CRISPR-cas systems for genome editing

A synthetic composition comprising:

• (a) a Cas protein or functional fragment thereof, comprising an amino acid sequence at least 90% identical to SEQ ID NO:
• 38 or SEQ ID NO:368, wherein the Cas protein or the functional fragment thereof comprises a tri-split RuvC domain and at least one zinc finger motif, and wherein the Cas protein or the functional fragment thereof recognizes a PAM sequence comprising a NNNNCCD nucleotide sequence, wherein N=cytosine, thymine, adenine, or guanine;
• C=cytosine;
• and D=thymine, adenine, or guanine;
• (b) a target double-stranded DNA polynucleotide that comprises the PAM sequence, wherein the target double-stranded DNA polynucleotide is heterologous to the source of the Cas protein;
• and
• (c) a guide polynucleotide comprising a variable targeting domain that comprises a region of complementarity to the target double-stranded DNA polynucleotide;
• wherein the Cas protein recognizes the PAM sequence on the target double-stranded DNA polynucleotide, wherein the guide polynucleotide and the Cas protein form a complex that binds to the target double-stranded DNA polynucleotide;
• wherein the Cas protein comprises DNA binding activity and lacks double-strand DNA cleavage activity.

A method of introducing a targeted edit in a target polynucleotide, the method comprising:

• providing a composition comprising:
• (a) a Cas endonuclease or a functional fragment thereof, comprising a polypeptide at least 90% identical to SEQID NO:
• 38 or SEQ ID NO:368, wherein the Cas endonuclease or functional fragment comprises at least one zinc finger domain, a tri-split RuvC domain, and the following amino acid motifs:
• GxxxG, ExL, CxnC, and Cxn(C or H);
• wherein G=Glycine, E=Glutamate, C=Cysteine, H=Histidine, L=Leucine, x=any amino acid and wherein n=an integer between 0 and 11, and wherein the Cas endonuclease or the functional fragment thereof recognizes a PAM sequence comprising a NNNNCCD nucleotide sequence, wherein N=cytosine, thymine, adenine, or guanine;
• C=cytosine;
• and D=thymine, adenine, or guanine, wherein the Cas endonuclease recognizes the PAM sequence on the target polynucleotide, and wherein the Cas endonuclease comprises DNA binding activity;
• and
• (b) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a portion of the target polynucleotide, wherein the guide polynucleotide and Cas endonuclease form a guide polynucleotide-Cas endonuclease complex that can recognize and bind to the target polynucleotide,
• wherein the guide polynucleotide-Cas endonuclease complex is heterologous to Clostridium novyi.