBR2738 GCATCTTCCAGAATAAAGTCA 21 SpyCas9  257 TGFBR2739 TGATGTGAGATTTTCCACCTG 21 Cas12a 1172

[0241] In some embodiments the gRNA for use in the disclosure is a gRNA targeting CISH (CISH gRNA). In some embodiments, the gRNA targeting CISH is one or more of the gRNAs described in Table 5.

TABLE-US-00005 TABLE 5 Exemplary CISH gRNAs gRNA Targeting Domain Sequence Name (DNA) Length Enzyme SEQ ID NO: CISH0873 CAACCGTCTGGTGGCCGACG 20 SpyCas9  258 CISHO874 CAGGATCGGGGCTGTCGCTT 20 SpyCas9  259 CISH0875 TCGGGCCTCGCTGGCCGTAA 20 SpyCas9  260 CISH0876 GAGGTAGTCGGCCATGCGCC 20 SpyCas9  261 CISH0877 CAGGTGTTGTCGGGCCTCGC 20 SpyCas9  262 CISH0878 GGAGGTAGTCGGCCATGCGC 20 SpyCas9  263 CISH0879 GGCATACTCAATGCGTACAT 20 SpyCas9  264 CISH0880 CCGCCTTGTCATCAACCGTC 20 SpyCas9  265 CISHO881 AGGATCGGGGCTGTCGCTTC 20 SpyCas9  266 CISHO882 CCTTGTCATCAACCGTCTGG 20 SpyCas9  267 CISHO883 TACTCAATGCGTACATTGGT 20 SpyCas9  268 CISHO884 GGGTTCCATTACGGCCAGCG 20 SpyCas9  269 CISHO885 GGCACTGCTTCTGCGTACAA 20 SpyCas9  270 CISHO886 GGTTGATGACAAGGCGGCAC 20 SpyCas9  271 CISHO887 TGCTGGGGCCTTCCTCGAGG 20 SpyCas9  272 CISHO888 TTGCTGGCTGTGGAGCGGAC 20 SpyCas9  273 CISHO889 TTCTCCTACCTTCGGGAATC 20 SpyCas9  274 CISH0890 GACTGGCTTGGGCAGTTCCA 20 SpyCas9  275 CISHO891 CATGCAGCCCTTGCCTGCTG 20 SpyCas9  276 CISHO892 AGCAAAGGACGAGGTCTAGA 20 SpyCas9  277 CISHO893 GCCTGCTGGGGCCTTCCTCG 20 SpyCas9  278 CISHO894 CAGACTCACCAGATTCCCGA 20 SpyCas9  279 CISHO895 ACCTCGTCCTTTGCTGGCTG 20 SpyCas9  280 CISHO896 CTCACCAGATTCCCGAAGGT 20 SpyCas9  281 CISH7048 TACGCAGAAGCAGTGCCCGC 20 AsCpf1  282 CISH7049 AGGTGTACAGCAGTGGCTGG 20 AsCpf1  283 CISH7050 GGTGTACAGCAGTGGCTGGT 20 AsCpf1  284 CISH7051 CGGATGTGGTCAGCCTTGTG 20 AsCpf1  285 CISH7052 CACTGACAGCGTGAACAGGT 20 AsCpf1  286 CISH7053 ACTGACAGCGTGAACAGGTA 20 AsCpf1  287 CISH7054 GCTCACTCTCTGTCTGGGCT 20 AsCpf1  288 CISH7055 CTGGCTGTGGAGCGGACTGG 20 AsCpf1  289 CISH7056 GCTCTGACTGTACGGGGCAA 20 AsCpf1 RR  290 CISH7057 AGCTCTGACTGTACGGGGCA 20 AsCpf1 RR  291 CISH7058 ACAGTACCCCTTCCAGCTCT 20 AsCpf1 RR  292 CISH7059 CGTCGGCCACCAGACGGTTG 20 AsCpf1 RR  293 CISH7060 CCAGCCACTGCTGTACACCT 20 AsCpf1 RR  294 CISH7061 ACCCCGGCCCTGCCTATGCC 20 AsCpf1 RR  295 CISH7062 GGTATCAGCAGTGCAGGAGG 20 AsCpf1 RR  296 CISH7063 GATGTGGTCAGCCTTGTGCA 20 AsCpf1 RR  297 CISH7064 GGATGTGGTCAGCCTTGTGC 20 AsCpf1 RR  298 CISH7065 GGCCACGCATCCTGGCCTTT 20 AsCpf1 RR  299 CISH7066 GAAAGGCCAGGATGCGTGGC 20 AsCpf1 RR  300 CISH7067 ACTGCTTGTCCAGGCCACGC 20 AsCpf1 RR  301 CISH7068 TCTGGACTCCAACTGCTTGT 20 AsCpf1 RR  302 CISH7069 GTCTGGACTCCAACTGCTTG 20 AsCpf1 RR  303 CISH7070 GCTTCCGTCTGGACTCCAAC 20 AsCpf1 RR  304 CISH7071 GACGGAAGCTGGAGTCGGCA 20 AsCpf1 RR  305 CISH7072 CGCTGTCAGTGAAAACCACT 20 AsCpf1 RR  306 CISH7073 CTGACAGCGTGAACAGGTAG 20 AsCpf1 RR  307 CISH7074 TTACGGCCAGCGAGGCCCGA 20 AsCpf1 RR  308 CISH7075 ATTACGGCCAGCGAGGCCCG 20 AsCpf1 RR  309 CISH7076 GGAATCTGGTGAGTCTGAGG 20 AsCpf1 RR  310 CISH7077 CCCTCAGACTCACCAGATTC 20 AsCpf1 RR  311 CISH7078 CGAAGGTAGGAGAAGGTCTT 20 AsCpf1 RR  312 CISH7079 GAAGGTAGGAGAAGGTCTTG 20 AsCpf1 RR  313 CISH7080 GCACCTTTGGCTCACTCTCT 20 AsCpf1 RR  314 CISH7081 TCGAGGAGGTGGCAGAGGGT 20 AsCpf1 RR  315 CISH7082 TGGAACTGCCCAAGCCAGTC 20 AsCpf1 RR  316 CISH7083 AGGGACGGGGCCCACAGGGG 20 AsCpf1 RR  317 CISH7084 GGGACGGGGCCCACAGGGGC 20 AsCpf1 RR  318 CISH7085 CTCCACAGCCAGCAAAGGAC 20 AsCpf1 RR  319 CISH7086 CAGCCAGCAAAGGACGAGGT 20 AsCpf1 RR  320 CISH7087 CTGCCTTCTAGACCTCGTCC 20 AsCpf1 RR  321 CISH7088 CCTAAGGAGGATGCGCCTAG 20 AsCpf1 RVR  322 CISH7089 TGGCCTCCTGCACTGCTGAT 20 AsCpf1 RVR  323 CISH7090 AGCAGTGCAGGAGGCCACAT 20 AsCpf1 RVR  324 CISH7091 CCGACTCCAGCTTCCGTCTG 20 AsCpf1 RVR  325 CISH7092 GGGGTTCCATTACGGCCAGC 20 AsCpf1 RVR  326 CISH7093 CACAGCAGATCCTCCTCTGG 20 AsCpf1 RVR  327 CISH7094 ATTGCCCCGTACAGTCAGAG 20 SauCas9  328 CISH7095 CCCGTACAGTCAGAGCTGGA 20 SauCas9  329 CISH7096 TGGTGGAGGAGCAGGCAGTG 20 SauCas9  330 CISH7097 TCCTTAGGCATAGGCAGGGC 20 SauCas9  331 CISH7098 CGGCCCTGCCTATGCCTAAG 20 SauCas9  332 CISH7099 TAGGCATAGGCAGGGCCGGG 20 SauCas9  333 CISH7100 AGGCAGGGCCGGGGTGGGAG 20 SauCas9  334 CISH7101 GCAGGATCGGGGCTGTCGCT 20 SauCas9  335 CISH7102 CTGCACAAGGCTGACCACAT 20 SauCas9  336 CISH7103 TGCACAAGGCTGACCACATC 20 SauCas9  337 CISH7104 CTGACCACATCCGGAAAGGC 20 SauCas9  338 CISH7105 GGCCACGCATCCTGGCCTTT 20 SauCas9  339 CISH7106 GCGTGGCCTGGACAAGCAGT 20 SauCas9  340 CISH7107 GACAAGCAGTTGGAGTCCAG 20 SauCas9  341 CISH7108 GTTGGAGTCCAGACGGAAGC 20 SauCas9  342 CISH7109 ATGCGTACATTGGTGGGGCC 20 SauCas9  343 CISH7110 TGGCCCCACCAATGTACGCA 20 SauCas9  344 CISH7111 GCTACCTGTTCACGCTGTCA 20 SauCas9  345 CISH7112 TGACAGCGTGAACAGGTAGC 20 SauCas9  346 CISH7113 GTCGGGCCTCGCTGGCCGTA 20 SauCas9  347 CISH7114 GCACTTGCCTAGGCTGGTAT 20 SauCas9  348 CISH7115 GGGAATCTGGTGAGTCTGAG 20 SauCas9  349 CISH7116 CTCACCAGATTCCCGAAGGT 20 SauCas9  350 CISH7117 CTCCTACCTTCGGGAATCTG 20 SauCas9  351 CISH7118 CAAGACCTTCTCCTACCTTC 20 SauCas9  352 CISH7119 CCAAGACCTTCTCCTACCTT 20 SauCas9  353 CISH7120 GCCAAGACCTTCTCCTACCT 20 SauCas9  354 CISH7121 TATGCACAGCAGATCCTCCT 20 SauCas9  355 CISH7122 CAAAGGTGCTGGACCCAGAG 20 SauCas9  356 CISH7123 GGCTCACTCTCTGTCTGGGC 20 SauCas9  357 CISH7124 AGGGTACCCCAGCCCAGACA 20 SauCas9  358 CISH7125 AGAGGGTACCCCAGCCCAGA 20 SauCas9  359 CISH7126 GTACCCTCTGCCACCTCCTC 20 SauCas9  360 CISH7127 CCTTCCTCGAGGAGGTGGCA 20 SauCas9  361 CISH7128 ATGACTGGCTTGGGCAGTTC 20 SauCas9  362 CISH7129 GGCCCCTGTGGGCCCCGTCC 20 SauCas9  363 CISH7130 AGGACGAGGTCTAGAAGGCA 20 SauCas9  364 CISH7131 ACTGACAGCGTGAACAGGTAG 21 Cas12a 1173

[0242] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting B2M (B2M gRNA). In some embodiments, the gRNA targeting B2M is one or more of the gRNAs described in Table 6.

