CRISPR ENZYMES AND SYSTEMS WITH MODIFIED PAM SPECIFICITY
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Aspects of the invention in particular relate to Cpf1 mutants having altered PAM specificity.
1 - 23 . (canceled)
24 . A mutated Cpf1 polypeptide comprising a mutated amino acid residue corresponding to amino acid position S542 of AsCpf1, wherein the mutated amino acid residue corresponding to amino acid position S542 of AsCpf1 is R.
25 . The mutated Cpf1 polypeptide according to claim 24 , wherein the mutated Cpf1 polypeptide further comprises one or more mutated amino acid residues corresponding to amino acid positions K548, M604, K607, or T167 of AsCpf1.
26 . The mutated Cpf1 polypeptide according to claim 25 , wherein the mutated amino acid residue corresponding to amino acid position K548 of AsCpf1 is R or V.
27 . The mutated Cpf1 polypeptide according to claim 25 , wherein the mutated amino acid residue corresponding to amino acid position K607 of AsCpf1 is R.
28 . The mutated Cpf1 polypeptide according to claim 24 , wherein the mutated Cpf1 polypeptide further comprises one or more mutated amino acid residues corresponding to amino acid positions N547, K550, N551, or N552 of AsCpf1.
29 . The mutated Cpf1 polypeptide according to claim 28 , wherein the mutated amino acid residue corresponding to amino acid position N552 of AsCpf1 is R.
30 . The mutated Cpf1 polypeptide according to claim 24 , wherein said mutated Cpf1 polypeptide is a mutated AsCpf1 comprising S542R mutation.
31 . The mutated Cpf1 polypeptide according to claim 30 , wherein said mutated AsCpf1 further comprises K548R or K548V mutation.
32 . The mutated Cpf1 polypeptide according to claim 30 , wherein said mutated AsCpf1 comprises one of the following combinations of mutations: S542R and K548R; S542R and K548V; S542R and K607R; or S542R, K548V and N552R.
33 . The mutated Cpf1 polypeptide according to claim 24 , wherein said mutated Cpf1 polypeptide is a mutated LbCpf1 comprising G532R mutation.
34 . The mutated Cpf1 polypeptide according to claim 33 , wherein said mutated LbCpf1 comprises K538R or K538V mutation.
35 . The mutated Cpf1 polypeptide according to claim 33 , wherein said mutated LbCpf1 comprises one of the following combinations of mutations: G532R and K595R; G532R and K538V; or G532R, K538V and Y542R.
36 . The mutated Cpf1 polypeptide according to claim 24 , wherein the mutated Cpf1 polypeptide further comprises a mutated amino acid residue corresponding to amino acid position E174 or M537 of AsCpf1.
37 . The mutated Cpf1 polypeptide according to claim 37 , wherein the mutated amino acid residue corresponding to amino acid position M537 of AsCpf1 is R.
38 . The mutated Cpf1 polypeptide according to claim 24 , wherein the mutated Cpf1 polypeptide is fused to one or more nuclear localization signals (NLSs).
39 . A polynucleotide encoding the mutated Cpf1 polypeptide according to claim 24 .
40 . The polynucleotide according to claim 39 , wherein said polynucleotide is codon optimized for expression in a eukaryotic cell, and wherein said polynucleotide is an mRNA.
41 . An expression vector comprising the polynucleotide according to claim 39 .
42 . A CRISPR-Cas system comprising (a) the mutated Cpf1 polypeptide according to claim 24 or a polynucleotide encoding the mutated Cpf1 polypeptide and (b) a gRNA capable of forming a CRISPR-Cpf1 complex with the mutated Cpf1 polypeptide.
43 . An isolated eukaryotic host cell comprising the mutated Cpf1 polypeptide according to claim 24 or a polynucleotide encoding the mutated Cpf1 polypeptide.