ENGINEERED CELLS FOR THERAPY

BACKGROUND

There remains a need for engineered cells for therapeutic interventions, as well as for methods of culturing stem cells, such as embryonic stem cells and induced pluripotent cells, such that pluripotency is maintained.

SUMMARY

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 A2a receptor (ADORA2A). In some embodiments, 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.

In some embodiments, 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. In some embodiments, the TGF beta receptor is a TGF beta receptor II (TGFβRII).

In some embodiments, 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.

In some embodiments, the disclosure features a differentiated cell, wherein the differentiated cell is a daughter cell of a pluripotent human stem cell described herein. In some embodiments, the differentiated cell is an immune cell. In some embodiments, the differentiated cell is a lymphocyte. In some embodiments, the differentiated cell is a natural killer cell. In some embodiments, the stem cell is a human induced pluripotent stem cell (iPSC), and wherein the differentiated daughter cell is an iNK cell. In some embodiments, 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.

In some embodiments, any of the cells described herein comprises one or more additional genomic edits. In some embodiments, 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 (e.g., non-naturally occurring 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.

In another aspect, the disclosure features 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). In some embodiments, 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). In some embodiments, 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.

In some embodiments, 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. In some embodiments, TGF beta receptor is a TGF beta receptor II (TGFβRII).

In some embodiments, 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.

In some embodiments, the disclosure features a differentiated cell, wherein the differentiated cell is a daughter cell of a human iPSC described herein. In some embodiments, the differentiated cell is an immune cell. In some embodiments, the differentiated cell is a lymphocyte. In some embodiments, the differentiated cell is a natural killer cell. In some embodiments, the differentiated daughter cell is an iNK cell. In some embodiments, 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.

In some embodiments, any of the cells described herein comprises one or more additional genomic edits. In some embodiments, 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 (e.g., non-naturally occurring 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.

In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence according to any one of SEQ ID NO: 258-364, 1155, and 1162. In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 258-364, 1155, and 1162. In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, and (ii) a 5′ extension sequence depicted in Table 3. In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence according to any one of SEQ ID NO: 29-257, 1157, and 1161. In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 29-257, 1157, and 1161. In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) a 5′ extension sequence depicted in Table 3. In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence according to any one of SEQ ID NO: 827-1143, 1159, and 1163. In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 827-1143, 1159, and 1163. In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) a 5′ extension sequence depicted in Table 3. In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence according to any one of SEQ ID NO: 258-364, 1155, and 1162. In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 258-364, 1155, and 1162. In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, and (ii) a 5′ extension sequence depicted in Table 3. In some embodiments, a genomic edit resulting in loss of function of CISH in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148), and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence according to any one of SEQ ID NO: 29-257, 1157, and 1161. In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 29-257, 1157, and 1161. In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148), and (ii) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) a 5′ extension sequence depicted in Table 3. In some embodiments, a genomic edit resulting in loss of function of TGFβRII in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148), and (ii) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence according to any one of SEQ ID NO: 827-1143, 1159, and 1163. In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 827-1143, 1159, and 1163. In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) a 5′ extension sequence depicted in Table 3. In some embodiments, a genomic edit resulting in loss of function of ADORA2A in any of the cells described herein was produced using a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or human induced pluripotent stem cell) with one or more of: an RNA-guided nuclease and a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, 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 or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, 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 or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 827-1143, 1159, and 1163.

In some embodiments, the method comprises contacting the cell with one or more of: (1) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, and (ii) a 5′ extension sequence depicted in Table 3; (2) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) a 5′ extension sequence depicted in Table 3; and (3) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) a 5′ extension sequence depicted in Table 3.

In some embodiments, the method comprises contacting the cell with one or more of: (1) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; (2) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; and (3) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In some embodiments, the RNA-guided nuclease is a Cas12a variant. In some embodiments, the Cas12a variant comprises one or more amino acid substitutions selected from M537R, F870L, and H800A. In some embodiments, the Cas12a variant comprises amino acid substitutions M537R, F870L, and H800A. In some embodiments, the Cas12a variant comprises an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148.