TABLE-US-00006 TABLE 6 Exemplary B2M gRNAs gRNA Targeting Domain SEQ ID gRNA name Target sequence (DNA) Length Enzyme NO: B2M1 TATAAGTGGAGGCGTCGCGC 20 SpyCas9 365 B2M2 GGGCACGCGTTTAATATAAG 20 SpyCas9 366 B2M3 ACTCACGCTGGATAGCCTCC 20 SpyCas9 367 B2M4 GGCCGAGATGTCTCGCTCCG 20 SpyCas9 368 B2M5 CACGCGTTTAATATAAGTGG 20 SpyCas9 369 B2M6 AAGTGGAGGCGTCGCGCTGG 20 SpyCas9 370 B2M7 GAGTAGCGCGAGCACAGCTA 20 SpyCas9 371 B2M8 AGTGGAGGCGTCGCGCTGGC 20 SpyCas9 372 B2M9 GCCCGAATGCTGTCAGCTTC 20 SpyCas9 373 B2M10 CGCGAGCACAGCTAAGGCCA 20 SpyCas9 374 B2M11 CTCGCGCTACTCTCTCTTTC 20 SpyCas9 375 B2M12 GGCCACGGAGCGAGACATCT 20 SpyCas9 376 B2M13 CGTGAGTAAACCTGAATCTT 20 SpyCas9 377 B2M14 AGTCACATGGTTCACACGGC 20 SpyCas9 378 B2M15 AAGTCAACTTCAATGTCGGA 20 SpyCas9 379 B2M16 CAGTAAGTCAACTTCAATGT 20 SpyCas9 380 B2M17 ACCCAGACACATAGCAATTC 20 SpyCas9 381 B2M18 GCATACTCATCTTTTTCAGT 20 SpyCas9 382 B2M19 ACAGCCCAAGATAGTTAAGT 20 SpyCas9 383 B2M20 GGCATACTCATCTTTTTCAG 20 SpyCas9 384 B2M21 TTCCTGAAGCTGACAGCATT 20 SpyCas9 385 B2M22 TCACGTCATCCAGCAGAGAA 20 SpyCas9 386 B2M23 CAGCCCAAGATAGTTAAGTG 20 SpyCas9 387 B2M-c1 AAUUCUCUCUCCAUUCUU 18 AsCpf1 388 B2M-c2 AAUUCUCUCUCCAUUCUUC 19 AsCpf1 389 B2M-c3 AAUUCUCUCUCCAUUCUUCA 20 AsCpf1 390 B2M-c4 AAUUCUCUCUCCAUUCUUCAG 21 AsCpf1 391 B2M-c5 AAUUCUCUCUCCAUUCUUCAGU 22 AsCpf1 392 B2M-c6 AAUUCUCUCUCCAUUCUUCAGUA 23 AsCpf1 393 B2M-c7 AAUUCUCUCUCCAUUCUUCAGUAA 24 AsCpf1 394 B2M-c8 ACUUUCCAUUCUCUGCUG 18 AsCpf1 395 B2M-c9 ACUUUCCAUUCUCUGCUGG 19 AsCpf1 396 B2M-c10 ACUUUCCAUUCUCUGCUGGA 20 AsCpf1 397 B2M-c11 ACUUUCCAUUCUCUGCUGGAU 21 AsCpf1 398 B2M-c12 ACUUUCCAUUCUCUGCUGGAUG 22 AsCpf1 399 B2M-c13 ACUUUCCAUUCUCUGCUGGAUGA 23 AsCpf1 400 B2M-c14 ACUUUCCAUUCUCUGCUGGAUGAC 24 AsCpf1 401 B2M-c15 AGCAAGGACUGGUCUUUC 18 AsCpf1 402 B2M-c16 AGCAAGGACUGGUCUUUCU 19 AsCpf1 403 B2M-c17 AGCAAGGACUGGUCUUUCUA 20 AsCpf1 404 B2M-c18 AGCAAGGACUGGUCUUUCUAU 21 AsCpf1 405 B2M-c19 AGCAAGGACUGGUCUUUCUAUC 22 AsCpf1 406 B2M-c20 AGCAAGGACUGGUCUUUCUAUCU 23 AsCpf1 407 B2M-c21 AGCAAGGACUGGUCUUUCUAUCUC 24 AsCpf1 408 B2M-c22 AGUGGGGGUGAAUUCAGU 18 AsCpf1 409 B2M-c23 AGUGGGGGUGAAUUCAGUG 19 AsCpf1 410 B2M-c24 AGUGGGGGUGAAUUCAGUGU 20 AsCpf1 411 B2M-c25 AGUGGGGGUGAAUUCAGUGUA 21 AsCpf1 412 B2M-c26 AGUGGGGGUGAAUUCAGUGUAG 22 AsCpf1 413 B2M-c27 AGUGGGGGUGAAUUCAGUGUAGU 23 AsCpf1 414 B2M-c28 AGUGGGGGUGAAUUCAGUGUAGUA 24 AsCpf1 415 B2M-c29 AUCCAUCCGACAUUGAAG 18 AsCpf1 416 B2M-c30 AUCCAUCCGACAUUGAAGU 19 AsCpf1 417 B2M-c31 AUCCAUCCGACAUUGAAGUU 20 AsCpf1 418 B2M-c32 AUCCAUCCGACAUUGAAGUUG 21 AsCpf1 419 B2M-c33 AUCCAUCCGACAUUGAAGUUGA 22 AsCpf1 420 B2M-c34 AUCCAUCCGACAUUGAAGUUGAC 23 AsCpf1 421 B2M-c35 AUCCAUCCGACAUUGAAGUUGACU 24 AsCpf1 422 B2M-c36 CAAUUCUCUCUCCAUUCU 18 AsCpf1 423 B2M-c37 CAAUUCUCUCUCCAUUCUU 19 AsCpf1 424 B2M-c38 CAAUUCUCUCUCCAUUCUUC 20 AsCpf1 425 B2M-c39 CAAUUCUCUCUCCAUUCUUCA 21 AsCpf1 426 B2M-c40 CAAUUCUCUCUCCAUUCUUCAG 22 AsCpf1 427 B2M-c41 CAAUUCUCUCUCCAUUCUUCAGU 23 AsCpf1 428 B2M-c42 CAAUUCUCUCUCCAUUCUUCAGUA 24 AsCpf1 429 B2M-c43 CAGUGGGGGUGAAUUCAG 18 AsCpf1 430 B2M-c44 CAGUGGGGGUGAAUUCAGU 19 AsCpf1 431 B2M-c45 CAGUGGGGGUGAAUUCAGUG 20 AsCpf1 432 B2M-c46 CAGUGGGGGUGAAUUCAGUGU 21 AsCpf1 433 B2M-c47 CAGUGGGGGUGAAUUCAGUGUA 22 AsCpf1 434 B2M-c48 CAGUGGGGGUGAAUUCAGUGUAG 23 AsCpf1 435 B2M-c49 CAGUGGGGGUGAAUUCAGUGUAGU 24 AsCpf1 436 B2M-c50 CAUUCUCUGCUGGAUGAC 18 AsCpf1 437 B2M-c51 CAUUCUCUGCUGGAUGACG 19 AsCpf1 438 B2M-c52 CAUUCUCUGCUGGAUGACGU 20 AsCpf1 439 B2M-c53 CAUUCUCUGCUGGAUGACGUG 21 AsCpf1 440 B2M-c54 CAUUCUCUGCUGGAUGACGUGA 22 AsCpf1 441 B2M-c55 CAUUCUCUGCUGGAUGACGUGAG 23 AsCpf1 442 B2M-c56 CAUUCUCUGCUGGAUGACGUGAGU 24 AsCpf1 443 B2M-c57 CCCGAUAUUCCUCAGGUA 18 AsCpf1 444 B2M-c58 CCCGAUAUUCCUCAGGUAC 19 AsCpf1 445 B2M-c59 CCCGAUAUUCCUCAGGUACU 20 AsCpf1 446 B2M-c60 CCCGAUAUUCCUCAGGUACUC 21 AsCpf1 447 B2M-c61 CCCGAUAUUCCUCAGGUACUCC 22 AsCpf1 448 B2M-c62 CCCGAUAUUCCUCAGGUACUCCA 23 AsCpf1 449 B2M-c63 CCCGAUAUUCCUCAGGUACUCCAA 24 AsCpf1 450 B2M-c64 CCGAUAUUCCUCAGGUAC 18 AsCpf1 451 B2M-c65 CCGAUAUUCCUCAGGUACU 19 AsCpf1 452 B2M-c66 CCGAUAUUCCUCAGGUACUC 20 AsCpf1 453 B2M-c67 CCGAUAUUCCUCAGGUACUCC 21 AsCpf1 454 B2M-c68 CCGAUAUUCCUCAGGUACUCCA 22 AsCpf1 455 B2M-c69 CCGAUAUUCCUCAGGUACUCCAA 23 AsCpf1 456 B2M-c70 CCGAUAUUCCUCAGGUACUCCAAA 24 AsCpf1 457 B2M-c71 CUCACGUCAUCCAGCAGA 18 AsCpf1 458 B2M-c72 CUCACGUCAUCCAGCAGAG 19 AsCpf1 459 B2M-c73 CUCACGUCAUCCAGCAGAGA 20 AsCpf1 460 B2M-c74 CUCACGUCAUCCAGCAGAGAA 21 AsCpf1 461 B2M-c75 CUCACGUCAUCCAGCAGAGAAU 22 AsCpf1 462 B2M-c76 CUCACGUCAUCCAGCAGAGAAUG 23 AsCpf1 463 B2M-c77 CUCACGUCAUCCAGCAGAGAAUGG 24 AsCpf1 464 B2M-c78 CUGAAUUGCUAUGUGUCU 18 AsCpf1 465 B2M-c79 CUGAAUUGCUAUGUGUCUG 19 AsCpf1 466 B2M-c80 CUGAAUUGCUAUGUGUCUGG 20 AsCpf1 467 B2M-c81 CUGAAUUGCUAUGUGUCUGGG 21 AsCpf1 468 B2M-c82 CUGAAUUGCUAUGUGUCUGGGU 22 AsCpf1 469 B2M-c83 CUGAAUUGCUAUGUGUCUGGGUU 23 AsCpf1 470 B2M-c84 CUGAAUUGCUAUGUGUCUGGGUUU 24 AsCpf1 471 B2M-c85 GAGUACCUGAGGAAUAUC 18 AsCpf1 472 B2M-c86 GAGUACCUGAGGAAUAUCG 19 AsCpf1 473 B2M-c87 GAGUACCUGAGGAAUAUCGG 20 AsCpf1 474 B2M-c88 GAGUACCUGAGGAAUAUCGGG 21 AsCpf1 475 B2M-c89 GAGUACCUGAGGAAUAUCGGGA 22 AsCpf1 476 B2M-c90 GAGUACCUGAGGAAUAUCGGGAA 23 AsCpf1 477 B2M-c91 GAGUACCUGAGGAAUAUCGGGAAA 24 AsCpf1 478 B2M-c92 UAUCUCUUGUACUACACU 18 AsCpf1 479 B2M-c93 UAUCUCUUGUACUACACUG 19 AsCpf1 480 B2M-c94 UAUCUCUUGUACUACACUGA 20 AsCpf1 481 B2M-c95 UAUCUCUUGUACUACACUGAA 21 AsCpf1 482 B2M-c96 UAUCUCUUGUACUACACUGAAU 22 AsCpf1 483 B2M-c97 UAUCUCUUGUACUACACUGAAUU 23 AsCpf1 484 B2M-c98 UAUCUCUUGUACUACACUGAAUUC 24 AsCpf1 485 B2M-c99 UCAAUUCUCUCUCCAUUC 18 AsCpf1 486 B2M-c100 UCAAUUCUCUCUCCAUUCU 19 AsCpf1 487 B2M-c101 UCAAUUCUCUCUCCAUUCUU 20 AsCpf1 488 B2M-c102 UCAAUUCUCUCUCCAUUCUUC 21 AsCpf1 489 B2M-c103 UCAAUUCUCUCUCCAUUCUUCA 22 AsCpf1 490 B2M-c104 UCAAUUCUCUCUCCAUUCUUCAG 23 AsCpf1 491 B2M-c105 UCAAUUCUCUCUCCAUUCUUCAGU 24 AsCpf1 492 B2M-c106 UCACAGCCCAAGAUAGUU 18 AsCpf1 493 B2M-c107 UCACAGCCCAAGAUAGUUA 19 AsCpf1 494 B2M-c108 UCACAGCCCAAGAUAGUU AA 20 AsCpf1 495 B2M-c109 UCACAGCCCAAGAUAGUUAAG 21 AsCpf1 496 B2M-c110 UCACAGCCCAAGAUAGUUAAGU 22 AsCpf1 497 B2M-c111 UCACAGCCCAAGAUAGUUAAGUG 23 AsCpf1 498 B2M-c112 UCACAGCCCAAGAUAGUUAAGUGG 24 AsCpf1 499 B2M-c113 UCAGUGGGGGUGAAUUCA 18 AsCpf1 500 B2M-c114 UCAGUGGGGGUGAAUUCAG 19 AsCpf1 501 B2M-c115 UCAGUGGGGGUGAAUUCAGU 20 AsCpf1 502 B2M-c116 UCAGUGGGGGUGAAUUCAGUG 21 AsCpf1 503 B2M-c117 UCAGUGGGGGUGAAUUCAGUGU 22 AsCpf1 504 B2M-c118 UCAGUGGGGGUGAAUUCAGUGUA 23 AsCpf1 505 B2M-c119 UCAGUGGGGGUGAAUUCAGUGUAG 24 AsCpf1 506 B2M-c120 UGGCCUGGAGGCUAUCCA 18 AsCpf1 507 B2M-c121 UGGCCUGGAGGCUAUCCAG 19 AsCpf1 508 B2M-c122 UGGCCUGGAGGCUAUCCAGC 20 AsCpf1 509 B2M-c123 UGGCCUGGAGGCUAUCCAGCG 21 AsCpf1 510 B2M-c124 UGGCCUGGAGGCUAUCCAGCGU 22 AsCpf1 511 B2M-c125 UGGCCUGGAGGCUAUCCAGCGUG 23 AsCpf1 512 B2M-c126 UGGCCUGGAGGCUAUCCAGCGUGA 24 AsCpf1 513 B2M-c127 AUAGAUCGAGACAUGUAA 18 AsCpf1 514 B2M-c128 AUAGAUCGAGACAUGUAAG 19 AsCpf1 515 B2M-c129 AUAGAUCGAGACAUGUAAGC 20 AsCpf1 516 B2M-c130 AUAGAUCGAGACAUGUAAGCA 21 AsCpf1 517 B2M-c131 AUAGAUCGAGACAUGUAAGCAG 22 AsCpf1 518 B2M-c132 AUAGAUCGAGACAUGUAAGCAGC 23 AsCpf1 519 B2M-c133 AUAGAUCGAGACAUGUAAGCAGCA 24 AsCpf1 520 B2M-c134 CAUAGAUCGAGACAUGUA 18 AsCpf1 521 B2M-c135 CAUAGAUCGAGACAUGUAA 19 AsCpf1 522 B2M-c136 CAUAGAUCGAGACAUGUAAG 20 AsCpf1 523 B2M-c137 CAUAGAUCGAGACAUGUAAGC 21 AsCpf1 524 B2M-c138 CAUAGAUCGAGACAUGUAAGCA 22 AsCpf1 525 B2M-c139 CAUAGAUCGAGACAUGUAAGCAG 23 AsCpf1 526 B2M-c140 CAUAGAUCGAGACAUGUAAGCAGC 24 AsCpf1 527 B2M-c141 CUCCACUGUCUUUUUCAU 18 AsCpf1 528 B2M-c142 CUCCACUGUCUUUUUCAUA 19 AsCpf1 529 B2M-c143 CUCCACUGUCUUUUUCAUAG 20 AsCpf1 530 B2M-c144 CUCCACUGUCUUUUUCAUAGA 21 AsCpf1 531 B2M-c145 CUCCACUGUCUUUUUCAUAGAU 22 AsCpf1 532 B2M-c146 CUCCACUGUCUUUUUCAUAGAUC 23 AsCpf1 533 B2M-c147 CUCCACUGUCUUUUUCAUAGAUCG 24 AsCpf1 534 B2M-c148 UCAUAGAUCGAGACAUGU 18 AsCpf1 535 B2M-c149 UCAUAGAUCGAGACAUGUA 19 AsCpf1 536 B2M-c150 UCAUAGAUCGAGACAUGUAA 20 AsCpf1 537 B2M-c151 UCAUAGAUCGAGACAUGUAAG 21 AsCpf1 538 B2M-c152 UCAUAGAUCGAGACAUGUAAGC 22 AsCpf1 539 B2M-c153 UCAUAGAUCGAGACAUGUAAGCA 23 AsCpf1 540 B2M-c154 UCAUAGAUCGAGACAUGUAAGCAG 24 AsCpf1 541 B2M-c155 UCCACUGUCUUUUUCAUA 18 AsCpf1 542 B2M-c156 UCCACUGUCUUUUUCAUAG 19 AsCpf1 543 B2M-c157 UCCACUGUCUUUUUCAUAGA 20 AsCpf1 544 B2M-c158 UCCACUGUCUUUUUCAUAGAU 21 AsCpf1 545 B2M-c159 UCCACUGUCUUUUUCAUAGAUC 22 AsCpf1 546 B2M-c160 UCCACUGUCUUUUUCAUAGAUCG 23 AsCpf1 547 B2M-c161 UCCACUGUCUUUUUCAUAGAUCGA 24 AsCpf1 548 B2M-c162 UCUCCACUGUCUUUUUCA 18 AsCpf1 549 B2M-c163 UCUCCACUGUCUUUUUCAU 19 AsCpf1 550 B2M-c164 UCUCCACUGUCUUUUUCAUA 20 AsCpf1 551 B2M-c165 UCUCCACUGUCUUUUUCAUAG 21 AsCpf1 552 B2M-c166 UCUCCACUGUCUUUUUCAUAGA 22 AsCpf1 553 B2M-c167 UCUCCACUGUCUUUUUCAUAGAU 23 AsCpf1 554 B2M-c168 UCUCCACUGUCUUUUUCAUAGAUC 24 AsCpf1 555 B2M-c169 UUCUCCACUGUCUUUUUC 18 AsCpf1 556 B2M-c170 UUCUCCACUGUCUUUUUCA 19 AsCpf1 557 B2M-c171 UUCUCCACUGUCUUUUUCAU 20 AsCpf1 558 B2M-c172 UUCUCCACUGUCUUUUUCAUA 21 AsCpf1 559 B2M-c173 UUCUCCACUGUCUUUUUCAUAG 22 AsCpf1 560 B2M-c174 UUCUCCACUGUCUUUUUCAUAGA 23 AsCpf1 561 B2M-c175 UUCUCCACUGUCUUUUUCAUAGAU 24 AsCpf1 562 B2M-c176 UUUCUCCACUGUCUUUUU 18 AsCpf1 563 B2M-c177 UUUCUCCACUGUCUUUUUC 19 AsCpf1 564 B2M-c178 UUUCUCCACUGUCUUUUUCA 20 AsCpf1 565 B2M-c179 UUUCUCCACUGUCUUUUUCAU 21 AsCpf1 566 B2M-c180 UUUCUCCACUGUCUUUUUCAUA 22 AsCpf1 567 B2M-c181 UUUCUCCACUGUCUUUUUCAUAG 23 AsCpf1 568 B2M-c182 UUUCUCCACUGUCUUUUUCAUAGA 24 AsCpf1 569 B2M-c183 UUUUCUCCACUGUCUUUU 18 AsCpf1 570 B2M-c184 UUUUCUCCACUGUCUUUUU 19 AsCpf1 571 B2M-c185 UUUUCUCCACUGUCUUUUUC 20 AsCpf1 572 B2M-c186 UUUUCUCCACUGUCUUUUUCA 21 AsCpf1 573 B2M-c187 UUUUCUCCACUGUCUUUUUCAU 22 AsCpf1 574 B2M-c188 UUUUCUCCACUGUCUUUUUCAUA 23 AsCpf1 575 B2M-c189 UUUUCUCCACUGUCUUUUUCAUAG 24 AsCpf1 576

[0243] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting PD1. gRNAs targeting B2M and PD1 for use in the disclosure are further described in WO2015161276 and WO2017152015 by Welstead et al.; both incorporated in their entirety herein by reference.

[0244] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting NKG2A (NKG2A gRNA). In some embodiments, the gRNA targeting NKG2A is one or more of the gRNAs described in Table 7.

TABLE-US-00007 TABLE 7 Exemplary NKG2A gRNAs SEQ gRNA Targeting Domain ID Name Sequence (DNA) Length Enzyme NO: NKG2A55 GAGGTAAAGCGTTTGCATTTG 21 AsCpf1 577 NKG2A56 CCTCTAAAGCTTATGCTTACA 21 AsCpf1 578 NKG2A57 AGTCGATTTACTTGTAGCACT 21 AsCpf1 579 NKG2A58 CTTGTAGCACTGCACAGTTAA 21 AsCpf1 580 NKG2A59 TCCATTACAGGATAAAAGACT 21 AsCpf1 581 NKG2A60 CTCCATTACAGGATAAAAGAC 21 AsCpf1 582 NKG2A61 TCTCCATTACAGGATAAAAGA 21 AsCpf1 583 NKG2A62 ATCCTGTAATGGAGAAAAATC 21 AsCpf1 584 NKG2A63 TCCTGTAATGGAGAAAAATCC 21 AsCpf1 585 NKG2A136 AAACATGAGTAAGTTGTTTTG 21 AsCpf1 586 NKG2A137 GCTTTCAAACATGAGTAAGTT 21 AsCpf1 587 NKG2A138 AAAGCCAAACCATTCATTGTC 21 AsCpf1 588 NKG2A139 GTAACAGCAGTCATCATCCAT 21 AsCpf1 589 NKG2A140 ACCATCCTCATGGATTGGTGT 21 AsCpf1 590 NKG2A141 TGTCCATCATTTCACCATCCT 21 AsCpf1 591 NKG2A142 GAAATTTCTGTCCATCATTTC 21 AsCpf1 592 NKG2A143 AGAAATTTCTGTCCATCATTT 21 AsCpf1 593 NKG2A144 TTTTAGAAATTTCTGTCCATC 21 AsCpf1 594 NKG2A145 CTTTTAGAAATTTCTGTCCAT 21 AsCpf1 595 NKG2A146 TTTTCTTTTAGAAATTTCTGT 21 AsCpf1 596 NKG2A147 TAAAAGAAAAGAAAGAATTTT 21 AsCpf1 597 NKG2A270 AAACATTTACATCTTACCATT 21 AsCpf1 598 NKG2A271 CATCTTACCATTTCTTCTTCA 21 AsCpf1 599 NKG2A272 TATAGATAATGAAGAAGAAAT 21 AsCpf1 600 NKG2A273 TTCTTCATTATCTATAGAAAG 21 AsCpf1 601 NKG2A274 CTGGCCTGTACTTCGAAGAAC 21 AsCpf1 602 NKG2A275 CTTACCAATGTAGTAACAACT 21 AsCpf1 603 NKG2A276 GCACGTCATTGTGGCCATTGT 21 AsCpf1 604 NKG2A277 TTTAGCACGTCATTGTGGCCA 21 AsCpf1 605 NKG2A414 CCATCAGCTCCAGAGAAGCTC 21 AsCpf1 606 NKG2A415 TCTCCCTGCAGATTTACCATC 21 AsCpf1 607 NKG2A437 AAATGCTTTACCTTTGCAGTG 21 AsCpf1 608 NKG2A438 AATGCTTTACCTTTGCAGTGA 21 AsCpf1 609 NKG2A439 CCTTTGCAGTGATAGGTTTTG 21 AsCpf1 610 NKG2A440 CAGTGATAGGTTTTGTCATTC 21 AsCpf1 611 NKG2A441 AAGGGAATGACAAAACCTATC 21 AsCpf1 612 NKG2A442 CAAGGGAATGACAAAACCTAT 21 AsCpf1 613 NKG2A443 GTCATTCCCTTGAAAATCCTG 21 AsCpf1 614 NKG2A444 TCATTCCCTTGAAAATCCTGA 21 AsCpf1 615 NKG2A445 TGAAGGTTTAATTCCGCATAG 21 AsCpf1 616 NKG2A446 GAAGGTTTAATTCCGCATAGG 21 AsCpf1 617 NKG2A447 AAGGTTTAATTCCGCATAGGT 21 AsCpf1 618 NKG2A448 ATTCCGCATAGGTTATTTCCT 21 AsCpf1 619 NKG2A449 GCAACTGAACAGGAAATAACC 21 AsCpf1 620 NKG2A450 AGCAACTGAACAGGAAATAAC 21 AsCpf1 621 NKG2A451 CTGTTCAGTTGCTAAAATGGA 21 AsCpf1 622 NKG2A452 TATTGCCTTTAGGTTTTCGTT 21 AsCpf1 623 NKG2A453 ATTGCCTTTAGGTTTTCGTTG 21 AsCpf1 624 NKG2A454 TTGCCTTTAGGTTTTCGTTGC 21 AsCpf1 625 NKG2A455 GGTTTTCGTTGCTGCCTCTTT 21 AsCpf1 626 NKG2A456 CGTTGCTGCCTCTTTGGGTTT 21 AsCpf1 627 NKG2A457 GTTGCTGCCTCTTTGGGTTTG 21 AsCpf1 628 NKG2A458 GGTTTGGGGGCAGATTCAGGT 21 AsCpf1 629 NKG2A459 GGGGCAGATTCAGGTCTGAGT 21 AsCpf1 630 NKG2A460 GCAACTGAACAGGAAATAACC 21 Cas12a 1176

[0245] In some embodiments, the gRNA for use in the disclosure is a gRNA targeting TIGIT (TIGIT gRNA). In some embodiments, the gRNA targeting TIGIT is one or more of the gRNAs described in Table 8.

