Methods and compositions for modification of protospacer adjacent motif specificity of CAS12A

A method of editing a target nucleic acid, comprising

• contacting the target nucleic acid with:
• (a) a LbCas12a polypeptide having a mutation, wherein the mutation is an arginine at position 532 and at 595, and a guide nucleic acid,
• (b) a LbCas12a polypeptide having a mutation, wherein the mutation is an arginine at position 532 and 542 and a valine at position 538, each of which position in the LbCas12a polypeptide of (a) and/or (b) is in reference to position numbering of the amino acid sequence of SEQ ID NO:180, and a guide nucleic acid,
• wherein the LbCas12a polypeptide of (a) recognizes a PAM sequence in the target nucleic acid of
• TTAA, TTAC, GTCT, ACTA, ACTC, ACTG, ATTA, ATTC,
• ATTG, CCTA, CCTC, CCTG, CTTA, CTTC, CTTG, GCTA,
• GCTC, GCTG, GTTA, GTTC, and/or GTTG,
• and the LbCas12a polypeptide of (b) recognizes a PAM sequence in the target nucleic acid of
• AACC, TACC, AATC, GATA, AATA, AATG, GATG, and/or
• GATC,
• and/or
• (c) a fusion protein and a guide nucleic acid, the fusion protein comprising (i) the LbCas12a polypeptide of (a) and (b), (ii) the LbCas12a polypeptide of (a) and a polypeptide of interest, or (iii) the LbCas12a polypeptide of (b) and a polypeptide of interest;
• thereby editing the target nucleic acid.

A method of editing a target nucleic acid, comprising contacting a cell or a cell free system comprising the target nucleic acid with:

• (a) a LbCas12a polypeptide having a mutation, wherein the mutation is an arginine at position 532 and at 595, and a guide nucleic acid,
• (b) a LbCas12a polypeptide having a mutation, wherein the mutation is an arginine at position 532 and 542 and a valine at position 538, each of which position in the LbCas12a polypeptide of (a) and/or (b) is in reference to position numbering of the amino acid sequence of SEQ ID NO:180, and a guide nucleic acid,
• wherein the LbCas12a polypeptide of (a) recognizes a PAM sequence in the target nucleic acid of
• TTAA, TTAC, GTCT, ACTA, ACTC, ACTG, ATTA, ATTC,
• ATTG, CCTA, CCTC, CCTG, CTTA, CTTC, CTTG, GCTA,
• GCTC, GCTG, GTTA, GTTC, and/or GTTG,
• and the LbCas12a polypeptide of (b) recognizes a PAM sequence in the target nucleic acid of
• AACC, TACC, AATC, GATA, AATA, AATG, GATG, and/or
• GATC,
• or an expression cassette or vector comprising the same and a guide nucleic acid, or an expression cassette or vector comprising the same;
• and/or
• (c) a nucleic acid construct encoding a complex comprising a fusion protein and a guide nucleic acid, the fusion protein comprising (i) the LbCas12a polypeptide of (a) and (b), (ii) the LbCas12a polypeptide of (a) and a polypeptide of interest, or (iii) the LbCas12a polypeptide of (b) and a polypeptide of interest, or an expression cassette or vector comprising the same, thereby editing the target nucleic acid.