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with one or more of: a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 258-364, 1155, and 1162; a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 29-257, 1157, and 1161; and/or a ribonucleoprotein (RNP) complex comprising (i) an RNA-guided nuclease (e.g., a Cas12a variant, e.g., a Cas12a variant comprising 1, 2, or 3 of the amino acid substitutions selected from M537R, F870L, and H800A, e.g., a Cas12a variant comprising an amino acid sequence having 90%, 95%, or 100% identity to SEQ ID NO:1148) and (ii) a guide RNA comprising a targeting domain sequence comprising or consisting of a nucleotide sequence that is identical to, or differs by no more than 1, 2, or 3 nucleotides from, any one of SEQ ID NO: 827-1143, 1159, and 1163.

In some embodiments, the method comprises contacting the cell with one or more of: (1) an RNP comprising a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, and (ii) a 5′ extension sequence depicted in Table 3; (2) an RNP comprising a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) a 5′ extension sequence depicted in Table 3; and (3) an RNP comprising a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) a 5′ extension sequence depicted in Table 3.

In some embodiments, the method comprises contacting the cell with one or more of: (1) an RNP comprising a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; (2) an RNP comprising a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; and (3) an RNP comprising a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence.

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with (i) a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1155 or 1162; a guide RNA comprises a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161; and a guide RNA comprises a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163; and (ii) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof).

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with (1) an RNP comprising (i) a guide RNA comprising a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1155 or 1162; and (ii) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof); (2) an RNP comprising (i) a guide RNA comprises a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof); and (3) an RNP comprising (i) a guide RNA comprises a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof).

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with (1) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, and (ii) a 5′ extension sequence depicted in Table 3; (2) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) a 5′ extension sequence depicted in Table 3; (3) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) a 5′ extension sequence depicted in Table 3; and (4) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof).

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with (1) an RNP comprising (a) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, and (ii) a 5′ extension sequence depicted in Table 3; and (b) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof); (2) an RNP comprising (a) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, and (ii) a 5′ extension sequence depicted in Table 3; and (b) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof); and (3) an RNP comprising (a) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, and (ii) a 5′ extension sequence depicted in Table 3; and (b) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof).

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with (1) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; (2) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence (3) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; and (4) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof).

In another aspect, the disclosure features a method of making a cell, e.g., a cell described herein, the method comprising contacting a cell (e.g., a pluripotent human stem cell or a human induced pluripotent stem cell) with (1) an RNP comprising (a) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:1155 or 1162, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; and (b) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof); (2) an RNP comprising (a) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1157 or 1161, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; and (b) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof); and (3) an RNP comprising (a) a guide RNA comprising (i) a targeting domain sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1159 or 1163, (ii) a scaffold sequence comprising or consisting of the nucleotide sequence of SEQ ID NO: 1153 located 5′ of the targeting domain sequence, and (iii) the nucleotide sequence of SEQ ID NO:1154 at the 5′ of the scaffold sequence; and (b) an RNA-guided nuclease comprising an amino acid sequence having 90%, 95%, or 100% identity to one of SEQ ID NO:1144-1151 (or a portion thereof).

In another aspect, the disclosure features a pluripotent human stem cell, wherein the stem cell comprises a disruption in the transforming growth factor beta (TGF beta) signaling pathway. In some embodiments, the stem cell comprises a genetic modification that results in a loss of function of an agonist of the TGF beta signaling pathway. In some embodiments, the genetic modification is a genomic edit. In some embodiments, the stem cell comprises a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor. In some embodiments, the TGF beta receptor is a TGF beta receptor II (TGFβRII).

In some embodiments, the stem cell further comprises a loss of function of an antagonist of interleukin signaling. In some embodiments, the stem cell further comprises a genomic modification that results in the loss of function of an antagonist of interleukin signaling. In some embodiments, the antagonist of interleukin signaling is an antagonist of the IL-15 signaling pathway and/or of the IL-2 signaling pathway.

In some embodiments, the stem cell comprises a loss of function of Cytokine Inducible SH2 Containing Protein (CISH). In some embodiments, the stem cell comprises a genomic modification that results in the loss of function of CISH.