TABLE-US-00008 TABLE 8 TIGIT gRNAs gRNA Targeting Domain SEQ ID Name Sequence (DNA) Length Enzyme NO: TIGIT4170 TCTGCAGAAATGTTCCCCGT 20 AsCpf1 631 TIGIT4171 TGCAGAGAAAGGTGGCTCTA 20 AsCpf1 632 TIGIT4172 TAATGCTGACTTGGGGTGGC 20 AsCpf1 633 TIGIT4173 TAGGACCTCCAGGAAGATTC 20 AsCpf1 634 TIGIT4174 TAGTCAACGCGACCACCACG 20 AsCpf1 635 TIGIT4175 TCCTGAGGTCACCTTCCACA 20 AsCpf1 636 TIGIT4176 TATTGTGCCTGTCATCATTC 20 AsCpf1 637 TIGIT4177 TGACAGGCACAATAGAAACAA 21 SauCas9 638 TIGIT4178 GACAGGCACAATAGAAACAAC 21 SauCas9 639 TIGIT4179 AAACAACGGGGAACATTTCTG 21 SauCas9 640 TIGIT4180 ACAACGGGGAACATTTCTGCA 21 SauCas9 641 TIGIT4181 TGATAGAGCCACCTTTCTCTG 21 SauCas9 642 TIGIT4182 GGGTCACTTGTGCCGTGGTGG 21 SauCas9 643 TIGIT4183 GGCACAAGTGACCCAGGTCAA 21 SauCas9 644 TIGIT4184 GTCCTGCTGCTCCCAGTTGAC 21 SauCas9 645 TIGIT4185 TGGCCATTTGTAATGCTGACT 21 SauCas9 646 TIGIT4186 TGGCACATCTCCCCATCCTTC 21 SauCas9 647 TIGIT4187 CATCTCCCCATCCTTCAAGGA 21 SauCas9 648 TIGIT4188 CCACTCGATCCTTGAAGGATG 21 SauCas9 649 TIGIT4189 GGCCACTCGATCCTTGAAGGA 21 SauCas9 650 TIGIT4190 CCTGGGGCCACTCGATCCTTG 21 SauCas9 651 TIGIT4191 GACTGGAGGGTGAGGCCCAGG 21 SauCas9 652 TIGIT4192 ATCGTTCACGGTCAGCGACTG 21 SauCas9 653 TIGIT4193 GTCGCTGACCGTGAACGATAC 21 SauCas9 654 TIGIT4194 CGCTGACCGTGAACGATACAG 21 SauCas9 655 TIGIT4195 GCATCTATCACACCTACCCTG 21 SauCas9 656 TIGIT4196 CCTACCCTGATGGGACGTACA 21 SauCas9 657 TIGIT4197 TACCCTGATGGGACGTACACT 21 SauCas9 658 TIGIT4198 CCCTGATGGGACGTACACTGG 21 SauCas9 659 TIGIT4199 TTCTCCCAGTGTACGTCCCAT 21 SauCas9 660 TIGIT4200 GGAGAATCTTCCTGGAGGTCC 21 SauCas9 661 TIGIT4201 CATGGCTCCAAGCAATGGAAT 21 SauCas9 662 TIGIT4202 CGCGGCCATGGCTCCAAGCAA 21 SauCas9 663 TIGIT4203 TCGCGGCCATGGCTCCAAGCA 21 SauCas9 664 TIGIT4204 CATCGTGGTGGTCGCGTTGAC 21 SauCas9 665 TIGIT4205 AAAGCCCTCAGAATCCATTCT 21 SauCas9 666 TIGIT4206 CATTCTGTGGAAGGTGACCTC 21 SauCas9 667 TIGIT4207 TTCTGTGGAAGGTGACCTCAG 21 SauCas9 668 TIGIT4208 CCTGAGGTCACCTTCCACAGA 21 SauCas9 669 TIGIT4209 TTCTCCTGAGGTCACCTTCCA 21 SauCas9 670 TIGIT4210 AGGAGAAAATCAGCTGGACAG 21 SauCas9 671 TIGIT4211 GGAGAAAATCAGCTGGACAGG 21 SauCas9 672 TIGIT4212 GCCCCAGTGCTCCCTCACCCC 21 SauCas9 673 TIGIT4213 TGGACACAGCTTCCTGGGGGT 21 SauCas9 674 TIGIT4214 TCTGCCTGGACACAGCTTCCT 21 SauCas9 675 TIGIT4215 AGCTGCACCTGCTGGGCTCTG 21 SauCas9 676 TIGIT4216 GCTGGGCTCTGTGGAGAGCAG 21 SauCas9 677 TIGIT4217 TGGGCTCTGTGGAGAGCAGCG 21 SauCas9 678 TIGIT4218 CTGCATGACTACTTCAATGTC 21 SauCas9 679 TIGIT4219 AATGTCCTGAGTTACAGAAGC 21 SauCas9 680 TIGIT4220 TGGGTAACTGCAGCTTCTTCA 21 SauCas9 681 TIGIT4221 GACAGGCACAATAGAAACAA 20 SpyCas9 682 TIGIT4222 ACAGGCACAATAGAAACAAC 20 SpyCas9 683 TIGIT4223 CAGGCACAATAGAAACAACG 20 SpyCas9 684 TIGIT4224 GGGAACATTTCTGCAGAGAA 20 SpyCas9 685 TIGIT4225 AACATTTCTGCAGAGAAAGG 20 SpyCas9 686 TIGIT4226 ATGTCACCTCTCCTCCACCA 20 SpyCas9 687 TIGIT4227 CTTGTGCCGTGGTGGAGGAG 20 SpyCas9 688 TIGIT4228 GGTCACTTGTGCCGTGGTGG 20 SpyCas9 689 TIGIT4229 CACCACGGCACAAGTGACCC 20 SpyCas9 690 TIGIT4230 CTGGGTCACTTGTGCCGTGG 20 SpyCas9 691 TIGIT4231 GACCTGGGTCACTTGTGCCG 20 SpyCas9 692 TIGIT4232 CACAAGTGACCCAGGTCAAC 20 SpyCas9 693 TIGIT4233 ACAAGTGACCCAGGTCAACT 20 SpyCas9 694 TIGIT4234 CCAGGTCAACTGGGAGCAGC 20 SpyCas9 695 TIGIT4235 CTGCTGCTCCCAGTTGACCT 20 SpyCas9 696 TIGIT4236 CCTGCTGCTCCCAGTTGACC 20 SpyCas9 697 TIGIT4237 GGAGCAGCAGGACCAGCTTC 20 SpyCas9 698 TIGIT4238 CATTACAAATGGCCAGAAGC 20 SpyCas9 699 TIGIT4239 GGCCATTTGTAATGCTGACT 20 SpyCas9 700 TIGIT4240 GCCATTTGTAATGCTGACTT 20 SpyCas9 701 TIGIT4241 CCATTTGTAATGCTGACTTG 20 SpyCas9 702 TIGIT4242 TTTGTAATGCTGACTTGGGG 20 SpyCas9 703 TIGIT4243 CCCCAAGTCAGCATTACAAA 20 SpyCas9 704 TIGIT4244 GCACATCTCCCCATCCTTCA 20 SpyCas9 705 TIGIT4245 CCCATCCTTCAAGGATCGAG 20 SpyCas9 706 TIGIT4246 CACTCGATCCTTGAAGGATG 20 SpyCas9 707 TIGIT4247 CCACTCGATCCTTGAAGGAT 20 SpyCas9 708 TIGIT4248 GCCACTCGATCCTTGAAGGA 20 SpyCas9 709 TIGIT4249 TTCAAGGATCGAGTGGCCCC 20 SpyCas9 710 TIGIT4250 TGGGGCCACTCGATCCTTGA 20 SpyCas9 711 TIGIT4251 GATCGAGTGGCCCCAGGTCC 20 SpyCas9 712 TIGIT4252 AGTGGCCCCAGGTCCCGGCC 20 SpyCas9 713 TIGIT4253 GTGGCCCCAGGTCCCGGCCT 20 SpyCas9 714 TIGIT4254 GAGGCCCAGGCCGGGACCTG 20 SpyCas9 715 TIGIT4255 TGAGGCCCAGGCCGGGACCT 20 SpyCas9 716 TIGIT4256 GTGAGGCCCAGGCCGGGACC 20 SpyCas9 717 TIGIT4257 TGGAGGGTGAGGCCCAGGCC 20 SpyCas9 718 TIGIT4258 CTGGAGGGTGAGGCCCAGGC 20 SpyCas9 719 TIGIT4259 GCGACTGGAGGGTGAGGCCC 20 SpyCas9 720 TIGIT4260 CGGTCAGCGACTGGAGGGTG 20 SpyCas9 721 TIGIT4261 GTTCACGGTCAGCGACTGGA 20 SpyCas9 722 TIGIT4262 CGTTCACGGTCAGCGACTGG 20 SpyCas9 723 TIGIT4263 TATCGTTCACGGTCAGCGAC 20 SpyCas9 724 TIGIT4264 TCGCTGACCGTGAACGATAC 20 SpyCas9 725 TIGIT4265 CGCTGACCGTGAACGATACA 20 SpyCas9 726 TIGIT4266 GCTGACCGTGAACGATACAG 20 SpyCas9 727 TIGIT4267 GTACTCCCCTGTATCGTTCA 20 SpyCas9 728 TIGIT4268 ATCTATCACACCTACCCTGA 20 SpyCas9 729 TIGIT4269 TCTATCACACCTACCCTGAT 20 SpyCas9 730 TIGIT4270 TACCCTGATGGGACGTACAC 20 SpyCas9 731 TIGIT4271 ACCCTGATGGGACGTACACT 20 SpyCas9 732 TIGIT4272 AGTGTACGTCCCATCAGGGT 20 SpyCas9 733 TIGIT4273 TCCCAGTGTACGTCCCATCA 20 SpyCas9 734 TIGIT4274 CTCCCAGTGTACGTCCCATC 20 SpyCas9 735 TIGIT4275 GTACACTGGGAGAATCTTCC 20 SpyCas9 736 TIGIT4276 CACTGGGAGAATCTTCCTGG 20 SpyCas9 737 TIGIT4277 CTGAGCTTTCTAGGACCTCC 20 SpyCas9 738 TIGIT4278 AGGTTCCAGATTCCATTGCT 20 SpyCas9 739 TIGIT4279 AAGCAATGGAATCTGGAACC 20 SpyCas9 740 TIGIT4280 GATTCCATTGCTTGGAGCCA 20 SpyCas9 741 TIGIT4281 TGGCTCCAAGCAATGGAATC 20 SpyCas9 742 TIGIT4282 GCGGCCATGGCTCCAAGCAA 20 SpyCas9 743 TIGIT4283 TGGAGCCATGGCCGCGACGC 20 SpyCas9 744 TIGIT4284 AGCCATGGCCGCGACGCTGG 20 SpyCas9 745 TIGIT4285 GACCACCAGCGTCGCGGCCA 20 SpyCas9 746 TIGIT4286 GCAGATGACCACCAGCGTCG 20 SpyCas9 747 TIGIT4287 CATCTGCACAGCAGTCATCG 20 SpyCas9 748 TIGIT4288 CTGCACAGCAGTCATCGTGG 20 SpyCas9 749 TIGIT4289 AGCCCTCAGAATCCATTCTG 20 SpyCas9 750 TIGIT4290 CTCAGAATCCATTCTGTGGA 20 SpyCas9 751 TIGIT4291 TTCCACAGAATGGATTCTGA 20 SpyCas9 752 TIGIT4292 CTTCCACAGAATGGATTCTG 20 SpyCas9 753 TIGIT4293 ATTCTGTGGAAGGTGACCTC 20 SpyCas9 754 TIGIT4294 TGAGGTCACCTTCCACAGAA 20 SpyCas9 755 TIGIT4295 GACCTCAGGAGAAAATCAGC 20 SpyCas9 756 TIGIT4296 CAGGAGAAAATCAGCTGGAC 20 SpyCas9 757 TIGIT4297 GTCCAGCTGATTTTCTCCTG 20 SpyCas9 758 TIGIT4298 GAGAAAATCAGCTGGACAGG 20 SpyCas9 759 TIGIT4299 AATCAGCTGGACAGGAGGAA 20 SpyCas9 760 TIGIT4300 CCCAGTGCTCCCTCACCCCC 20 SpyCas9 761 TIGIT4301 CTGGGGGTGAGGGAGCACTG 20 SpyCas9 762 TIGIT4302 CCTGGGGGTGAGGGAGCACT 20 SpyCas9 763 TIGIT4303 TCCTGGGGGTGAGGGAGCAC 20 SpyCas9 764 TIGIT4304 ACACAGCTTCCTGGGGGTGA 20 SpyCas9 765 TIGIT4305 GACACAGCTTCCTGGGGGTG 20 SpyCas9 766 TIGIT4306 ACCCCCAGGAAGCTGTGTCC 20 SpyCas9 767 TIGIT4307 GCCTGGACACAGCTTCCTGG 20 SpyCas9 768 TIGIT4308 TGCCTGGACACAGCTTCCTG 20 SpyCas9 769 TIGIT4309 CTGCCTGGACACAGCTTCCT 20 SpyCas9 770 TIGIT4310 TCTGCCTGGACACAGCTTCC 20 SpyCas9 771 TIGIT4311 CAGGCAGAAGCTGCACCTGC 20 SpyCas9 772 TIGIT4312 AGGCAGAAGCTGCACCTGCT 20 SpyCas9 773 TIGIT4313 CAGCAGGTGCAGCTTCTGCC 20 SpyCas9 774 TIGIT4314 GCTGCACCTGCTGGGCTCTG 20 SpyCas9 775 TIGIT4315 TGCTCTCCACAGAGCCCAGC 20 SpyCas9 776 TIGIT4316 CTGGGCTCTGTGGAGAGCAG 20 SpyCas9 777 TIGIT4317 TGGGCTCTGTGGAGAGCAGC 20 SpyCas9 778 TIGIT4318 GGGCTCTGTGGAGAGCAGCG 20 SpyCas9 779 TIGIT4319 CTGTGGAGAGCAGCGGGGAG 20 SpyCas9 780 TIGIT4320 ATTGAAGTAGTCATGCAGCT 20 SpyCas9 781 TIGIT4321 TGTCCTGAGTTACAGAAGCC 20 SpyCas9 782 TIGIT4322 GTCCTGAGTTACAGAAGCCT 20 SpyCas9 783 TIGIT4323 TACCCAGGCTTCTGTAACTC 20 SpyCas9 784 TIGIT4324 TGAAGAAGCTGCAGTTACCC 20 SpyCas9 785 TIGIT4325 TGCAGCTTCTTCACAGAGAC 20 SpyCas9 786 TIGIT5053 GTTGTTTCTATTGTGCCTGT 20 AsCpf1 RR 787 TIGIT5054 CGTTGTTTCTATTGTGCCTG 20 AsCpf1 RR 788 TIGIT5055 CCGTTGTTTCTATTGTGCCT 20 AsCpf1 RR 789 TIGIT5056 CCACGGCACAAGTGACCCAG 20 AsCpf1 RR 790 TIGIT5057 AGTTGACCTGGGTCACTTGT 20 AsCpf1 RR 791 TIGIT5058 AAGTCAGCATTACAAATGGC 20 AsCpf1 RR 792 TIGIT5059 CATCCTTCAAGGATCGAGTG 20 AsCpf1 RR 793 TIGIT5060 ATCCTTCAAGGATCGAGTGG 20 AsCpf1 RR 794 TIGIT5061 AGGATCGAGTGGCCCCAGGT 20 AsCpf1 RR 795 TIGIT5062 AGGTCCCGGCCTGGGCCTCA 20 AsCpf1 RR 796 TIGIT5063 GGCCTGGGCCTCACCCTCCA 20 AsCpf1 RR 797 TIGIT5064 CGGTCAGCGACTGGAGGGTG 20 AsCpf1 RR 798 TIGIT5065 GTCGCTGACCGTGAACGATA 20 AsCpf1 RR 799 TIGIT5066 TGTATCGTTCACGGTCAGCG 20 AsCpf1 RR 800 TIGIT5067 CTGTATCGTTCACGGTCAGC 20 AsCpf1 RR 801 TIGIT5068 ATCAGGGTAGGTGTGATAGA 20 AsCpf1 RR 802 TIGIT5069 AGTGTACGTCCCATCAGGGT 20 AsCpf1 RR 803 TIGIT5070 GGAAGATTCTCCCAGTGTAC 20 AsCpf1 RR 804 TIGIT5071 TGGAGGTCCTAGAAAGCTCA 20 AsCpf1 RR 805 TIGIT5072 AGCAATGGAATCTGGAACCT 20 AsCpf1 RR 806 TIGIT5073 AGATTCCATTGCTTGGAGCC 20 AsCpf1 RR 807 TIGIT5074 GATTCCATTGCTTGGAGCCA 20 AsCpf1 RR 808 TIGIT5075 ATTGCTTGGAGCCATGGCCG 20 AsCpf1 RR 809 TIGIT5076 TTGCTTGGAGCCATGGCCGC 20 AsCpf1 RR 810 TIGIT5077 CAGAATGGATTCTGAGGGCT 20 AsCpf1 RR 811 TIGIT5078 ACAGAATGGATTCTGAGGGC 20 AsCpf1 RR 812 TIGIT5079 TTCTGTGGAAGGTGACCTCA 20 AsCpf1 RR 813 TIGIT5080 GCTGATTTTCTCCTGAGGTC 20 AsCpf1 RR 814 TIGIT5081 TCCTGTCCAGCTGATTTTCT 20 AsCpf1 RR 815 TIGIT5082 TTCCTCCTGTCCAGCTGATT 20 AsCpf1 RR 816 TIGIT5083 TGGGGGTGAGGGAGCACTGG 20 AsCpf1 RR 817 TIGIT5084 AGTGCTCCCTCACCCCCAGG 20 AsCpf1 RR 818 TIGIT5085 TCACCCCCAGGAAGCTGTGT 20 AsCpf1 RR 819 TIGIT5086 CAGGAAGCTGTGTCCAGGCA 20 AsCpf1 RR 820 TIGIT5087 AGGAAGCTGTGTCCAGGCAG 20 AsCpf1 RR 821 TIGIT5088 GGCAGAAGCTGCACCTGCTG 20 AsCpf1 RR 822 TIGIT5089 CAGAGCCCAGCAGGTGCAGC 20 AsCpf1 RR 823 TIGIT5090 GCTGCTCTCCACAGAGCCCA 20 AsCpf1 RR 824 TIGIT5091 CGCTGCTCTCCACAGAGCCC 20 AsCpf1 RR 825 TIGIT5092 ATGTCCTGAGTTACAGAAGC 20 AsCpf1 RR 826 TIGIT5093 TGCAGAGAAAGGTGGCTCTAT 21 Cas12a 1175

[0246] In some embodiments the gRNA for use in the disclosure is a gRNA targeting ADORA2a (ADORA2a gRNA). In some embodiments, the gRNA targeting ADORA2a is one or more of the gRNAs described in Table 9.

