Thermostable cas9 nucleases

A method of binding, cleaving, marking or modifying a double stranded target polynucleotide, wherein the double stranded target polynucleotide comprises a target nucleic acid strand comprising a target nucleic acid sequence, and a non-target nucleic acid strand comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence, said method comprising:

• a) designing at least one targeting RNA molecule, wherein the targeting RNA molecule recognizes the target sequence in the target strand, and the non-target strand further comprises a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′;
• b) forming a ribonucleoprotein complex comprising the targeting RNA molecule and a Cas protein, wherein the isolated Cas protein has an amino acid sequence of SEQ ID NO:
• 1 or a sequence of at least 77% identity therewith;
• and
• c) the ribonucleoprotein complex binding, cleaving, marking or modifying the target polynucleotide, and wherein said method is not used in human cells.

A transformed non-human cell, having a double stranded target polynucleotide comprising a target nucleic acid sequence, wherein the double stranded target polynucleotide comprises a target nucleic acid strand, comprising said target nucleic acid sequence, and a non-target nucleic acid strand, comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence, said cell comprising:

• a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) protein having an amino acid sequence of SEQ ID NO:
• 1 or a sequence of at least 77% identity therewith;
• at least one targeting RNA molecule which recognizes the target nucleic acid sequence in the target nucleic acid strand, wherein the non-target strand further comprises a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′;
• and
• an expression vector comprising a nucleic acid encoding at least one of said Cas protein and said targeting RNA molecule.

A nucleoprotein complex comprising a Cas protein, at least one targeting RNA molecule which recognizes a target nucleic acid sequence in a double stranded target polynucleotide, and the target polynucleotide, wherein

• the Cas protein has an amino acid sequence of SEQ ID NO:
• 1 or a sequence of at least 77% identity therewith;
• the double stranded target polynucleotide comprises a target nucleic acid strand, comprising said target nucleic acid sequence, and a non-target nucleic acid strand, comprising a protospacer nucleic acid sequence complementary to the target nucleic acid sequence and a protospacer adjacent motif (PAM) sequence directly adjacent the 3′ end of the protospacer sequence, wherein the PAM sequence comprises 5′-NNNNCNN-3′;
• wherein the nucleoprotein complex is not in a human cell.