In some embodiments, the stem cell comprises one or more additional genetic modifications. In some embodiments, the stem cell: (1) comprises at least one genetic modification 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 (e.g., non-naturally occurring 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 genetic modification 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.

In some embodiments, the stem cell comprises a genetic modification in a TGFβRII gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:29-257, 1157, and 1161. In some embodiments, the stem cell comprises a genetic modification in a CISH gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:258-364, 1155, and 1162. In some embodiments, the stem cell comprises a genetic modification in a ADORA2A gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:827-1143, 1159, and 1163. In some embodiments, the stem cell comprises a genetic modification in a TIGIT gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:631-826. In some embodiments, the stem cell comprises a genetic modification in a B2M gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:365-576. In some embodiments, the stem cell comprises a genetic modification in a NKG2A gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:577-630.

In another aspect, the disclosure features a differentiated cell, wherein the differentiated cell is a daughter cell of a pluripotent human stem cell described herein. In some embodiments, the differentiated cell is an immune cell. In some embodiments, the differentiated cell is a lymphocyte. In some embodiments, the differentiated cell is a natural killer cell. In some embodiments, the stem cell is a human induced pluripotent stem cell (iPSC), and wherein the differentiated daughter cell is an induced Natural Killer (iNK) cell.

In some embodiments, the differentiated 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.

In some embodiments, the differentiated stem cell comprises one or more additional genetic modifications. In some embodiments, the differentiated stem cell: (1) comprises at least one genetic modification 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 (e.g., non-naturally occurring 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 genetic modification 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.

In some embodiments, the differentiated stem cell comprises a genetic modification in a TGFβRII gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:29-257, 1157, and 1161. In some embodiments, the differentiated stem cell comprises a genetic modification in a CISH gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:258-364, 1155, and 1162. In some embodiments, the differentiated stem cell comprises a genetic modification in a ADORA2A gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:827-1143, 1159, and 1163. In some embodiments, the differentiated stem cell comprises a genetic modification in a TIGIT gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:631-826. In some embodiments, the differentiated stem cell comprises a genetic modification in a B2M gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:365-576. In some embodiments, the differentiated stem cell comprises a genetic modification in a NKG2A gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:577-630.

In another aspect, the disclosure features a method of culturing a pluripotent human stem cell, comprising culturing the stem cell in a medium comprising activin. In some embodiments, the pluripotent human stem cell is an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the pluripotent human stem cell does not express TGFβRII. In some embodiments, the pluripotent human stem cell is genetically engineered not to express TGFβRII. In some embodiments, the pluripotent human stem cell is genetically engineered to knock out a gene encoding TGFβRII.

In some embodiments, the activin is activin A. In some embodiments, the medium does not comprise TGFβ.

In some embodiments, 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). In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, the pluripotent human stem cell, or its progeny, is further differentiated into a natural killer (NK) cell.

In some embodiments, the pluripotent human stem cell is differentiated into an NK cell in a medium comprising human serum. In some embodiments, the medium comprises NKMACS+human serum (e.g., 5%, 10%, 15%, 20% or more human serum). In some embodiments, the NK cells exhibit improved cellular expansion, increased NK maturity (as exhibited by increased marker expression (e.g., CD45, CD56, CD16, and/or KIR)), and/or increased cytotoxicity, relative to an NK cell differentiated in a media without serum.

In some embodiments, the pluripotent human stem cell (1) comprises at least one genetic modification 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 (e.g., non-naturally occurring 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 genetic modification 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.

In some embodiments, the pluripotent human stem cell comprises a genetic modification in a TGFβRII gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:29-257, 1157, and 1161. In some embodiments, the pluripotent human stem cell comprises a genetic modification in a CISH gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:258-364, 1155, and 1162. In some embodiments, the pluripotent human stem cell comprises a genetic modification in a ADORA2A gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:827-1143, 1159, and 1163. In some embodiments, the pluripotent human stem cell comprises a genetic modification in a TIGIT gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:631-826. In some embodiments, the pluripotent human stem cell comprises a genetic modification in a B2M gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:365-576. In some embodiments, the pluripotent human stem cell comprises a genetic modification in a NKG2A gene made using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:577-630.