TABLE-US-00009 TABLE 9 ADORA2a gRNAs gRNA Targeting Domain SEQ ID Name Sequence (DNA) Length Enzyme NO: ADORA2A337 GAGCACACCCACTGCGATGT 20 SpyCas9 827 ADORA2A338 GATGGCCAGGAGACTGAAGA 20 SpyCas9 828 ADORA2A339 CTGCTCACCGGAGCGGGATG 20 SpyCas9 829 ADORA2A340 GTCTGTGGCCATGCCCATCA 20 SpyCas9 830 ADORA2A341 TCACCGGAGCGGGATGCGGA 20 SpyCas9 831 ADORA2A342 GTGGCAGGCAGCGCAGAACC 20 SpyCas9 832 ADORA2A343 AGCACACCAGCACATTGCCC 20 SpyCas9 833 ADORA2A344 CAGGTTGCTGTTGAGCCACA 20 SpyCas9 834 ADORA2A345 CTTCATTGCCTGCTTCGTCC 20 SpyCas9 835 ADORA2A346 GTACACCGAGGAGCCCATGA 20 SpyCas9 836 ADORA2A347 GATGGCAATGTAGCGGTCAA 20 SpyCas9 837 ADORA2A348 CTCCTCGGTGTACATCACGG 20 SpyCas9 838 ADORA2A349 CGAGGAGCCCATGATGGGCA 20 SpyCas9 839 ADORA2A350 GGGCTCCTCGGTGTACATCA 20 SpyCas9 840 ADORA2A351 CTTTGTGGTGTCACTGGCGG 20 SpyCas9 841 ADORA2A352 CCGCTCCGGTGAGCAGGGCC 20 SpyCas9 842 ADORA2A353 GGGTTCTGCGCTGCCTGCCA 20 SpyCas9 843 ADORA2A354 GGACGAAGCAGGCAATGAAG 20 SpyCas9 844 ADORA2A355 GTGCTGATGGTGATGGCAAA 20 SpyCas9 845 ADORA2A356 AGCGCAGAACCCGGTGCTGA 20 SpyCas9 846 ADORA2A357 GAGCTCCATCTTCAGTCTCC 20 SpyCas9 847 ADORA2A358 TGCTGATGGTGATGGCAAAG 20 SpyCas9 848 ADORA2A359 GGCGGCGGCCGACATCGCAG 20 SpyCas9 849 ADORA2A360 AATGAAGAGGCAGCCGTGGC 20 SpyCas9 850 ADORA2A361 GGGCAATGTGCTGGTGTGCT 20 SpyCas9 851 ADORA2A362 CATGCCCATCATGGGCTCCT 20 SpyCas9 852 ADORA2A363 AATGTAGCGGTCAATGGCGA 20 SpyCas9 853 ADORA2A364 AGTAGTTGGTGACGTTCTGC 20 SpyCas9 854 ADORA2A365 AGCGGTCAATGGCGATGGCC 20 SpyCas9 855 ADORA2A366 CGCATCCCGCTCCGGTGAGC 20 SpyCas9 856 ADORA2A367 GCATCCCGCTCCGGTGAGCA 20 SpyCas9 857 ADORA2A368 TGGGCAATGTGCTGGTGTGC 20 SpyCas9 858 ADORA2A369 CAACTACTTTGTGGTGTCAC 20 SpyCas9 859 ADORA2A370 CGCTCCGGTGAGCAGGGCCG 20 SpyCas9 860 ADORA2A371 GATGGTGATGGCAAAGGGGA 20 SpyCas9 861 ADORA2A372 GGTGTACATCACGGTGGAGC 20 SpyCas9 862 ADORA2A373 GAACGTCACCAACTACTTTG 20 SpyCas9 863 ADORA2A374 CAGTGACACCACAAAGTAGT 20 SpyCas9 864 ADORA2A375 GGCCATCCTGGGCAATGTGC 20 SpyCas9 865 ADORA2A376 CCCGGCCCTGCTCACCGGAG 20 SpyCas9 866 ADORA2A377 CACCAGCACATTGCCCAGGA 20 SpyCas9 867 ADORA2A378 TTTGCCATCACCATCAGCAC 20 SpyCas9 868 ADORA2A379 CTCCACCGTGATGTACACCG 20 SpyCas9 869 ADORA2A380 GGAGCTGGCCATTGCTGTGC 20 SpyCas9 870 ADORA2A381 CAGGATGGCCAGCACAGCAA 20 SpyCas9 871 ADORA2A382 GAACCCGGTGCTGATGGTGA 20 SpyCas9 872 ADORA2A383 TGGAGCTCTGCGTGAGGACC 20 SpyCas9 873 ADORA2A384 CCCGCTCCGGTGAGCAGGGC 20 SpyCas9 874 ADORA2A385 AGGCAATGAAGAGGCAGCCG 20 SpyCas9 875 ADORA2A386 CCGGCCCTGCTCACCGGAGC 20 SpyCas9 876 ADORA2A387 GCGGCGGCCGACATCGCAGT 20 SpyCas9 877 ADORA2A388 GGTGCTGATGGTGATGGCAA 20 SpyCas9 878 ADORA2A389 CTACTTTGTGGTGTCACTGG 20 SpyCas9 879 ADORA2A390 TACACCGAGGAGCCCATGAT 20 SpyCas9 880 ADORA2A391 TCTGTGGCCATGCCCATCAT 20 SpyCas9 881 ADORA2A392 ATTGCTGTGCTGGCCATCCT 20 SpyCas9 882 ADORA2A393 CGTGAGGACCAGGACGAAGC 20 SpyCas9 883 ADORA2A394 TTGCCATCACCATCAGCACC 20 SpyCas9 884 ADORA2A395 GGATGCGGATGGCAATGTAG 20 SpyCas9 885 ADORA2A396 TTGCCATCCGCATCCCGCTC 20 SpyCas9 886 ADORA2A397 TGAAGATGGAGCTCTGCGTG 20 SpyCas9 887 ADORA2A398 CATTGCTGTGCTGGCCATCC 20 SpyCas9 888 ADORA2A399 TGCTGGTGTGCTGGGCCGTG 20 SpyCas9 889 ADORA2A820 GGCTCCTCGGTGTACATCACG 21 SauCas9 890 ADORA2A821 GAGCTCTGCGTGAGGACCAGG 21 SauCas9 891 ADORA2A822 GATGGAGCTCTGCGTGAGGAC 21 SauCas9 892 ADORA2A823 CCAGCACACCAGCACATTGCC 21 SauCas9 893 ADORA2A824 AGGACCAGGACGAAGCAGGCA 21 SauCas9 894 ADORA2A825 TGCCATCCGCATCCCGCTCCG 21 SauCas9 895 ADORA2A826 GTGTGGCTCAACAGCAACCTG 21 SauCas9 896 ADORA2A827 AGCTCCACCGTGATGTACACC 21 SauCas9 897 ADORA2A828 GTAGCGGTCAATGGCGATGGC 21 SauCas9 898 ADORA2A829 CGGTGCTGATGGTGATGGCAA 21 SauCas9 899 ADORA2A830 CCCTGCTCACCGGAGCGGGAT 21 SauCas9 900 ADORA2A831 GTGACGTTCTGCAGGTTGCTG 21 SauCas9 901 ADORA2A832 GCTCCACCGTGATGTACACCG 21 SauCas9 902 ADORA2A833 ACTGAAGATGGAGCTCTGCGT 21 SauCas9 903 ADORA2A834 CCAGCTCCACCGTGATGTACA 21 SauCas9 904 ADORA2A835 CCTTTGCCATCACCATCAGCA 21 SauCas9 905 ADORA2A836 CCGGTGCTGATGGTGATGGCA 21 SauCas9 906 ADORA2A837 CCTGGGCAATGTGCTGGTGTG 21 SauCas9 907 ADORA2A838 AGGCAGCCGTGGCAGGCAGCG 21 SauCas9 908 ADORA2A839 GCGATGGCCAGGAGACTGAAG 21 SauCas9 909 ADORA2A840 CGATGGCCAGGAGACTGAAGA 21 SauCas9 910 ADORA2A841 TCCCGCTCCGGTGAGCAGGGC 21 SauCas9 911 ADORA2A842 TGCTTCGTCCTGGTCCTCACG 21 SauCas9 912 ADORA2A843 ACCAGGACGAAGCAGGCAATG 21 SauCas9 913 ADORA2A844 ATGTACACCGAGGAGCCCATG 21 SauCas9 914 ADORA2A845 TCGTCTGTGGCCATGCCCATC 21 SauCas9 915 ADORA2A846 TCAATGGCGATGGCCAGGAGA 21 SauCas9 916 ADORA2A847 GGTGCTGATGGTGATGGCAAA 21 SauCas9 917 ADORA2A848 TAGCGGTCAATGGCGATGGCC 21 SauCas9 918 ADORA2A849 TCCGCATCCCGCTCCGGTGAG 21 SauCas9 919 ADORA2A850 CTGGCGGCGGCCGACATCGCA 21 SauCas9 920 ADORA2A851 GCCATTGCTGTGCTGGCCATC 21 SauCas9 921 ADORA2A852 ATCCCGCTCCGGTGAGCAGGG 21 SauCas9 922 ADORA2A853 AGACTGAAGATGGAGCTCTGC 21 SauCas9 923 ADORA2A854 CCCCGGCCCTGCTCACCGGAG 21 SauCas9 924 ADORA2A855 ATGGTGATGGCAAAGGGGATG 21 SauCas9 925 ADORA2A856 GCTCCTCGGTGTACATCACGG 21 SauCas9 926 ADORA2A248 TGTCGATGGCAATAGCCAAG 20 SpyCas9 927 ADORA2A249 AGAAGTTGGTGACGTTCTGC 20 SpyCas9 928 ADORA2A250 TTCGCCATCACCATCAGCAC 20 SpyCas9 929 ADORA2A251 GAAGAAGAGGCAGCCATGGC 20 SpyCas9 930 ADORA2A252 CACAAGCACGTTACCCAGGA 20 SpyCas9 931 ADORA2A253 CAACTTCTTCGTGGTATCTC 20 SpyCas9 932 ADORA2A254 CAGGATGGCCAGCACAGCAA 20 SpyCas9 933 ADORA2A255 AATTCCACTCCGGTGAGCCA 20 SpyCas9 934 ADORA2A256 AGCGCAGAAGCCAGTGCTGA 20 SpyCas9 935 ADORA2A257 GTGCTGATGGTGATGGCGAA 20 SpyCas9 936 ADORA2A258 GGAGCTGGCCATTGCTGTGC 20 SpyCas9 937 ADORA2A259 AATAGCCAAGAGGCTGAAGA 20 SpyCas9 938 ADORA2A260 CTCCTCGGTGTACATCATGG 20 SpyCas9 939 ADORA2A261 GGACAAAGCAGGCGAAGAAG 20 SpyCas9 940 ADORA2A262 TCTGGCGGCGGCTGACATCG 20 SpyCas9 941 ADORA2A263 TGGGTAACGTGCTTGTGTGC 20 SpyCas9 942 ADORA2A264 GATGTACACCGAGGAGCCCA 20 SpyCas9 943 ADORA2A265 TAACCCCTGGCTCACCGGAG 20 SpyCas9 944 ADORA2A266 TCACCGGAGTGGAATTCGGA 20 SpyCas9 945 ADORA2A267 GCGGCGGCTGACATCGCGGT 20 SpyCas9 946 ADORA2A268 GATGGTGATGGCGAATGGGA 20 SpyCas9 947 ADORA2A269 GGCTTCTGCGCTGCCTGCCA 20 SpyCas9 948 ADORA2A270 ATTCCACTCCGGTGAGCCAG 20 SpyCas9 949 ADORA2A271 GGTGTACATCATGGTGGAGC 20 SpyCas9 950 ADORA2A272 ATTGCTGTGCTGGCCATCCT 20 SpyCas9 951 ADORA2A273 CTCCACCATGATGTACACCG 20 SpyCas9 952 ADORA2A274 GGCGGCGGCTGACATCGCGG 20 SpyCas9 953 ADORA2A275 TACACCGAGGAGCCCATGGC 20 SpyCas9 954 ADORA2A276 GGGTAACGTGCTTGTGTGCT 20 SpyCas9 955 ADORA2A277 CAGGTTGCTGTTGATCCACA 20 SpyCas9 956 ADORA2A278 TGAAGATGGAACTCTGCGTG 20 SpyCas9 957 ADORA2A279 GATGGCGATGTATCTGTCGA 20 SpyCas9 958 ADORA2A280 CTTCTTCGCCTGCTTTGTCC 20 SpyCas9 959 ADORA2A281 AGGCGAAGAAGAGGCAGCCA 20 SpyCas9 960 ADORA2A282 TGCTTGTGTGCTGGGCCGTG 20 SpyCas9 961 ADORA2A283 GAAGCCAGTGCTGATGGTGA 20 SpyCas9 962 ADORA2A284 CGTGAGGACCAGGACAAAGC 20 SpyCas9 963 ADORA2A285 TGGAACTCTGCGTGAGGACC 20 SpyCas9 964 ADORA2A286 CATTGCTGTGCTGGCCATCC 20 SpyCas9 965 ADORA2A287 TTCTCCCGCCATGGGCTCCT 20 SpyCas9 966 ADORA2A288 TGGCTCACCGGAGTGGAATT 20 SpyCas9 967 ADORA2A289 TGCTGATGGTGATGGCGAAT 20 SpyCas9 968 ADORA2A290 CTTCGTGGTATCTCTGGCGG 20 SpyCas9 969 ADORA2A291 AGCACACAAGCACGTTACCC 20 SpyCas9 970 ADORA2A292 GGGCTCCTCGGTGTACATCA 20 SpyCas9 971 ADORA2A293 GTACACCGAGGAGCCCATGG 20 SpyCas9 972 ADORA2A294 GAACGTCACCAACTTCTTCG 20 SpyCas9 973 ADORA2A295 TCGCCATCCGAATTCCACTC 20 SpyCas9 974 ADORA2A296 GAGTTCCATCTTCAGCCTCT 20 SpyCas9 975 ADORA2A297 GAATTCCACTCCGGTGAGCC 20 SpyCas9 976 ADORA2A298 CAGAGATACCACGAAGAAGT 20 SpyCas9 977 ADORA2A299 CTTCTTCGTGGTATCTCTGG 20 SpyCas9 978 ADORA2A695 CAGTGCTGATGGTGATGGCGA 21 SauCas9 979 ADORA2A696 CGAATTCCACTCCGGTGAGCC 21 SauCas9 980 ADORA2A697 CCGAATTCCACTCCGGTGAGC 21 SauCas9 981 ADORA2A698 GCTGAAGATGGAACTCTGCGT 21 SauCas9 982 ADORA2A699 CGTGCTTGTGTGCTGGGCCGT 21 SauCas9 983 ADORA2A700 GTGAGGACCAGGACAAAGCAG 21 SauCas9 984 ADORA2A701 TCGATGGCAATAGCCAAGAGG 21 SauCas9 985 ADORA2A702 CATCGACAGATACATCGCCAT 21 SauCas9 986 ADORA2A703 GTACACCGAGGAGCCCATGGC 21 SauCas9 987 ADORA2A704 GCTCCACCATGATGTACACCG 21 SauCas9 988 ADORA2A705 AAGCCAGTGCTGATGGTGATG 21 SauCas9 989 ADORA2A706 CACCGCGATGTCAGCCGCCGC 21 SauCas9 990 ADORA2A707 AGGCTGAAGATGGAACTCTGC 21 SauCas9 991 ADORA2A708 GCCGCCGCCAGAGATACCACG 21 SauCas9 992 ADORA2A709 AGCTCCACCATGATGTACACC 21 SauCas9 993 ADORA2A710 AGGCAGCCATGGCAGGCAGCG 21 SauCas9 994 ADORA2A711 CCTGGCTCACCGGAGTGGAAT 21 SauCas9 995 ADORA2A712 CCAGCTCCACCATGATGTACA 21 SauCas9 996 ADORA2A713 ACCAGGACAAAGCAGGCGAAG 21 SauCas9 997 ADORA2A714 CCTGGGTAACGTGCTTGTGTG 21 SauCas9 998 ADORA2A715 AGGACCAGGACAAAGCAGGCG 21 SauCas9 999 ADORA2A716 TCAGCCGCCGCCAGAGATACC 21 SauCas9 1000 ADORA2A717 GGCTCCTCGGTGTACATCATG 21 SauCas9 1001 ADORA2A718 CTGGCGGCGGCTGACATCGCG 21 SauCas9 1002 ADORA2A719 GATGGAACTCTGCGTGAGGAC 21 SauCas9 1003 ADORA2A720 GCTCCTCGGTGTACATCATGG 21 SauCas9 1004 ADORA2A721 TGTACACCGAGGAGCCCATGG 21 SauCas9 1005 ADORA2A722 GCCATTGCTGTGCTGGCCATC 21 SauCas9 1006 ADORA2A723 CAATAGCCAAGAGGCTGAAGA 21 SauCas9 1007 ADORA2A724 ATGGTGATGGCGAATGGGATG 21 SauCas9 1008 ADORA2A725 ATGTACACCGAGGAGCCCATG 21 SauCas9 1009 ADORA2A726 GTGTGGATCAACAGCAACCTG 21 SauCas9 1010 ADORA2A727 TGCTTTGTCCTGGTCCTCACG 21 SauCas9 1011 ADORA2A728 GTAACCCCTGGCTCACCGGAG 21 SauCas9 1012 ADORA2A729 CCAGCACACAAGCACGTTACC 21 SauCas9 1013 ADORA2A730 TATCTGTCGATGGCAATAGCC 21 SauCas9 1014 ADORA2A731 GCAATAGCCAAGAGGCTGAAG 21 SauCas9 1015 ADORA2A732 AGTGCTGATGGTGATGGCGAA 21 SauCas9 1016 ADORA2A733 ACACCGAGGAGCCCATGGCGG 21 SauCas9 1017 ADORA2A734 CGCCATCCGAATTCCACTCCG 21 SauCas9 1018 ADORA2A4111 TGGTGTCACTGGCGGCGGCC 20 AsCpf1 1019 ADORA2A4112 CCATCACCATCAGCACCGGG 20 AsCpf1 1020 ADORA2A4113 CCATCGGCCTGACTCCCATG 20 AsCpf1 1021 ADORA2A4114 GCTGACCGCAGTTGTTCCAA 20 AsCpf1 1022 ADORA2A4115 AGGATGTGGTCCCCATGAAC 20 AsCpf1 1023 ADORA2A4116 CCTGTGTGCTGGTGCCCCTG 20 AsCpf1 1024 ADORA2A4117 CGGATCTTCCTGGCGGCGCG 20 AsCpf1 1025 ADORA2A4118 CCCTCTGCTGGCTGCCCCTA 20 AsCpf1 1026 ADORA2A4119 TTCTGCCCCGACTGCAGCCA 20 AsCpf1 1027 ADORA2A4120 AAGGCAGCTGGCACCAGTGC 20 AsCpf1 1028 ADORA2A4121 TAAGGGCATCATTGCCATCTG 21 SauCas9 1029 ADORA2A4122 CGGCCTGACTCCCATGCTAGG 21 SauCas9 1030 ADORA2A4123 GCAGTTGTTCCAACCTAGCAT 21 SauCas9 1031 ADORA2A4124 CCGCAGTTGTTCCAACCTAGC 21 SauCas9 1032 ADORA2A4125 CAAGAACCACTCCCAGGGCTG 21 SauCas9 1033 ADORA2A4126 CTTGGCCCTCCCCGCAGCCCT 21 SauCas9 1034 ADORA2A4127 CACTTGGCCCTCCCCGCAGCC 21 SauCas9 1035 ADORA2A4128 GGCCAAGTGGCCTGTCTCTTT 21 SauCas9 1036 ADORA2A4129 TTCATGGGGACCACATCCTCA 21 SauCas9 1037 ADORA2A4130 TGAAGTACACCATGTAGTTCA 21 SauCas9 1038 ADORA2A4131 CTGGTGCCCCTGCTGCTCATG 21 SauCas9 1039 ADORA2A4132 GCTCATGCTGGGTGTCTATTT 21 SauCas9 1040 ADORA2A4133 CTTCAGCTGTCGTCGCGCCGC 21 SauCas9 1041 ADORA2A4134 CGCGACGACAGCTGAAGCAGA 21 SauCas9 1042 ADORA2A4135 GATGGAGAGCCAGCCTCTGCC 21 SauCas9 1043 ADORA2A4136 GCGTGGCTGCAGTCGGGGCAG 21 SauCas9 1044 ADORA2A4137 ACGATGGCCAGGTACATGAGC 21 SauCas9 1045 ADORA2A4138 CTCTCCCACACCAATTCGGTT 21 SauCas9 1046 ADORA2A4139 GATTCACAACCGAATTGGTGT 21 SauCas9 1047 ADORA2A4140 GGGATTCACAACCGAATTGGT 21 SauCas9 1048 ADORA2A4141 CGTAGATGAAGGGATTCACAA 21 SauCas9 1049 ADORA2A4142 GGATACGGTAGGCGTAGATGA 21 SauCas9 1050 ADORA2A4143 TCATCTACGCCTACCGTATCC 21 SauCas9 1051 ADORA2A4144 CGGATACGGTAGGCGTAGATG 21 SauCas9 1052 ADORA2A4145 GCGGAAGGTCTGGCGGAACTC 21 SauCas9 1053 ADORA2A4146 AATGATCTTGCGGAAGGTCTG 21 SauCas9 1054 ADORA2A4147 GACGTGGCTGCGAATGATCTT 21 SauCas9 1055 ADORA2A4148 TTGCTGCCTCAGGACGTGGCT 21 SauCas9 1056 ADORA2A4149 CAAGGCAGCTGGCACCAGTGC 21 SauCas9 1057 ADORA2A4150 CGGGCACTGGTGCCAGCTGCC 21 SauCas9 1058 ADORA2A4151 CTTGGCAGCTCATGGCAGTGA 21 SauCas9 1059 ADORA2A4152 CCGTCTCAACGGCCACCCGCC 21 SauCas9 1060 ADORA2A4153 CACACTCCTGGCGGGTGGCCG 21 SauCas9 1061 ADORA2A4154 TGCCGTTGGCCCACACTCCTG 21 SauCas9 1062 ADORA2A4155 CCATTGGGCCTCCGCTCAGGG 21 SauCas9 1063 ADORA2A4156 CATAGCCATTGGGCCTCCGCT 21 SauCas9 1064 ADORA2A4157 AATGGCTATGCCCTGGGGCTG 21 SauCas9 1065 ADORA2A4158 ATGCCCTGGGGCTGGTGAGTG 21 SauCas9 1066 ADORA2A4159 GCCCTGGGGCTGGTGAGTGGA 21 SauCas9 1067 ADORA2A4160 TGGTGAGTGGAGGGAGTGCCC 21 SauCas9 1068 ADORA2A4161 GAGGGAGTGCCCAAGAGTCCC 21 SauCas9 1069 ADORA2A4162 AGGGAGTGCCCAAGAGTCCCA 21 SauCas9 1070 ADORA2A4163 GTCTGGGAGGCCCGTGTTCCC 21 SauCas9 1071 ADORA2A4164 CATGGCTAAGGAGCTCCACGT 21 SauCas9 1072 ADORA2A4165 GAGCTCCTTAGCCATGAGCTC 21 SauCas9 1073 ADORA2A4166 GCTCCTTAGCCATGAGCTCAA 21 SauCas9 1074 ADORA2A4167 GGCCTAGATGACCCCCTGGCC 21 SauCas9 1075 ADORA2A4168 CCCCCTGGCCCAGGATGGAGC 21 SauCas9 1076 ADORA2A4169 CTCCTGCTCCATCCTGGGCCA 21 SauCas9 1077 ADORA2A4416 CCGTGATGTACACCGAGGAG 20 AsCpf1 RR 1078 ADORA2A4417 CTTTGCCATCACCATCAGCA 20 AsCpf1 RR 1079 ADORA2A4418 TTTGCCATCACCATCAGCAC 20 AsCpf1 RR 1080 ADORA2A4419 TTGCCTGCTTCGTCCTGGTC 20 AsCpf1 RR 1081 ADORA2A4420 TCCTGGTCCTCACGCAGAGC 20 AsCpf1 RR 1082 ADORA2A4421 TCTTCAGTCTCCTGGCCATC 20 AsCpf1 RR 1083 ADORA2A4422 GTCTCCTGGCCATCGCCATT 20 AsCpf1 RR 1084 ADORA2A4423 ACCTAGCATGGGAGTCAGGC 20 AsCpf1 RR 1085 ADORA2A4424 AACCTAGCATGGGAGTCAGG 20 AsCpf1 RR 1086 ADORA2A4425 ATGCTAGGTTGGAACAACTG 20 AsCpf1 RR 1087 ADORA2A4426 GCAGCCCTGGGAGTGGTTCT 20 AsCpf1 RR 1088 ADORA2A4427 CGCAGCCCTGGGAGTGGTTC 20 AsCpf1 RR 1089 ADORA2A4428 AGGGCTGCGGGGAGGGCCAA 20 AsCpf1 RR 1090 ADORA2A4429 TGGGGACCACATCCTCAAAG 20 AsCpf1 RR 1091 ADORA2A4430 CATGAACTACATGGTGTACT 20 AsCpf1 RR 1092 ADORA2A4431 ATGAACTACATGGTGTACTT 20 AsCpf1 RR 1093 ADORA2A4432 ACTTCTTTGCCTGTGTGCTG 20 AsCpf1 RR 1094 ADORA2A4433 TGCTGCTCATGCTGGGTGTC 20 AsCpf1 RR 1095 ADORA2A4434 CAAATAGACACCCAGCATGA 20 AsCpf1 RR 1096 ADORA2A4435 GCTGTCGTCGCGCCGCCAGG 20 AsCpf1 RR 1097 ADORA2A4436 TGGCGGCGCGACGACAGCTG 20 AsCpf1 RR 1098 ADORA2A4437 TCTGCTTCAGCTGTCGTCGC 20 AsCpf1 RR 1099 ADORA2A4438 GGCAGAGGCTGGCTCTCCAT 20 AsCpf1 RR 1100 ADORA2A4439 CGGCAGAGGCTGGCTCTCCA 20 AsCpf1 RR 1101 ADORA2A4440 CCGGCAGAGGCTGGCTCTCC 20 AsCpf1 RR 1102 ADORA2A4441 CACTGCAGAAGGAGGTCCAT 20 AsCpf1 RR 1103 ADORA2A4442 TGCTGCCAAGTCACTGGCCA 20 AsCpf1 RR 1104 ADORA2A4443 ACAATGATGGCCAGTGACTT 20 AsCpf1 RR 1105 ADORA2A4444 TACACATCATCAACTGCTTC 20 AsCpf1 RR 1106 ADORA2A4445 CTTTCTTCTGCCCCGACTGC 20 AsCpf1 RR 1107 ADORA2A4446 GACTGCAGCCACGCCCCTCT 20 AsCpf1 RR 1108 ADORA2A4447 TCTCTGGCTCATGTACCTGG 20 AsCpf1 RR 1109 ADORA2A4448 CAACCGAATTGGTGTGGGAG 20 AsCpf1 RR 1110 ADORA2A4449 ACACCAATTCGGTTGTGAAT 20 AsCpf1 RR 1111 ADORA2A4450 GTTGTGAATCCCTTCATCTA 20 AsCpf1 RR 1112 ADORA2A4451 TTCATCTACGCCTACCGTAT 20 AsCpf1 RR 1113 ADORA2A4452 TCTACGCCTACCGTATCCGC 20 AsCpf1 RR 1114 ADORA2A4453 CGAGTTCCGCCAGACCTTCC 20 AsCpf1 RR 1115 ADORA2A4454 GCCAGACCTTCCGCAAGATC 20 AsCpf1 RR 1116 ADORA2A4455 CCAGACCTTCCGCAAGATCA 20 AsCpf1 RR 1117 ADORA2A4456 GCAAGATCATTCGCAGCCAC 20 AsCpf1 RR 1118 ADORA2A4457 CAAGATCATTCGCAGCCACG 20 AsCpf1 RR 1119 ADORA2A4458 CAGCCACGTCCTGAGGCAGC 20 AsCpf1 RR 1120 ADORA2A4459 AGGCAGCTGGCACCAGTGCC 20 AsCpf1 RR 1121 ADORA2A4460 TCACTGCCATGAGCTGCCAA 20 AsCpf1 RR 1122 ADORA2A4461 TCTCAACGGCCACCCGCCAG 20 AsCpf1 RR 1123 ADORA2A4462 CTCAGGGTGGGGAGCACTGC 20 AsCpf1 RR 1124 ADORA2A4463 CACCCTGAGCGGAGGCCCAA 20 AsCpf1 RR 1125 ADORA2A4464 ACCCTGAGCGGAGGCCCAAT 20 AsCpf1 RR 1126 ADORA2A4465 AGGGCATAGCCATTGGGCCT 20 AsCpf1 RR 1127 ADORA2A4466 CTCACCAGCCCCAGGGCATA 20 AsCpf1 RR 1128 ADORA2A4467 TCCACTCACCAGCCCCAGGG 20 AsCpf1 RR 1129 ADORA2A4468 TGGGACTCTTGGGCACTCCC 20 AsCpf1 RR 1130 ADORA2A4469 CTGGGACTCTTGGGCACTCC 20 AsCpf1 RR 1131 ADORA2A4470 CCTGGGACTCTTGGGCACTC 20 AsCpf1 RR 1132 ADORA2A4471 AGGGGAACACGGGCCTCCCA 20 AsCpf1 RR 1133 ADORA2A4472 CGTCTGGGAGGCCCGTGTTC 20 AsCpf1 RR 1134 ADORA2A4473 AGACGTGGAGCTCCTTAGCC 20 AsCpf1 RR 1135 ADORA2A4474 TTGAGCTCATGGCTAAGGAG 20 AsCpf1 RR 1136 ADORA2A4475 CTGGCCTAGATGACCCCCTG 20 AsCpf1 RR 1137 ADORA2A4476 TGGCCTAGATGACCCCCTGG 20 AsCpf1 RR 1138 ADORA2A4477 TCCTGGGCCAGGGGGTCATC 20 AsCpf1 RR 1139 ADORA2A4478 CTGGCCCAGGATGGAGCAGG 20 AsCpf1 RR 1140 ADORA2A4479 TGGCCCAGGATGGAGCAGGA 20 AsCpf1 RR 1141 ADORA2A4480 CGCGAGTTCCGCCAGACCTT 20 AsCpf1 RVR 1142 ADORA2A4481 CCCTGGGGCTGGTGAGTGGA 20 AsCpf1 RVR 1143 ADORA2A4482 CCATCGGCCTGACTCCCATGC 21 Cas12a 1174

