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 an engineered protein and a guide nucleic acid, the engineered protein comprising:
• (a) a first polypeptide that is a first portion of a modified protein, wherein the modified protein comprises an amino acid sequence having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO:180 (LbCas12a) and
• a second polypeptide that is heterologous to the first polypeptide and is not a Type V CRISPR-Cas effector polypeptide,
• wherein the first polypeptide and second polypeptide are different from each other, and
• wherein the engineered protein comprises a mutation, and (i) the mutation is an arginine at position 680 and at 743 with reference to position numbering of the amino acid sequence of SEQ ID NO:184 (RR), or (ii) the mutation is an arginine at position 680 and 690 and a valine at position 686 with reference to position numbering of the amino acid sequence of SEQ ID NO:185 (RVR),
• wherein the engineered protein of (a)(i) recognizes a PAM sequence in the target nucleic acid of TTAA, TTAC, ACCA, ACCC, ACCG, ATCA, ATCC, ATCG, CCCA, CCCG, CTCA, CTCC, CTCG, GCCA, GCCC, GCCG, GTCA, GTCC, GTCG, GTCT, TCCT, TTCT, ACTA, ACTC, ACTG, ATTA, ATTC, ATTG, CCTA, CCTC, CCTG, CTTA, CTTC, CTTG, GCTA, GCTC, GCTG, GTTA, GTTC, GTTG, TCTA, TCTC, TCTG, TCTT, TTTT, CCCC, TCCA, TCCC, TCCG, TTCA, TTCC, and/or TTCG, and the engineered protein of (a)(ii) recognizes a PAM sequence in the target nucleic acid of AACC, TACC, AATC, GATA, AATA, AATG, GATG, GATC, TATA, TATC, and/or TATG, and/or
• (b) a fusion protein and a guide nucleic acid, the fusion protein comprising the engineered protein of (a) and a polypeptide of interest, thereby editing the target nucleic acid.