In some embodiments, the method further comprises (1) genetically modifying the pluripotent human stem cell such that the pluripotent human stem cell expresses a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a non-naturally occurring 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) genetically modifying the pluripotent human stem cell to lose 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.

In some embodiments, the method further comprises genetically modifying a TGFβRII gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:29-257, 1157, and 1161. In some embodiments, the method further comprises genetically modifying a CISH gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:258-364, 1155, and 11162. In some embodiments, the method further comprises genetically modifying a ADORA2A gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:827-1143, 1159, and 1163. In some embodiments, the method further comprises genetically modifying a TIGIT gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:631-826. In some embodiments, the method further comprises genetically modifying a B2M gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:365-576. In some embodiments, the method further comprises genetically modifying a NKG2A gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:577-630.

In another aspect, the disclosure features 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. In some embodiments, the stem cell comprises a genetic modification that results in a loss of function of an agonist of the TGF beta signaling pathway. In some embodiments, the genetic modification is a genomic edit. In some embodiments, the stem cell comprises a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor. In some embodiments, the TGF beta receptor is a TGF beta receptor II (TGFβRII).

In some embodiments, the pluripotent human stem cell: (1) comprises at least one genetic modification 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 (e.g., non-naturally occurring 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 genetic modification 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.

In another aspect, the method comprises 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.

In some embodiments, the iNK is differentiated from a pluripotent human stem cell cultured in a medium comprising activin. In some embodiments, the method further comprises culturing the pluripotent human stem cell in a medium comprising activin before and/or during the differentiating step.

In some embodiments, the method further comprises disrupting the transforming growth factor beta (TGF beta) signaling pathway in the pluripotent human stem cell. In some embodiments, the stem cell comprises a genetic modification that results in a loss of function of an agonist of the TGF beta signaling pathway. In some embodiments, the genetic modification is a genomic edit. In some embodiments, the stem cell comprises a loss of function of a TGF beta receptor or a dominant-negative variant of a TGF beta receptor. In some embodiments, the TGF beta receptor is a TGF beta receptor II (TGFβRID.

In some embodiments, the method further comprises (1) genetically modifying the pluripotent human stem cell such that the pluripotent human stem cell expresses a nucleic acid sequence encoding: (i) a chimeric antigen receptor (CAR); (ii) a FcγRIII (CD16) or a variant (e.g., non-naturally occurring 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) genetically modifying the pluripotent human stem cell to lose 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.

In some embodiments, the method further comprises genetically modifying a TGFβRII gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:29-257, 1157, and 1161. In some embodiments, the method further comprises genetically modifying a CISH gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:258-364, 1155, and 1162. In some embodiments, the method further comprises genetically modifying a ADORA2A gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:827-1143, 1159, and 1163. In some embodiments, the method further comprises genetically modifying a TIGIT gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:631-826. In some embodiments, the method further comprises genetically modifying a B2M gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:365-576. In some embodiments, the method further comprises genetically modifying a NKG2A gene using an RNA-guided nuclease and a gRNA molecule comprising a targeting domain sequence that is the same as, or differs by no more than 3 nucleotides from, any one of SEQ ID NOs:577-630.