[0247] It will be understood that the exemplary gRNAs disclosed herein are provided to illustrate non-limiting embodiments embraced by the present disclosure. Additional suitable gRNA sequences will be apparent to the skilled artisan based on the present disclosure, and the disclosure is not limited in this respect.

[0248] RNA-Guided Nucleases

[0249] RNA-guided nucleases according to the present disclosure include, but are not limited to, naturally-occurring Class 2 CRISPR nucleases such as Cas9, and Cpf1, as well as other nucleases derived or obtained therefrom. In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and/or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Cpf1), species (e.g., S. pyogenes vs. S. aureus) or variation (e.g., full-length vs. truncated or split; naturally-occurring PAM specificity vs. engineered PAM specificity, etc.) of RNA-guided nuclease.

[0250] The PAM sequence takes its name from its sequential relationship to the “protospacer” sequence that is complementary to gRNA targeting domains (or “spacers”). Together with protospacer sequences, PAM sequences define target regions or sequences for specific RNA-guided nuclease/gRNA combinations.

[0251] Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. In general, Cas9s recognize PAM sequences that are 3′ of the protospacer. Cpf1, on the other hand, generally recognizes PAM sequences that are 5′ of the protospacer.

[0252] In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT or NNGRRV, wherein the N residues are immediately 3′ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. F. novicida Cpf1 recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described by Shmakov et al., 2015, Molecular Cell 60, 385-397, Nov. 5, 2015. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of reference molecules (for instance, in the case of an engineered RNA-guided nuclease, the reference molecule may be the naturally occurring variant from which the RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to the engineered RNA-guided nuclease).

[0253] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered variants have been produced that generate only SSBs (discussed above) Ran & Hsu, et al., Cell 154(6), 1380-1389, Sep. 12, 2013 (“Ran”)), or that that do not cut at all.

[0254] Cas9

[0255] Crystal structures have been determined for S. pyogenes Cas9 (Jinek et al., Science 343(6176), 1247997, 2014 (“Jinek 2014”), and for S. aureus Cas9 in complex with a unimolecular guide RNA and a target DNA (Nishimasu 2014; Anders et al., Nature. 2014 Sep. 25; 513(7519):569-73 (“Anders 2014”); and Nishimasu 2015).

[0256] A naturally occurring Cas9 protein comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which comprise particular structural and/or functional domains. The REC lobe comprises an arginine-rich bridge helix (BH) domain, and at least one REC domain (e.g., a REC1 domain and, optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. While not wishing to be bound by any theory, mutational analyses suggest specific functional roles for the BH and REC domains: the BH domain appears to play a role in gRNA:DNA recognition, while the REC domain is thought to interact with the repeat:anti-repeat duplex of the gRNA and to mediate the formation of the Cas9/gRNA complex.

[0257] The NUC lobe comprises a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. It may be formed from two or more split RuvC motifs (such as RuvC I, RuvCII, and RuvCIII in S. pyogenes and S. aureus). The HNH domain, meanwhile, is structurally similar to HNN endonuclease motifs, and cleaves the complementary (i.e., top) strand of the target nucleic acid. The PI domain, as its name suggests, contributes to PAM specificity.

[0258] While certain functions of Cas9 are linked to (but not necessarily fully determined by) the specific domains set forth above, these and other functions may be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe. For instance, in S. pyogenes Cas9, as described in Nishimasu 2014, the repeat:antirepeat duplex of the gRNA falls into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem loop structure also interact with amino acids in multiple domains (PI, BH and REC1), as do some nucleotides in the second and third stem loops (RuvC and PI domains).

[0259] Cpf1

[0260] The crystal structure of Acidaminococcus sp. Cpf1 in complex with crRNA and a dsDNA target including a TTTN PAM sequence has been solved by Yamano et al. (Cell. 2016 May 5; 165(4): 949-962 (“Yamano”), incorporated by reference herein). Cpf1, like Cas9, has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe. The REC lobe includes REC1 and REC2 domains, which lack similarity to any known protein structures. The NUC lobe, meanwhile, includes three RuvC domains (RuvC-I, -II and -III) and a BH domain. However, in contrast to Cas9, the Cpf1 REC lobe lacks an HNH domain, and includes other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II and —III), and a nuclease (Nuc) domain.

[0261] While Cas9 and Cpf1 share similarities in structure and function, it should be appreciated that certain Cpf1 activities are mediated by structural domains that are not analogous to any Cas9 domains. For instance, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the HNH domain of Cas9. Additionally, the non-targeting portion of Cpf1 gRNA (the handle) adopts a pseudoknot structure, rather than a stem loop structure formed by the repeat:antirepeat duplex in Cas9 gRNAs.

[0262] Nuclease Variants

[0263] The RNA-guided nucleases described herein have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that RNA-guided nucleases can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.

[0264] Turning first to modifications that alter cleavage activity, mutations that reduce or eliminate the activity of domains within the NUC lobe have been described above. Exemplary mutations that may be made in the RuvC domains, in the Cas9 HNH domain, or in the Cpf1 Nuc domain are described in Ran & Hsu, et al., (Cell 154(6), 1380-1389, Sep. 12, 2013), and Yamano, et al. (Cell. 2016 May 5; 165(4): 949-962); as well as in WO 2016/073990 by Cotta-Ramusino, the entire contents of each of which are incorporated herein by reference. In general, mutations that reduce or eliminate activity in one of the two nuclease domains result in RNA-guided nucleases with nickase activity, but it should be noted that the type of nickase activity varies depending on which domain is inactivated. As one example, inactivation of a RuvC domain or of a Cas9 HNH domain results in a nickase.

[0265] Modifications of PAM specificity relative to naturally occurring Cas9 reference molecules has been described by Kleinstiver et al. for both S. pyogenes (Kleinstiver et al., Nature. 2015 Jul. 23; 523(7561):481-5); and S. aureus (Kleinstiver et al., Nat Biotechnol. 2015 December; 33(12): 1293-1298). Kleinstiver et al. have also described modifications that improve the targeting fidelity of Cas9 (Nature, 2016 Jan. 28; 529, 490-495). Each of these references is incorporated by reference herein.

[0266] RNA-guided nucleases have been split into two or more parts, as described by Zetsche et al. (Nat Biotechnol. 2015 February; 33(2):139-42, incorporated by reference), and by Fine et al. (Sci Rep. 2015 Jul. 1; 5:10777, incorporated by reference).

[0267] RNA-guided nucleases can be, in certain embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the nuclease while still retaining gRNA association, target and PAM recognition, and cleavage activities. In certain embodiments, RNA guided nucleases are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound nucleases and linkers are described by Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which is incorporated by reference herein.

[0268] RNA-guided nucleases also optionally include a tag, such as, but not limited to, a nuclear localization signal, to facilitate movement of RNA-guided nuclease protein into the nucleus. In certain embodiments, the RNA-guided nuclease can incorporate C- and/or N-terminal nuclear localization signals. Nuclear localization sequences are known in the art and are described in Maeder and elsewhere.

[0269] The foregoing list of modifications is intended to be exemplary in nature, and the skilled artisan will appreciate, in view of the instant disclosure, that other modifications may be possible or desirable in certain applications. For brevity, therefore, exemplary systems, methods and compositions of the present disclosure are presented with reference to particular RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used may be modified in ways that do not alter their operating principles. Such modifications are within the scope of the present disclosure.

[0270] Exemplary suitable nuclease variants include, but are not limited to, AsCpf1 variants comprising an M537R substitution, an H800A substitution, and/or an F870L substitution, or any combination thereof (numbering scheme according to AsCpf1 wild-type sequence). In some embodiments, an ASCpfl variant comprises an M537R substitution, an H800A substitution, and an F870L substitution. Other suitable modifications of the AsCpf1 amino acid sequence are known to those of ordinary skill in the art. Some exemplary sequences of wild-type AsCpf1 and AsCpf1 variants are provided below:

TABLE-US-00010 His-AsCpf1-sNLS-sNLS H800A amino acid sequence (SEQ ID NO: 1144): MGHHHHHHGSTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYK ELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAI HDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFD KFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFE NVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNL AIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNEN VLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKS AKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQE EKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNY ATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRY KALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFI EPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKT TSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKD FAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEK MLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRR FTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDST GKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIV DLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKV GGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHES RKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDA KGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDD SHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADA NGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGSPKKKRKVGSPK KKRKV Cpf1 variant 1 amino acid sequence (SEQ ID NO: 1145): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRK VGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH Cpf1 variant 2 amino acid sequence (SEQ ID NO: 1146): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRK VGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH Cpf1 variant 3 amino acid sequence (SEQ ID NO: 1147): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRK VGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH Cpf1 variant 4 amino acid sequence (SEQ ID NO: 1148): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRK V Cpf1 variant 5 amino acid sequence (SEQ ID NO: 1149): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRK V Cpf1 variant 6 amino acid sequence (SEQ ID NO: 1150): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGRSSDDEATADSQHAAPPKKKRK VGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH Cpf1 variant 7 amino acid sequence (SEQ ID NO: 1151): MGRDPGKPIPNPLLGLDSTAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQ GKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDS YRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNG KVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFP KFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYN QLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFIL EEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCD HWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQK TSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNN GAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCS TQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQK GYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRI AEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNG QAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDL SDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLK EHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQA WSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQ FEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTS KIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRG LPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALL EEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDL NGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWL AYIQELRNPKKKRKVKLAAALEHHHHHH Exemplary AsCpf1 wild-type amino acid sequence (SEQ ID NO: 1152): MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYK TYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDN LTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYE NRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVS TSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAH IIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFN ELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLK HEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEK FKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDL NNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSS QYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQ KTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHV PITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLN TIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAV VVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQL TDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRI VPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALI RSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKG QLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

[0271] Additional suitable nucleases and nuclease variants will be apparent to the skilled artisan based on the present disclosure in view of the knowledge in the art. Exemplary suitable nucleases may include, but are not limited to, those provided in Table 2 herein.

[0272] Nucleic Acids Encoding RNA-Guided Nucleases

[0273] Nucleic acids encoding RNA-guided nucleases, e.g., Cas9, Cpf1 or functional fragments thereof, are provided herein. Exemplary nucleic acids encoding RNA-guided nucleases have been described previously (see, e.g., Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

[0274] In some cases, a nucleic acid encoding an RNA-guided nuclease can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified. In certain embodiments, an mRNA encoding an RNA-guided nuclease will have one or more (e.g., all) of the following properties: it can be capped; polyadenylated; and substituted with 5-methylcytidine and/or pseudouridine.

[0275] Synthetic nucleic acid sequences can also be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein. Examples of codon optimized Cas9 coding sequences are presented in Cotta-Ramusino.

[0276] In addition, or alternatively, a nucleic acid encoding an RNA-guided nuclease may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0277] As an example, the nucleic acid sequence for Cpf1 variant 4 is set forth below as SEQ ID NO: 1177

TABLE-US-00011 ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACT GATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAG CACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCA GATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTA TCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTA ATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGT CACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACA GCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCA CCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGC ACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACAT TTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAG CCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAG CTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGC AGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATG AAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTG AGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCA GAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCAC TGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAA ACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCG TATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGA AACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCA TTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCC GACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGG GTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTT AGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGC CCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTC CGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTG AAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAG CTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGG ATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAG ACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGA GATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAA AAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGAT TTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCA GTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTC AGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAG ATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTG GACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGG AACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCAGCTCGTCTGGGTGAAAAAATG CTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTA TGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGA ATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAA TTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAA CCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTG GTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGT AGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACG TGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATC TGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTG GAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCA GCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTG AAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAA ATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCC GCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAAC ATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCAC TTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGA TATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTA AACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCG GCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACAT TCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTC GTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCG GTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGA TGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGA AAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATC CAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGCAGC ACCGCCTAAAAAGAAACGTAAAGTT

[0278] Activin

[0279] The TGF-β superfamily consists of more than 45 members including activins, inhibins, myostatin, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and nodal (see, e.g., Morianos et al., Journal of Autoimmunity 104:102314 (2019)). Activins are found either as homodimers or heterodimers of βA or/and βB subunits linked with disulfide bonds. There are three functional isoforms of activins: activin-A (βAβA), activin B (βBβB) and activin AB (βAβB) (Xia et al., J. Endocrinol. 202:1-12 (2009)). The βC and βE subunits are found in mammals and the βB subunit in Xenopus laevis. Transcripts of the βA and βB subunits are detected in nearly every tissue in the human body and exhibit increased expression in the reproductive system, while the βC and βE subunits are predominantly expressed in the liver (Woodruff, Biochem. Pharmacol. 55:953-963 (1998)). Activin-A is a cytokine of approximately 25 kDa and represents the most extensively investigated protein among the family of activins. Activin-A was initially identified as a gonadal protein that induces the biosynthesis and secretion of the follicle-stimulating hormone from the pituitary (Hedger et al., Cytokine Growth Factor Rev. 24:285-295 (2013)). It is highly conserved among vertebrates, reaching up to 95% homology between species. Activin-A regulates fundamental biologic processes, such as, haematopoiesis, embryonic development, stem cell maintenance and pluripotency, tissue repair and fibrosis (Kariyawasam et al., Clin. Exp. Allergy 41:1505-1514 (2011)).

[0280] Activin, e.g., Activin A, is well known and commercially available (from, e.g., STEMCELL Technologies Inc., Cambridge, Mass.).

[0281] Culture Methods

[0282] In general, an ES cell (e.g., an ES cell genetically engineered not to express one or more TGFβ receptor, e.g., TGFβRII) can be cultured to maintain pluripotency by culturing such ES cells in media that contains activin, e.g., a particular, effective level of activin (e.g., during one or more stages of culture).

[0283] In some embodiments, ES cells described herein are cultured (e.g., at one or more stages of culture) in a medium that includes activin, e.g., an elevated level of activin, to maintain pluripotency of the cells. In some embodiments, a level of one or more ES markers (e.g., SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and/or Nanog) in a sample of cells from the culture is increased relative to the corresponding level(s) in a sample of cells cultured using the same medium that does not include activin, e.g., an elevated level of activin. In some embodiments, the increased level of one or more ES marker is higher than the corresponding level(s) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more, of the corresponding level.

[0284] As used herein, an “elevated level of activin” means a higher concentration of activin than is present in a standard medium, a starting medium, a medium used at one or more stages of culture, and/or in a medium in which ES cells are cultured. In some embodiments, activin is not present in a standard and/or starting medium, a medium used at one or more other stages of culture, and/or in a medium in which ES cells are cultured, and an “elevated level” is any amount of activin. A medium can include an elevated level of activin initially (i.e., at the start of a culture), and/or medium can be supplemented with activin to achieve an elevated level of activin at a particular time or times (e.g., at one or more stages) during culturing.

[0285] In some embodiments, an elevated level of activin is an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000% or more, relative to a level of activin in a standard medium, a starting medium, a medium during one or more stages of culture, and/or in a medium in which ES cells are cultured.

[0286] In some embodiments, an elevated level of activin is about 0.5 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL, 35 ng/mL, 40 ng/mL, 45 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL, or more, activin. In some embodiments, an elevated level of activin is about 0.5 ng/mL to about 20 ng/mL activin, about 0.5 ng/mL to about 10 ng/mL activin, about 4 ng/mL to about 10 ng/mL activin.

[0287] Cells can be cultured in a variety of cell culture media known in the art, which are modified according to the disclosure to include activin as described herein. Cell culture medium is understood by those of skill in the art to refer to a nutrient solution in which cells, such as animal or mammalian cells, are grown. A cell culture medium generally includes one or more of the following components: an energy source (e.g., a carbohydrate such as glucose); amino acids; vitamins; lipids or free fatty acids; and trace elements, e.g., inorganic compounds or naturally occurring elements in the micromolar range. Cell culture medium can also contain additional components, such as hormones and other growth factors (e.g., insulin, transferrin, epidermal growth factor, serum, and the like); signaling factors (e.g., interleukin 15 (IL-15), transforming growth factor beta (TGF-(3), and the like); salts (e.g., calcium, magnesium and phosphate); buffers (e.g., HEPES); nucleosides and bases (e.g., adenosine, thymidine, hypoxanthine); antibiotics (e.g., gentamycin); and cell protective agents (e.g., a Pluronic polyol (Pluronic F68)).

[0288] Media that has been prepared or commercially available can be modified according to the present disclosure for utilization in the methods described herein. Nonlimiting examples of such media include Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Ham's F10 Medium (Sigma); Dulbecco's Modified Eagles Medium (DMEM, Sigma); RPM 1-1640 Medium (Sigma); HyClone cell culture medium (HyClone, Logan, Utah); Power CHO2 (Lonza Inc., Allendale, N.J.); and chemically-defined (CD) media, which are formulated for particular cell types. In some embodiments, a culture medium is an E8 medium described in, e.g., Chen et al., Nat. Methods 8:424-429 (2011)). In some embodiments, a cell culture medium includes activin but lacks TGFβ.

[0289] Cell culture conditions (including pH, 02, CO2, agitation rate and temperature) suitable for ES cells are those that are known in the art, such as described in Schwartz et al., Methods Mol. Biol. 767:107-123 (2011) and Chen et al., Nat. Methods 8:424-429 (2011).

[0290] In some embodiments, cells are cultured in one or more stages, and cells can be cultured in medium having an elevated level of activin in one or more stages. For example, a culture method can include a first stage (e.g., using a medium having a reduced level of or no activin) and a second stage (e.g., using a medium having an elevated level of activin). In some embodiments, a culture method can include a first stage (e.g., using a medium having an elevated level of activin) and a second stage (e.g., using a medium having a reduced level of activin). In some embodiments, a culture method includes more than two stages, e.g., 3, 4, 5, 6, or more stages, and any stage can include medium having an elevated level of activin or a reduced level of activin. The length of culture is not limiting. For example, a culture method can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days. In some embodiments, a culture method includes at least two stages. For example, a first stage can include culturing cells in medium having a reduced level of activin (e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days), and a second stage can include culturing cells in medium having an elevated level of activin (e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days). In some embodiments, a first stage can include culturing cells in medium having an elevated level of activin (e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days), and a second stage can include culturing cells in medium having a reduced level of activin (e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days).

[0291] In particular methods, levels of one or more ES marker (e.g., SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and/or Nanog) expressed in a sample of cells from a cell culture are monitored during one or more times (e.g., one or more stages) of cell culture, thereby allowing adjustment (e.g., increasing or decreasing the amount of activin in the culture) stopping the culture, and/or harvesting the cells from the culture.

[0292] Methods of Characterization

[0293] Methods of characterizing cells including characterizing cellular phenotype are known to those of skill in the art. In some embodiments, one or more such methods may include, but not be limited to, for example, morphological analyses and flow cytometry. Cellular lineage and identity markers are known to those of skill in the art. One or more such markers may be combined with one or more characterization methods to determine a composition of a cell population or phenotypic identity of one or more cells. For example, in some embodiments, cells of a particular population will be characterized using flow cytometry. In some such embodiments, a sample of a population of cells will be evaluated for presence and proportion of one or more cell surface markers and/or one or more intracellular markers. As will be understood by those of skill in the art, such cell surface markers may be representative of different lineages. For example, pluripotent cells may be identified by one or more of any number of markers known to be associated with such cells, such as, for example, CD34. Further, in some embodiments, cells may be identified by markers that indicate some degree of differentiation. Such markers will be known to one of skill in the art. For example, in some embodiments, markers of differentiated cells may include those associated with differentiated hematopoietic cells such as, e.g., CD43, CD45 (differentiated hematopoietic cells). In some embodiments, markers of differentiated cells may be associated with NK cell phenotypes such as, e.g., CD56 (also known as neural cell adhesion molecule), NK cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII, cluster of differentiation 16 (CD16), natural killer group-2 member A (NKG2A), natural killer group-2 member D (NKG2D), CD69, a natural cytotoxicity receptor (e.g., NCR1, NCR2, NCR3, NKp30, NKp44, NKp46, and/or CD158b), killer immunoglobulin-like receptor (KIR), and CD94 (also known as killer cell lectin-like receptor subfamily D, member 1 (KLRD1)) etc. In some embodiments, markers may be T cell markers (e.g., CD3, CD4, CD8, etc.).

[0294] Methods of Use

[0295] A variety of diseases, disorders and/or conditions may be treated through use of technologies provided by the present disclosure. For example, in some embodiments, a disease, disorder and/or condition may be treated by introducing modified cells as described herein (e.g., edited iNK cells) to a subject. Examples of diseases that may be treated include, but not limited to, cancer, e.g., solid tumors, e.g., of the brain, prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovary, testes, bladder, kidney, head, neck, stomach, cervix, rectum, larynx, or esophagus; and hematological malignancies, e.g., acute and chronic leukemias, lymphomas, e.g., B-cell lymphomas including Hodgkin's and non-Hodgkin lymphomas, multiple myeloma and myelodysplastic syndromes.

[0296] In some embodiments, the present disclosure provides methods of treating a subject in need thereof by administering to the subject a composition comprising any of the cells described herein. In some embodiments, a therapeutic agent or composition may be administered before, during, or after the onset of a disease, disorder, or condition (including, e.g., an injury).

[0297] In particular embodiments, the subject has a disease, disorder, or condition, that can be treated by a cell therapy. In some embodiments, a subject in need of cell therapy is a subject with a disease, disorder and/or condition, whereby a cell therapy, e.g., a therapy in which a composition comprising a cell described herein, is administered to the subject, whereby the cell therapy treats at least one symptom associated with the disease, disorder, and/or condition. In some embodiments, a subject in need of cell therapy includes, but is not limited to, a candidate for bone marrow or stem cell transplant, a subject who has received chemotherapy or irradiation therapy, a subject who has or is at risk of having a hyperproliferative disorder or a cancer, e.g., a hyperproliferative disorder or a cancer of hematopoietic system, a subject having or at risk of developing a tumor, e.g., a solid tumor, and/or a subject who has or is at risk of having a viral infection or a disease associated with a viral infection.

[0298] Pharmaceutical Compositions

[0299] In some embodiments, the present disclosure provides pharmaceutical compositions comprising one or more genetically modified cells described herein, e.g., an edited iNK cell described herein. In some embodiments, a pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises isolated pluripotent stem cell-derived hematopoietic lineage cells comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% T cells, NK cells, NKT cells, CD34+HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+HE cells or HSCs. In some embodiments, a pharmaceutical composition comprises isolated pluripotent stem cell-derived hematopoietic lineage cells comprising about 95% to about 100% T cells, NK cells, NKT cells, CD34+HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+HE cells or HSCs.

[0300] In some embodiments, a pharmaceutical composition of the present disclosure comprises an isolated population of pluripotent stem cell-derived hematopoietic lineage cells, wherein the isolated population has less than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% T cells, NK cells, NKT cells, CD34+HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+HE cells or HSCs. In some embodiments, an isolated population of pluripotent stem cell-derived hematopoietic lineage cells has more than about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, or 30% T cells, NK cells, NKT cells, CD34+HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+HE cells or HSCs. In some embodiments, an isolated population of pluripotent stem cell-derived hematopoietic lineage cells has about 0.1% to about 1%, about 1% to about 3%, about 3% to about 5%, about 10%-about 15%, about 15%-20%, about 20%-25%, about 25%-30%, about 30%-35%, about 35%-40%, about 40%-45%, about 45%-50%, about 60%-70%, about 70%-80%, about 80%-90%, about 90%-95%, or about 95% to about 100% T cells, NK cells, NKT cells, CD34+HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+HE cells or HSCs.

[0301] In some embodiments, an isolated population of pluripotent stem cell-derived hematopoietic lineage cells comprises about 0.1%, about 1%, about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% T cells, NK cells, NKT cells, CD34+HE cells or HSCs, e.g., genetically modified (e.g., edited) T cells, NK cells, NKT cells, CD34+HE cells or HSCs.

[0302] As one of ordinary skill in the art would understand, both autologous and allogeneic cells can be used in adoptive cell therapies. Autologous cell therapies generally have reduced infection, low probability for GVHD, and rapid immune reconstitution relative to other cell therapies. Allogeneic cell therapies generally have an immune mediated graft-versus-malignancy (GVM) effect, and low rate of relapse relative to other cell therapies. Based on the specific condition(s) of the subject in need of the cell therapy, one of ordinary skill in the art would be able to determine which specific type of therapy(ies) to administer.

[0303] In some embodiments, a pharmaceutical composition comprises pluripotent stem cell-derived hematopoietic lineage cells that are allogeneic to a subject. In some embodiments, a pharmaceutical composition comprises pluripotent stem cell-derived hematopoietic lineage cells that are autologous to a subject. For autologous transplantation, the isolated population of pluripotent stem cell-derived hematopoietic lineage cells can be either a complete or partial HLA-match with patient subject. In some embodiments, the pluripotent stem cell-derived hematopoietic lineage cells are not HLA-matched to a subject.

[0304] In some embodiments, pluripotent stem cell-derived hematopoietic lineage cells can be administered to a subject without being expanded ex vivo or in vitro prior to administration. In particular embodiments, an isolated population of derived hematopoietic lineage cells is modulated and treated ex vivo using one or more agent to obtain immune cells with improved therapeutic potential. In some embodiments, the modulated population of derived hematopoietic lineage cells can be washed to remove the treatment agent(s), and the improved population can be administered to a subject without further expansion of the population in vitro. In some embodiments, an isolated population of derived hematopoietic lineage cells is expanded prior to modulating the isolated population with one or more agents.

[0305] In some embodiments, an isolated population of derived hematopoietic lineage cells can be genetically modified (e.g., by recombinant methods) to express TCR, CAR or other proteins. For genetically engineered derived hematopoietic lineage cells that express recombinant TCR or CAR, whether prior to or after genetic modification of the cells, the cells can be activated and expanded using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0306] Cancers

[0307] Any cancer can be treated using a composition described herein. Exemplary therapeutic targets of the present disclosure include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, eye, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, a cancer may specifically be of the following non-limiting histological type: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; B-cell lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0308] In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is RCC. In another embodiment, the cancer is non-small cell lung cancer (NSCLC).

[0309] In some embodiments, solid cancer indications that can be treated with iNK cells (e.g., genetically modified iNK cells, e.g., edited iNK cells) provided herein, either alone or in combination with one or more additional cancer treatment modality, include: bladder cancer, hepatocellular carcinoma, prostate cancer, ovarian/uterine cancer, pancreatic cancer, mesothelioma, melanoma, glioblastoma, HPV-associated and/or HPV-positive cancers such as cervical and HPV+ head and neck cancer, oral cavity cancer, cancer of the pharynx, thyroid cancer, gallbladder cancer, and soft tissue sarcomas. In some embodiments, hematological cancer indications that can be treated with the iNK cells (e.g., genetically modified iNK cells, e.g., edited iNK cells) provided herein, either alone or in combination with one or more additional cancer treatment modalities, include: ALL, CLL, NHL, DLBCL, AML, CML, and multiple myeloma (MM).

[0310] Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, tumors such as bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, metastatic tumors, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

[0311] Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

[0312] Examples of cellular proliferative and/or differentiative disorders involving the colon include, but are not limited to, tumors of the colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0313] Examples of cancers or neoplastic conditions, in addition to the ones described above, include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

[0314] Exemplary useful additional cancer treatment modalities include, but are not limited to: chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfanide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANET™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; cyclosporine, sirolimus, rapamycin, rapalogs, ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU, leucovovin; anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON®); anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX®); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); aptamers, described for example in U.S. Pat. No. 6,344,321, which is herein incorporated by reference in its entirety; anti HGF monoclonal antibodies (e.g., AV299 from Aveo, AMG102, from Amgen); truncated mTOR variants (e.g., CGEN241 from Compugen); protein kinase inhibitors that block mTOR induced pathways (e.g., ARQ197 from Arqule, XL880 from Exelexis, SGX523 from SGX Pharmaceuticals, MP470 from Supergen, PF2341066 from Pfizer); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl) benzenesulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0315] Other compounds that are effective in treating cancer are known in the art and described herein that are suitable for use with the compositions and methods of the present disclosure as additional cancer treatment modalities are described, for example, in the “Physicians' Desk Reference, 62nd edition. Oradell, N.J.: Medical Economics Co., 2008”, Goodman & Gilman's “The Pharmacological Basis of Therapeutics, Eleventh Edition. McGraw-Hill, 2005”, “Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000.”, and “The Merck Index, Fourteenth Edition. Whitehouse Station, N.J.: Merck Research Laboratories, 2006”, incorporated herein by reference in relevant parts.

[0316] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of:” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0317] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

[0318] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. The contents of database entries, e.g., NCBI nucleotide or protein database entries provided herein, are incorporated herein in their entirety. Where database entries are subject to change over time, the contents as of the filing date of the present application are incorporated herein by reference. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0319] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.

EXAMPLES

Example 1: Generating Edited iPSC Cells Using Cas12a and Testing Effect of Activin a on Pluripotency

[0320] To generate natural killer cells from pluripotent stem cells, a representative induced pluripotent stem cell (iPSC) line was generated and designated “PCS-201”. This line was generated by reprogramming adult male human primary dermal fibroblasts purchased from ATCC (ATCC® PCS-201-012) using a commercially available non-modified RNA reprogramming kit (Stemgent/Reprocell, USA). The reprogramming kit contains non-modified reprogramming mRNAs (OCT4, SOX2, KLF4, cMYC, NANOG, and LIN28) with immune evasion mRNAs (E3, K3, and B18R) and double-stranded microRNAs (miRNAs) from the 302/367 clusters. Fibroblasts were seeded in fibroblast expansion medium (DMEM/F12 with 10% FBS). The next day, media was switched to Nutristem medium and daily overnight transfections were performed for 4 days (day 1 to 4). Primary iPSC colonies appeared on day 7 and were picked on day 10-14. Picked colonies were expanded clonally to achieve a sufficient number of cells to establish a master cell bank. The parental line chosen from this process and used for the subsequent experiments passed standard quality controls, including confirmation of stemness marker expression, normal karyotype and pluripotency.