In another aspect, the disclosure features a method of culturing a stem cell, for example, a human stem cell, such as, e.g., a human embryonic stem cell, a human induced pluripotent stem cell, or a human pluripotent stem cell, comprising culturing the stem cell in a medium that comprises activin, e.g., activin A. In some embodiments, the stem cell is an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the stem cell comprises a modification, e.g., a genetic modification, that disrupts a TGF (transforming growth factor) signaling pathway in the stem cell. In some embodiments, the genetic modification is a modification that disrupts (e.g., reduces or abolishes) TGF beta signaling in the stem cell. For example, in some embodiments, the modification is a modification of a gene encoding a protein of the TGF beta signaling pathway, such as a TGF beta receptor. In some embodiments, the modification results in a loss of function and/or a loss of expression of the protein of the TGF beta signaling pathway. In some embodiments, the modification results in a knockout of the protein of the TGF beta signaling pathway. In some embodiments, the stem cell does not express a functional TGFβ receptor protein, e.g., the stem cell does not express a TGFβRII protein or does not express a functional TGFβRII protein. In some embodiments, the stem cell expresses a dominant negative variant of an agonist of a protein of the TGF beta signaling pathway, e.g., a dominant negative variant of TGFβRII. In some embodiments, the stem cell over-expresses an antagonist of the TGF beta signaling pathway. In some embodiments, the stem cell does not express TGFβRII. In some embodiments, the stem cell is genetically engineered not to express TGFβRII. In some embodiments, the stem cell is genetically engineered to knock out a gene encoding TGFβRII. In some embodiments, the genetic modification is a modification that enhances (e.g., maintains or increases) IL-15 signaling in the stem cell. For example, in some embodiments, the modification is a modification of a gene encoding a protein that acts on the IL-15 signaling pathway, such as Cytokine Inducible SH2 Containing Protein (CISH), a negative regulator of IL-15 signaling. In some embodiments, the modification results in a loss of function and/or a loss of expression of the protein that acts on the IL-15 signaling pathway. In some embodiments, the modification results in a knockout of the protein that acts on the IL-15 signaling. In some embodiments, the stem cell does not express a functional CISH gene, e.g., the stem cell does not express a CISH protein or does not express a functional CISH protein. In some embodiments, the stem cell does not express CISH. In some embodiments, the stem cell is genetically engineered not to express CISH. In some embodiments, the stem cell is genetically engineered to knock out a gene encoding CISH (i.e., CISH, cytokine-inducible SH2-containing protein). In some embodiments, the stem cell does not express TGFβRII or CISH. In some embodiments, the stem cell is genetically engineered not to express each of TGFβRII or CISH. In some embodiments, the stem cell is genetically engineered to knock out a gene encoding TGFβRII and a gene encoding CISH in the same cell (double KO). In some embodiments, the stem cell has been edited, e.g., via CRISPR/Cas editing or other suitable technology, to disrupt a gene encoding a gene product involved in TGF signaling, e.g., in TGF beta signaling, such as, for example, a gene encoding a TGF beta RII protein, or e.g., IL-15 signaling, such as, for example, a gene encoding a CIS protein, within the genome of the cell. In some embodiments, e.g., in embodiments, where two copies or alleles of the gene encoding a gene product involved in TGF signaling and/or IL-15 signaling is present in the cell, the cell is modified (e.g., edited), so that both copies or alleles are modified, e.g., in that expression of the gene, or of a functional gene product encoded by the gene, is disrupted, decreased, or abolished from both alleles.

In some embodiments, the activin is activin A. In some embodiments, the medium does not comprise TGFβ.

In some embodiments, 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). In some embodiments, at one or more times during or following the culturing step, the human stem cell maintains pluripotency (e.g., exhibits one or more measure of pluripotency). In some embodiments, at one or more times during or following the culturing step, the 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. In some embodiments, at a time during or following the culturing step, the human stem cell retains the capacity to differentiate into cells of endoderm, mesoderm, and ectoderm germ layers.

In another aspect, the disclosure features a cell culture comprising (i) an embryonic stem cell or an induced pluripotent stem cell and (ii) a cell culture medium comprising activin, wherein the embryonic stem cell or an induced pluripotent stem cell is genetically engineered not to express TGFβRII and/or CISH.

In some embodiments, an RNA-guided nuclease is a Cas12a variant. In some embodiments, the Cas12a variant comprises amino acid substitutions selected from M537R, F870L, and H800A. In some embodiments, the Cas12a variant comprises amino acid substitutions M537R, F870L, and H800A. In some embodiments, the Cas12a variant comprises an amino acid sequence according to SEQ ID NO: 1148.

BRIEF DESCRIPTION OF THE DRAWING

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.

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).

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).

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).

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

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

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.

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

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

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

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).

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.

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.

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

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.

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.

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.

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.

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.

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

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.

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.

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”).

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).

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.

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.

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).

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).

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.

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).

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

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.

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.