[0321] To generate edited iPSC cells, the PCS-201 (PCS) cells were electroporated with a Cas12a RNP designed to cut at the target gene of interest. Briefly, the cells were treated 24 hours prior to transfection with a ROCK inhibitor (Y27632). On the day of transfection, a single cell solution was generated using accutase and 500,000 PCS iPS cells were resuspended in the appropriate electroporation buffer and Cas12a RNP at a final concentration of 2 μM. When two RNPs were added simultaneously, the total RNP concentration was 4 μM (2+2). This solution was electroporated using a Lonza 4D electroporator system. Following electroporation, the cells were plated in 6-well plates in mTESR media containing CloneR (Stemcell Technologies). The cells were allowed to grow for 3-5 days with daily media changes, and the CloneR was removed from the media by 48 hours post electroporation. To pick single colonies, the expanded cells were plated at a low density in 10 cm plates after resuspending them in a single cell suspension. Rock inhibitor was used to support the cells during single cell plating for 3-5 days post plating depending on the size of the colonies on the plate. After 7-10 days, sufficiently sized colonies with acceptable morphology were picked and plated into 24-well plates. The picked colonies were expanded to sufficient numbers to allow harvesting of genomic DNA for subsequent analysis and for cell line cryopreservation. Editing was confirmed by NGS and selected clones were expanded further and banked. Ultimately, karyotyping, sternness flow, and differentiation assays were performed on a subset of selected clones.

[0322] Two target genes of interest were CISH and TGFβRII, both of which were hypothesized to enhance natural killer cell function. As the TGFβ:TGFβRII pathway is believed to be involved in the maintenance of pluripotency, it was hypothesized that a functional deletion of TGFβRII in iPSCs could lead to differentiation and prevent generation of TGFβRII edited iPSCs. Due to the convergence of Activin receptor signaling and TGFβRII signaling in regulating SMAD2/3 and other intracellular molecules, it was hypothesized that Activin A could replace TGFβ in commercially available pluripotent stem cell medias to generate edited lines. To test this hypothesis, the pluripotency of unedited and TGFβRII edited iPSCs grown with Activin A was assessed. Several different culture medias were utilized: “E6” (Essential 6™ Medium, #A1516401, ThermoFisher), which lacks TGFβ, “E7”, which was E6 supplemented with 100 ng/ml of bFGF (Peprotech, #100-18B), “E8” (Essential 8™ Medium, #A1517001, ThermoFisher), and “E7+ActA”, which was E6 supplemented with 100 ng/ml of bFGF and varying concentrations of Activin A (Peprotech #120-14P). Typically, E6 and E7 medias are typically insufficient to maintain the sternness and pluripotency of PSCs over multiple passages in culture.

[0323] In order to determine whether Activin A could maintain PCS iPSCs in the absence of exogenous TGFβ, unedited PCS iPSCs were plated on a LaminStem™ 521 (Biological Industries) coated 6-well plate and cultured in E6, E7, E8 or E7+ActA (with Activin A at two different concentrations—1 ng/ml and 4 ng/ml). After 2 passages, the cells were assessed for morphology and sternness marker expression. Morphology was assessed using a standard phase contrast setting on an inverted microscope. Colonies with defined edges and non-differentiated cells typical of iPSC colonies, were deemed to be stem like. To confirm the morphological observations, the expression of standard iPS cell sternness markers was measured using intracellular flow cytometry. Briefly, cells were dissociated, stained for extracellular markers, and then fixed overnight and permeabilized using the reagents and standard protocol from the Foxp3/Transcription Factor Staining Buffer Set (eBioscience™). Cells were stained for flow cytometric analysis with anti-human TRA-1-60-R_AF®488 (Biolegend®; Clone TRA-1-60-R), anti-Human Nanog AF®647 (BD Pharmingen™; Clone N31-355), and anti-Oct4 (Oct3) PE (Biolegend®; Clone 3A2A20),. Cells were recorded on a NovoCyte Quanteon Flow Cytometer (Agilent) and analyzed using FlowJo (FlowJo, LLC). As shown in FIG. 1, both 1 ng/mL and 4 ng/ml of Activin A was sufficient to maintain pluripotency with equivalent sternness marker expression to the cells grown in E8. As expected, cells grown in E6 and E7 (which lacked TGFβ) did not maintain sternness gene expression to the same degree as E8, indicating the loss of iPSC sternness in the absence of TGFβ or Activin A. These results suggest that Activin A can supplement iPSC sternness in the absence of TGFβ signaling.

[0324] Given the demonstration that Activin A could support iPSC sternness in the absence of TGFβ, TGFβRII knockout (“KO”) iPSCs, CISH KO iPSCs, and TGFβRII/CISH double knockout (“DKO”) iPSC lines were generated. Specifically, iPSCs were edited using an RNP having an engineered Cas12a with three amino acid substitutions (M537R, F870L, and H800A (SEQ ID NO: 1148)) and a gRNA specific for CISH or TGFβRII. To make CISH/TGFβRII DKO iPSCs, iPSCs were treated with an RNP targeting CISH and an RNP targeting TGFβRII simultaneously. The particular guide RNA sequences of Table 10 were used for editing of CISH and TGFβRII. Both guides were generated with a targeting domain consisting of RNA, an AsCpf1 scaffold of the sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 1153) located 5′ of the targeting domain, and a 25-mer DNA extension of the sequence ATGTGTTTTTGTCAAAAGACCTTTT (SEQ ID NO: 1154) at the 5′ terminus of the scaffold sequence.

TABLE-US-00012 TABLE 10 Guide RNA sequences gRNA Targeting  Full Length gRNA Target Domain Sequence Sequence CISH GGUGUACAGCAGUGGCU ATGTGTTTTTGTCAAAAGACCTT 7050 GGU TTrUrArArUrUrUrCrUrArCr (SEQ ID NO: 1155) UrCrUrUrGrUrArGrArUrGrG rUrGrUrArCrArGrCrArGrUr GrGrCrUrGrGrU (SEQ ID NO: 1156) TGFβRII UGAUGUGAGAUUUUCCA ATGTGTTTTTGTCAAAAGACCTT 24026 CCU TTrUrArArUrUrUrCrUrArCr (SEQ ID NO: 1157) UrCrUrUrGrUrArGrArUrUrG rArUrGrUrGrArGrArUrUrUr UrCrCrArCrCrU (SEQ ID NO: 1158)

[0325] The edited clones were generated as described above with a minor modification for the cells treated with TGFβRII RNPs. Briefly, TGFβRII-edited PCS iPSCs and TGFβRII/CISH edited PCS iPSCs were plated after electroporation at the 6-well stage in the mTESR supplemented with 10 ng/ml of Activin A in order to support the generation of edited clones. The cells were cultured with 10 ng/ml of Activin A through the cell colony picking and early expansion stages. Colonies assessed as having the correct single KO (CISH KO or TGFβRII KO) or double KO (CISH/TGFβRII DKO) were picked and expanded (clonal selection).

[0326] To determine the optimal concentration of Activin A for culturing of TGFβRII KO and TGFβRII/CISH DKO iPSCs, a slightly expanded concentration curve was tested as shown FIG. 2. Similar to the assessment performed previously, the iPSCs were cultured in a Matrigel-treated 6-well plate with concentrations of 1 ng/ml, 2 ng/ml, 4 ng/ml and 10 ng/ml Activin A. As shown in FIG. 2, TGFβRII KO or CISH/TGFβRII DKO cells cultured in E7 medium supplemented with 4 ng/mL Activin A for 19 days (over 5 passages) maintained a wild type morphology. FIG. 3 shows the morphology of TGFβRII KO PCS-201 hiPSC Clone 9.

[0327] As shown in FIG. 4A, the initial editing efficiency of the iPSCs treated simultaneously with the CISH and TGFβRII RNPs (prior to clonal selection) was high, with 95% of the CISH alleles edited and 78% of the TGFβRII alleles edited. Unedited iPSC controls did not have indels at either loci. iPSCs that were treated with CISH or TGFβRII RNPs individually showed 93% and 82% editing rates prior to clone selection (depicted in FIG. 4A). The KO cell lines (CISH KO iPSCs, TGFβRII KO iPSCs, and CISH/TGFβRII DKO iPSCs) were subsequently assessed for the presence of pluripotency markers Oct4, SSEA4, Nanog, and Tra-1-60 after culturing in the presence of supplemental Activin A. As shown in FIGS. 4B and 5, culturing the KO cell lines in Activin A maintained expression of these pluripotency markers.

[0328] The KO iPSC lines cultured in Activin A were next assessed for their capacity to differentiate using the STEMdiff™ Trilineage Differentiation Kit assay (from STEMCELL Technologies Inc., Vancouver, BC, CA) as depicted schematically in FIG. 6. As shown in FIG. 7A, culturing the single KO (TGFβRII KO iPSCs or CISH KO iPSCs) and DKO (TGFβRII/CISH DKO iPSCs) cell lines in media with supplemental Activin A maintained their ability to differentiate into early progenitors of all 3 germ layers, as shown by expression of ectoderm (OTX2), mesoderm (brachyury), and endoderm (GATA4) markers (FIG. 7A). The unedited PCS control cells were also able to express each of these markers.

[0329] The edited iPSCs were next karyotyped to determine whether the Cas12a editing caused large genetic abnormalities, such as translocations. As shown in FIG. 7B, the cells had normal karyotypes with no translocation between the cut sites.

[0330] To further support the results described above, an expanded Activin A concentration curve was performed on the unedited parental PSC line, an edited TGFβRII KO iPSC clone (C7), and an additional representative (unedited) cell line designated RUCDR (RUCDR Infinite Biologics group, Piscaway N.J.). At the outset, the iPSCs were seeded at 1e5 cells per well in a 1× LaminStem™ 521 (Biological Industries) coated 12-well plate. Cells were then passaged 10 times over ˜40-50 days using 0.5 mM EDTA in 1×PBS dissociation and Y-27632 (Biological Industries) until wells achieved >75% confluency. Cells were cultured in Essential 6™ Medium (Gibco), TeSR™-E7 ™, and TeSR™-E8™ (StemCell Technologies) for controls and titrated using TeSR™-E7™ supplemented with E. coli-derived recombinant human/murine/rat Activin A (PeproTech) spanning a 4-log concentration dosage (0.001-10 ng/mL). Following 5 and 10 passages, cells were dissociated and then fixed overnight and permeabilized using the reagents and standard protocol from the Foxp3/Transcription Factor Staining Buffer Set (eBioscience™). Cells were stained for flow cytometric analysis with anti-human TRA-1-60-R_AF®488 (Biolegend®; Clone TRA-1-60-R), anti Sox2 PerCP-Cy™5.5 (BD Pharmingen™; Clone 030-678), anti Human Nanog_AF®647 (BD Pharmingen™; Clone N31-355), anti-Oct4 (Oct3) PE (Biolegend®; Clone 3A2A20), and anti-human SSEA-4 PE/Dazzle™ 594 (Biolegend®; Clone MC-813-70). Cells were recorded on a NovoCyte Quanteon Flow Cytometer (Agilent) and analyzed using FlowJo (FlowJo, LLC). FIG. 7C shows the titration curves for the tested iPSC lines. The minimum concentration of Activin A required to maintain each line varied slightly, with the TGFβRII KO iPSCs requiring a higher baseline amount of Activin A as compared to the parental control (0.5 ng/ml vs 0.1 ng/ml). In all 3 cell lines, 4 ng/ml was well above the minimum amount of Activin A necessary to maintain stemness marker expression over an extended culture period. FIG. 7D shows the stemness marker expression in the cells culture with the base medias alone (no Activin A). As expected, the TGFβRII KO iPSCs did not maintain expression, while the two unedited lines were able to maintain stemness marker expression in E8.

Example 2: Differentiation of Edited CISH KO, TGFβRII KO, and CISH/TGFβRII DKO iPSCs into iNK Cells Exhibiting Enhanced Function

[0331] FIG. 8A depicts a schematic of an exemplary workflow for development of a CRISPR-Cas12a-edited iPSC platform for generation of enhanced CD56+ iNK cells. As shown in FIG. 8A, the CISH and TGFβRII genes are targeted in iPSCs via delivery of RNPs to the cells using electroporation to generate CISH/TGFβRII DKO iPSCs. iPSCs with the desired edits at both the CISH and TGFβRII genes can then be selected and expanded to create a master iPSC bank. Edited cells from the iPSC master bank can then be differentiated into CD56+ CISH/TGFβRII DKO iNK cells.

[0332] FIGS. 8B and 8C depict two exemplary schematics of the process of differentiating iPSCs into iNK cells. As shown in FIGS. 8B and 8C, edited cells (or unedited control cells) were differentiated using a two-phase process. First, in the “hematopoietic differentiation phase,” hiPSCs (edited and unedited) were cultured in StemDiff™ APEL2™ medium (StemCell Technologies) with SCF (40 ng/mL), BMP4 (20 ng/mL), and VEGF (20 ng/mL) from days 0-10, to produce spin embryoid bodies (SEBs). As shown in FIG. 8B, SEBs were then cultured from days 11-39 in StemDiff™ APEL2™ medium comprising IL-3 (5 ng/mL, only present for the first week of culture), IL-7 (20 ng/mL), IL-15 (10 ng/mL), SCF (20 ng/mL), and Flt3L (10 ng/mL) in an NK cell differentiation phase. CISH KO iPSCs, TGFβRII KO iPSCs, CISH/TGFβRII DKO iPSCs, and unedited wild-type iPSC lines (PCS), were differentiated into iNKs according to the schematic in FIG. 8B, and then characterized to assess whether they exhibited a phenotype congruent with NK cells (see FIGS. 9, 10, and 11A). CISH KO iPSCs, TGFβRII KO iPSCs, CISH/TGFβRII DKO iPSCs, and unedited wild-type iPSC lines, described in FIGS. 11B, 11C, 12B, 12C, and 13 were also differentiated into iNKs utilizing the alternative method shown in FIG. 8C, and then characterized to assess whether they exhibited a phenotype congruent with NK cells (see FIGS. 11B, 11C, 12B, 12C, and 13).

[0333] Specifically, the CISH KO iNKs, TGFβRII KO iNKs, CISH/TGFβRII DKO iNKs were assessed for exemplary phenotypic markers of (i) stem cells (CD34); and (ii) hematopoietic cells (CD43 and CD45) by flow cytometry. Briefly, two rows of embryoid bodies from a 96-well plate for each genotype were harvested for staining. Once a single cell solution was generated using Trypsin and mechanical disruption, the cells were stained for the human markers CD34, CD45, CD31, CD43, CD235a and CD41. As shown in FIG. 9, CISH KO iNKs, TGFβRII KO iNKs, CISH/TGFβRII DKO iNKs, and iNKs derived from wild-type parental clones (PCS) exhibited lower levels of CD34 relative to control cells, which were purified CD34+ HSCs. CD34 expression levels were similar across these iNK cell clones indicating that editing of the iPSCs did not affect differentiation to the CD34+ stage. FIG. 10 shows that CISH KO iNKs, TGFβRII KO iNKs, CISH/TGFβRII DKO iNKs, and iNKs derived from wild-type parental clones (PCS) exhibited similar surface expression profiles for CD43 and CD45. Thus, iNKS differentiated from edited and unedited iPSCs exhibited similar levels of markers for stem cells and hematopoietic cells, and both differentiated edited and unedited cells exhibited certain NK cell phenotypes based on marker expression profiles.

[0334] CISH KO iNKs, TGFβRII KO iNKs, CISH/TGFβRII DKO iNKs, iNKs derived from wild-type parental clones (WT), and NK cells derived from peripheral blood (PBNKs) were further assayed to determine their surface expression of CD56, a marker for NK cells. Briefly, cells were harvested on day 39 of differentiation, washed and resuspended in a flow staining buffer containing antibodies that recognize human CD56, CD16, NKp80, NKG2A, NKG2D, CD335, CD336, CD337, CD94, CD158. Cells events were recorded on a NovoCyte Quanteon Flow Cytometer (Agilent) and analyzed using FlowJo (FlowJo, LLC). FIG. 11A shows that iNK cells derived from edited iPSCs exhibited similar CD56+ surface expression relative to iNKs derived from unedited iPSC parental clones and PBNK cells (at day 39 in culture). FIG. 11B shows that iNK cells derived from edited iPSCs exhibited similar CD56+ and CD16+ surface expression relative to iNKs derived from unedited iPSC parental clones (at day 39 in culture). FIG. 11C shows that iNK cells derived from edited iPSCs exhibited similar CD56+, CD54+, KIR+, CD16+, CD94+, NKG2A+, NKG2D+, NCR1+, NCR2+, and NCR3+ surface expression relative to iNKs derived from unedited iPSC parental clones and PBNK cells (at day 39 in culture)

[0335] To confirm cell functionality, cells were assessed using a tumor cell cytotoxicity assay on the xCelligence platform. Briefly, tumor targets, SK-OV-3 tumor cells, were plated and grown to an optimal cell density in 96-well xCelligence plates. iNKs were then added to the tumor targets at different E:T ratios (1:4, 1:2, 1:1, 2:1. 4:1 and 8:1) in the presence of TGFβ. FIG. 12C shows that TGFβRII KO and CISH/TGFβRII DKO cells more effectively killed SK-OV-3 cells, as measured by percent cytolysis, relative to unedited iNK cells either in the presence or absence of TGF-β (at E:T ratios of 1:4, 1:2, 1:1, and 2:1).

[0336] While iNK cells generated using the alternative method described in FIG. 8B were CD56+ and capable of killing tumor targets in an in vitro cytotoxicity assay, the iNKs did not express many of the canonical markers associated with mature NK cells such as CD16, NKG2A, and KIRs. A K562 feeder cell line is typically used to expand and mature iNKs that are generated by similar differentiation methodologies. After expansion on feeders, the iNKs often express CD16, KIRs and other surface markers indicative of a more mature phenotype. In order to identify a feeder free approach to achieve more mature iNKs with enhanced functionality, an alternative media composition was tested for the stage of differentiation between day 11 and day 39. Instead of culturing cells between day 11 and day 39 in APEL2 (as shown in FIG. 8B), the spin embryoid bodies (SEBs) were cultured in NK MACS® media (MACS Miltenyi Biotec) with 15% human AB serum in the presence of the same cytokines as mentioned above. This protocol is depicted in FIG. 8C. In order to compare the two media compositions, Day 11 SEBs from WT PCS, TGFβRII KO iPSCs, CISH KO iPSCs, and DKO iPSCs were split into two conditions for the second half of the differentiation process, one with APEL2 base and the other with the NKMACS+serum base. At day 39, the cell yield, marker expression, and cytotoxicity levels were assessed. In all cases, the NKMACS+serum condition (depicted in FIG. 8C) outperformed the APEL2 condition (depicted in FIG. 8B). FIG. 8D shows that the NKMACS+serum condition yielded a greater fold expansion at the end of the 39 day process (nearly 300 fold expansion vs 100 fold expansion). When NK marker expression was analyzed by flow cytometry as described above, the iNKs cultured in NKMACS+serum were 34% CD16 positive and exhibited 20% KIR expression while the APEL2 conditions yielded cells that were essentially negative for both markers. This was the case for all genotypes tested. In order to visualize the markers relative to time or condition, flow cytometry data was gated and analyzed in FlowJo and heat maps were constructed (FIGS. 8E and 8F). Samples were first cleaned by gating for live cells (FSC-H vs. LIVE/DEAD™ Fixable Yellow) followed by immune cells (SSC-A vs. FSC-A), singlets (FSC-H vs. FSC-A) and the natural killer cell population (CD56 vs. CD45). The NK population, defined as CD45+56+ cells, was gated and each marker was analyzed along the X-axis in an analysis synonymous to a histogram/count plot (CD16+, CD94+, NKG2A+, NKG2D+, CD335+, CD336+, CD337+, NKp80+, panKIR+). Statistics for the aforementioned markers are visualized with a double-gradient heat map (GraphPad Prism 8) with the key set to the following parameters: black=0, medium intensity 30<x<50, maximum intensity=100. Based on this analysis, the expression kinetics and magnitude across all genotypes were improved by the NKMACS+serum condition. The cells were also assessed in a tumor cell cytotoxicity assay as described previously. The iNKs generated in the NKMACS+serum conditions were capable of killing at a lower E:T ratio than the cells differentiated in APEL2, indicating that the improved NK maturation had a positive impact on the functionality of the cells (FIG. 8G).

[0337] Analysis of additional differentiation markers in NKMACS+serum confirmed the presence of CD16 expression. FIG. 11B shows analysis of specific subpopulations (CD45 vs CD56 and CD56 vs CD16) derived from unedited or DKO iPSCs. Additionally, the cell surface marker profile of unedited iNK cells and CISH/TGFβRII DKO iNKs in FIG. 11C confirmed that the NK cell marker profile of the edited iNK cells was similar to that of unedited iNK cells. Taken together, these data show that Cas12a-edited single and double KO iPSC clones differentiate into iNK cells in a similar fashion as unedited iPSC clones, as defined by NK cell markers.

[0338] Additionally, certain edited iNK clonal cells (CISH single knockout “CISH_C2, C4, C5, and C8”, TGFβRII single knockout “TGFβRII-C7”, and TGFβRII/CISH double knockout “DKO-C1”), and parental clone iNK cells (“WT”) were cultured in the presence of 1 ng/mL or 10 ng/mL IL-15, and differentiation markers were assessed at day 25, day 32, and day 39 post-hiPSC differentiation. As shown in FIG. 14, surface expression phenotypes (measured as a percentage of the population) culturing in 10 ng/mL IL-15 resulted in a higher proportion of surface expression in the single knockouts, double knockouts, and the parental clonal line.

[0339] The edited iNK cells differentiated in NK MACS® medium+serum conditions were assessed for effector function in vitro using a range of molecular and functional analyses. First, a phosphoflow cytometry assay was performed to determine the phosphorylated state of STAT3 (pSTAT3) and SMAD2/3 (pSMAD2/3) in the day 39 iNK cells. CISH KO iNKs exhibited increased pSTAT3 upon IL-15 stimulation (FIG. 11D), and CISH/TGFβRII DKO iNKs exhibited decreased pSMAD2/3 levels upon TGF-β stimulation as compared to unedited iNK cells (FIG. 11E). These data suggest that CISH/TGFβRII DKO iNKs have enhanced sensitivity to IL-15 and resistance to TGF-β mediated immunosuppression. In addition, CISH/TGFβRII DKO iNKs were characterized for IFNγ and TNFα production using a phorbol myristate acetate and Ionomycin (PMA/IMN) stimulation assay. Briefly, cells were treated with 2 ng/ml of PMA and 0.125 μM of Ionomycin along with a protein transport inhibitor for 4 hours. The cells were harvested and stained using a standard intracellular staining protocol. The CISH/TGFβRII DKO iNKs produced significantly higher amounts of IFNγ and TFNa when stimulated with PMA/IMN (FIGS. 11F and 11G), providing evidence of enhanced cytokine production following stimulation relative to unedited control iNKs.

[0340] To test iNK tumor cell killing activity, a 3D solid tumor cell killing assay (depicted schematically in FIG. 12A) was utilized. In brief, spheroids were formed by seeding 5,000 NucLight Red labeled SK-OV-3 cells in 96 well ultra-low attachment plates. Spheroids were incubated at 37° C. before addition of effector cells (at different E:T ratios) and 10 ng/mL TGF-β, spheroids were subsequently imaged every 2 hours using the Incucyte S3 system for up to 120 hours. Data shown are normalized to the red object intensity at time of effector addition. Normalization of spheroid curves maintains the same efficacy patterns observed in non-normalized data. Using this assay, the cytotoxicity of iNKs differentiated from four CISH KO iPSC clones, two TGFβRII KO iPSC clones and one CISH/TGFβRII DKO iPSC clone were compared to control iNKs derived from the unedited parental iPSCs. As shown in FIG. 12B, edited iNK cells were capable of reducing the size of SK-OV-3 spheroids more effectively than unedited iNK control cells (averaged data from 6 assays). In particular the CISH/TGFβRII DKO iNK cells reduced the size of SK-OV-3 spheroids to a greater extent than unedited iNK cells at all E:T ratios greater than 0.01, and significantly at E:T ratios of 1 or higher. The TGFβRII KO clone 7 iNKs also exhibited significantly enhanced killing when compared to unedited iNK cells. While a number of single CISH KO clones did not show significant enhancement of killing at the 10:1 E:T ratio, the majority of clones did display a trend towards increased SK-OV-3 spheroid cell killing, with the greatest differential at the highest E:T ratio. To further elucidate the functionality of the edited iNKs, the cells were pushed to kill tumor targets repeatedly over a multiday period, herein described as an in vitro serial killing assay. At day 0 of the assay, 10×10.sup.6 Nalm6 tumor cells (a B cell leukemia cell line) and 2×10.sup.5 iNKs were plated in each well of a 96-well plate in the presence of IL-15 (10 ng/ml) and TGF-β (long/ml). At 48 hour intervals, a bolus of 5×10.sup.3 Nalm6 tumor cells (a B cell leukemia cell line) was added to re-challenge the iNK population. As shown in FIG. 13, the edited iNK cells (CISH/TGFβRII DKO iNK cells) exhibited continued killing of Nalm6 cells after multiple challenges with Nalm6 tumor cells, whereas unedited iNK cells were limited in their serial killing effect. The data supports the conclusion that the CISH and TGFβRII edits result in prolonged enhancement of cell killing.

[0341] Finally, edited iNK cells (CISH/TGFβRII DKO iNK cells) were assayed for their ability to kill tumor targets in an in vivo model. To this end, an established NOD scid gamma (NSG) xenograft model was utilized in an assay as depicted in FIG. 15A. Briefly, 1×10.sup.6 SK-OV-3 cells engineered to express luciferase were injected intraperitoneally (IP) at day 0. On day 3, the inoculated mice were imaged using an In vivo imaging system (IVIS) and randomized into 3 groups. The next day (day 4), 20×10.sup.6 unedited iNKs or CISH/TGFβRII DKO iNKs were administered by IP injection, while a third group was injected with vehicle as a control. Following inoculation of the animals with tumor cells, animals were imaged once a week to measure tumor burden over time. FIG. 15B depicts the bioluminescence of the tumors in the individual mice in the 3 different groups (n=9 in each group), vehicle, unedited iNKs, and CISH/TGFβRII DKO iNKs. The average tumor burden over time for these same animals is depicted in FIG. 15C. A two way anova analysis was performed on the data, and CISH/TGFβRII DKO iNK treated animals had significantly less tumor burden as measured by bioluminescence when compared to animals treated with unedited iNKs (p value: 0.0004). By 10 days post-tumor implantation, mice injected with the CISH/TGFβRII DKO iNKs exhibited a significant reduction in the size of their tumors relative to mice injected with the vehicle controls or the unedited iNKs. The overall reduction in tumor size is seen for several days, and at least until 35 days post-tumor implantation. These data show that the edited DKO iNKs were actively killing tumor cells in this in vivo model.

[0342] Overall, these results demonstrate that unedited and CISH/TGFβRII DKO iPSCs can be differentiated into iNK cells exhibiting canonical NK cell markers. Additionally, CISH/TGFβRII DKO iNK cells demonstrated enhanced anti-tumor activity against tumor cell lines derived from both solid and hematological malignancies.

Example 3: ADORA2A Edited iPSCs Give Rise to Edited iNKs with Enhanced Function

[0343] ADORA2A is another target gene of interest, the loss of which is hypothesized to affect NK cell function in a tumor microenvironment (TME). The ADORA2A gene encodes a receptor that responds to adenosine in the TME, resulting in the production of cAMP which functions to drive a number of inhibitory effects on NK cells. We hypothesized that knocking out the function of ADORA2A could enhance iNK cell function. Utilizing a similar approach to the one described in Examples 1 and 2, the PCS iPSC line was edited using a RNP having an engineered Cas12a with three amino acid substitutions (M537R, F870L, and H800A (SEQ ID NO: 1148)) and a gRNA specific to ADORA2A (except that 4 μM RNP was delivered to cells rather than 2 μM RNP). As described in Example 1, the gRNA was generated with a targeting domain consisting of RNA, an AsCpf1 scaffold of the sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 1153) located 5′ of the targeting domain, and a 25-mer DNA extension of the sequence ATGTGTTTTTGTCAAAAGACCTTTT (SEQ ID NO: 1154) at the 5′ terminus of the scaffold sequence. The ADORA2A gRNA sequence is shown in Table 11.

TABLE-US-00013 TABLE 11 Guide RNA sequence gRNA Targeting Full Length gRNA Target Domain Sequence Sequence ADORA2A CCAUCGGCCUGACUCCC ATGTGTTTTTGTCAAAAGACCTT 4113 AUG TTrUrArArUrUrUrCrUrArCr (SEQ ID NO: 1159) UrCrUrUrGrUrArGrArUrCrC rArUrCrGrGrCrCrUrGrArCr UrCrCrCrArUrG(SEQ ID NO: 1160)

[0344] The bulk editing rate by the Cas12a RNP prior to clonal selection was 49% as determined by next-generation sequencing (NGS). Nonetheless, several clones that had both ADORA2A alleles edited were identified, expanded and differentiated. To determine whether an ADORA2A edited iPSC could yield CD45.sup.+ CD56+ iNKs, both bulk and singled ADORA2A KO clones were differentiated using the NKMACS+serum protocol as described in Example 2 (FIG. 8C). As shown in FIG. 16A, edited iPSCs differentiated to iNKs with similar NK cell marker expression compared to unedited control iPSCs.

[0345] To confirm that Cas12a-mediated ADORA2A editing resulted in a functional deletion of the gene, cAMP accumulation in response to treatment with 5′-N-ethylcarboxamide adenosine (“NECA”, a more stable adenosine analog that acts as an ADORA2A agonist) was assessed in both the edited and unedited control iNKs. Edited cells with a functional knockout of ADORA2A would not be expected to accumulate as much cAMP in the cells in response to NECA relative to cells with functional ADORA2A. Briefly, iNK cells were treated with varying concentrations of NECA for 15 minutes. The iNK cells were then lysed, and the cAMP in the lysate was then measured using a CisBio cAMP kit. As shown in FIG. 16B, unedited iNKs had increased levels of cAMP accumulation as the concentration of NECA was increased (n=2). Conversely, the ADORA2A (“A2A KOs”) KO iNKs showed minimal production of cAMP at increasing concentrations of NECA, indicating that the Cas12a-induced edits functionally knocked out ADORA2A function. The bulk iNKs (top two A2A KO iNK lines in FIG. 16B) exhibited slightly higher levels of cAMP than the selected ADORA2A KO clones (lower four A2A KO iNK lines in FIG. 16B), as would be expected from the lower editing rates in the bulk population. Based on this molecular evidence of functional ablation of ADORA2A, the iNKs would be expected to be resistant to the inhibitory effects of adenosine in a tumor microenvironment.

[0346] The ADORA2A KO iNKs were also tested in an in vitro NALM6 serial killing assay as described in Example 2, with one main difference: 100 μM of NECA was added in place of TGFβ. The ADORA2A KO iNKs exhibited enhanced serial killing relative to the wild type iNKs in the presence of NECA, indicating that the ADORA2A KO iNKs were resistant to NECA inhibition (FIG. 16C). As a result, the ADORA2A KO iNK cells would be expected to have improved cytotoxicity against tumor cells in the presence of adenosine in the TME relative to unedited iNK cells.

Example 4: Generation of CISH/TGFβRII/ADORA2A Triple Edited (TKO) iPSCs and the Characterization of Differentiated TKO iNKs

[0347] In order to generate CISH, TGFβRII, and ADORA2A triple edited (TKO) iPSCs, two approaches were taken; 1) two step editing in which the CISH/TGFβRII DKO (CR) iPSC clone described in Examples 1 and 2 was edited at the ADORA2A locus via electroporation with an ADORA2A targeting RNP (as described in Example 3), and 2) simultaneous editing of PCS iPS cells with all 3 RNPs, one for each target gene. Both strategies utilized the editing protocol briefly described in Example 1. In the case of simultaneous editing, the total RNP concentration was 8 μM (Cish:2 μM+ TGFβRII:2 μM+ADORA2A:4 μM). Regardless of the approach, cells were plated, expanded and colonies were picked as described above. Using NGS to analyze gDNA harvested from the iPSCs, it was determined that the bulk editing rates were 96.70%, 97.17%, and 90.16% for CISH, TGFβRII and ADORA2A, respectively, when all target genes were edited simultaneously. Picked colonies that had Insertions and/or Deletions (InDels) at all 6 alleles were selected for further analysis.

[0348] Similar to the analysis described in Example 1, unedited iPSCs and the edited iPSCs were differentiated to iNKs using the NK MACS+Serum condition (described in FIG. 8C) and assessed by flow cytometry at different time points, including at day 25, day 32, and day 39 in culture. As shown in FIG. 17A, analysis of the different NK surface markers revealed no major differences between clones that were generated by the two-step editing method (CR+A 8) or the simultaneous editing method (CRA 6). Both TKO clones (CR+A 8 and CRA 6) showed similar expression profiles to the unedited iNKs (Wt) at each time point. When the TKO iNK cells were analyzed for their responsiveness to NECA (as described in Example 3), both TKO iNKs had little to no cAMP accumulation (FIG. 17B), demonstrating that ADORA2A was functionally knocked out. By contrast, the unedited iNKs demonstrated a NECA dose dependent increase in cAMP (FIG. 17B). These results indicate that the TKO iNKs would be expected to be resistant to the inhibitory effects of adenosine in the TME. Finally, the CISH/TGFβRII/ADORA2A TKO iNKs were assessed alongside CISH/TGFβRII DKO iNKs, ADORA2A single KO (SKO) iNKs, and unedited iNKs in a 3D tumor cell killing assay. This assay was performed as described in Example 2 with IL-15 and TGFβ but without NECA. Interestingly, both the TKO (CRA6) and DKO (CR) iNKs outperformed the unedited iNKs in killing the tumor cells, indicating that both multiplex edited iNKs have enhanced function over unedited control cells (FIG. 17C). These results show that knocking out ADORA2A does not negatively affect the ability of iNKs having CISH and TGFBRII KOs to kill tumor spheroid cells.

Example 5: Selection of CISH, TGFβRII, ADORA2A, TIGIT, and NKG2A Targeting gRNAs

[0349] The cutting efficiency of CISH, TGFBRII, ADORA2A, TIGIT, and NKG2A Cas12a guide RNAs were further tested. Guide RNAs were screened by complexing commercially synthesized gRNAs with Cas12a in vitro and delivering gRNA/Cas12a ribonucleoprotein (RNP) to IPSCs via electroporation. The iPSCs were edited using a RNP having an engineered Cas12a with three amino acid substitutions (M537R, F870L, and H800A (SEQ ID NO: 1148)). The gRNAs were generated with a targeting domain consisting of RNA, an AsCpf1 scaffold of the sequence UAAUUUCUACUCUUGUAGAU (SEQ ID NO: 1153) located 5′ of the targeting domain, and a 25-mer DNA extension of the sequence ATGTGTTTTTGTCAAAAGACCTTTT (SEQ ID NO: 1154) at the 5′ terminus of the scaffold sequence. Table 12 provides the targeting domains of the guide RNAs that were tested for editing activity.

TABLE-US-00014 TABLE 12 guide RNA sequences Target gRNA Targeting Domain Sequence TGFβRII UGAUGUGAGAUUUUCCACCUG (SEQ ID NO: 1161) CISH ACUGACAGCGUGAACAGGUAG (SEQ ID NO: 1162) ADORA2A CCAUCGGCCUGACUCCCAUGC (SEQ ID NO: 1163) ADORA2A CCAUCACCAUCAGCACCGGGU (SEQ ID NO: 1164) ADORA2A CCUGUGUGCUGGUGCCCCUGC (SEQ ID NO: 1165) TIGIT UGCAGAGAAAGGUGGCUCUAU (SEQ ID NO: 1166) TIGIT UCUGCAGAAAUGUUCCCCGUU (SEQ ID NO: 1167) TIGIT UAGGACCUCCAGGAAGAUUCU (SEQ ID NO: 1168) NKG2A GCAACUGAACAGGAAAUAACC (SEQ ID NO: 1169) NKG2A GUUGCUGCCUCUUUGGGUUUG (SEQ ID NO: 1170) NKG2A AAGGGAAUGACAAAACCUAUC (SEQ ID NO: 1171)

[0350] In brief, 100,000 iPSCs/well were transfected with the RNP of interest, cells were incubated at 37° C. for 72 hours, and then harvested for DNA characterization. iPSCs were transfected with gRNA/Cas12a RNPs at various concentrations. The percentage editing events were determined for eight different RNP concentrations ranging from negative control (0 mM), to 8 mM.

[0351] As shown in FIG. 18 panel 1, the TGFβRII gRNA (SEQ ID NO: 1161) exhibited an EC50 of ˜79 nM RNP. As shown in FIG. 18 panel 2, the CISH gRNA (SEQ ID NO: 1162) exhibited an EC50 of ˜50 nM RNP. As shown in FIG. 18 panel 3, an ADORA2A gRNA (SEQ ID NO: 1163) included in RNP2960 exhibited an EC50 of ˜63 nM RNP, while an ADORA2A gRNA (SEQ ID NO: 1164) included in RNP3109, or gRNA (SEQ ID NO: 1165) included in RNP3108 exhibited EC50 values of ˜493 nM and ˜280 nM RNP respectively. As shown in FIG. 18 panel 4, a TIGIT gRNA (SEQ ID NO: 1166) included in RNP2892 exhibited an EC50 of ˜29 nM RNP, while a TIGIT gRNA (SEQ ID NO: 1167) included in RNP3106, or gRNA (SEQ ID NO: 167) included in RNP3107 exhibited EC50 values of ˜1146 nM and ˜40 nM RNP respectively. As shown in FIG. 18 panel 5, a NKG2A gRNA (SEQ ID NO: 1169) included in RNP19142 exhibited an EC50 of —8 nM RNP, while a NKG2A gRNA (SEQ ID NO: 1170) included in RNP3069, or gRNA (SEQ ID NO: 1171) included in RNP2891 exhibited EC50 values of ˜12 nM and ˜13 nM RNP respectively.

EQUIVALENTS

[0352] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.