FIG. 15A is a schematic of an in-vivo tumor killing assay. Mice were intraperitoneally inoculated with 1×10⁶SKOV3-luc cells, mice are randomized, and 4 days later, 20×10⁶iNK 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).

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.

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.

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.

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.

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.

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.

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.

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).

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

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

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

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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

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.

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.

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.

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.

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).

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.

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.

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.

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 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

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.

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.

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.

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.

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 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)

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).

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.

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.

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.

Stem Cells

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.

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).

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.

iPSCs

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.

Genetically Engineered Stem Cells

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.

TGF Signaling

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.

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.

Additional Loss-of-Function Modifications

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Gain-of-Function Modifications

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).

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.

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).

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.

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.

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.

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).

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.

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.

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.

Generation of iNK Cells

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.

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.

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.

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.

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.

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.

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.

In certain embodiments, a somatic donor cell is a CD8⁺ T cell, a CD8⁺naïve T cell, a CD4⁺ central memory T cell, a CD8⁺ central memory T cell, a CD4⁺ effector memory T cell, a CD4⁺ effector memory T cell, a CD4⁺ T cell, a CD4⁺ 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⁺ T cell, a TH1 CD4⁺ T cell, a TH2 CD4⁺ T cell, a TH9 CD4⁺ T cell, a CD4⁺ Foxp3⁺ T cell, a CD4⁺ CD25⁺ CD127⁻ T cell, or a CD4⁺ CD25⁺ CD127⁻ Foxp3⁺ T cell.

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.

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.

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⁺ cell, CD34+CD90⁺ cell, CD34⁺ CD38⁻ cell, CD34⁺ CD90⁺ CD49f⁺ CD38⁻ CD45RA⁻ cell, CD105⁺ cell, CD31⁺, or CD133⁺ cell, or a CD34⁺ CD90⁺ CD133⁺ cell. In some embodiments, a donor cell is one or more of an umbilical cord blood CD34⁺ HSPC, umbilical cord venous endothelial cell, umbilical cord arterial endothelial cell, amniotic fluid CD34⁺ cell, amniotic fluid endothelial cell, placental endothelial cell, or placental hematopoietic CD34⁺ cell. In some embodiments, a donor cell is one or more of a mobilized peripheral blood hematopoietic CD34⁺ 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.

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

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.

Genome Editing Systems

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.

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.

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.

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.

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.

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).

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.

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.

Guide RNA (gRNA) Molecules

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.

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”)).

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.

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.

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.

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).

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.

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.

gRNA Design

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.

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.

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.

gRNA Modifications

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.

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

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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:

embedded image

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

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.

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).

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:

embedded image

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

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

embedded image

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

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.

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., NH₂, 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.

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., NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH₂)_n-amino (wherein amino can be, e.g., NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

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′→2′)).

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.

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.

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.

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.

Exemplary gRNAs

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.

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 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

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 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

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 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

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.

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 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

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 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

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 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

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.

RNA-Guided Nucleases

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.

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.

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.

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).

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.

Cas9

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).

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.

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.

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).

Cpf1

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.

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.

Nuclease Variants

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.

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.

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.

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).

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.

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.

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.

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:

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

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.

Nucleic Acids Encoding RNA-Guided Nucleases

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).

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.

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.

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.

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

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

Activin

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)).

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

Culture Methods

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).

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.

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.

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.

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.

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)).

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.

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).

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).

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.

Methods of Characterization

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.).

Methods of Use

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.

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).

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.

Pharmaceutical Compositions

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.

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.

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.

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.

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.

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.

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.

Cancers

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.

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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 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)

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).

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.

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.

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.

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.

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

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.

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).

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.

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)

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).

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).

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.

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.

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.

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, 10×10⁶Nalm6 tumor cells (a B cell leukemia cell line) and 2×10⁵iNKs 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 5×10³Nalm6 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.

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, 1×10⁶SK-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), 20×10⁶unedited 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.

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

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 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)

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⁺ 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.

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.

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

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.

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

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 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)

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.

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

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.

Number	Date	Country
63115592	Nov 2020	US
63025735	May 2020	US
62950063	Dec 2019	US

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