CRISPR enzymes and systems
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
1. A Type V non-naturally occurring, engineered composition comprising
a) a Type V Cas polypeptide comprising one or more catalytic motifs of a RuvC nuclease domain but not comprising a HNH domain, and one or more nucleic acid components comprising a heterologous guide sequence and a tracrRNA sequence that are capable of forming a complex with the Type V Cas polypeptide and directing site-specific binding of said complex to a target sequence; or
b) a Type V Cas polypeptide comprising one or more catalytic motifs of the RuvC nuclease domain and a Zn finger region with at least one Zn-binding cysteine, but not comprising a HNH domain, and one or more nucleic acid components comprising a heterologous guide sequence that is capable of forming a complex with the Type V Cas polypeptide and directing sequence-specific binding of said complex to a target sequence.
2. The composition of claim 1 , wherein the Type V Cas polypeptide comprises an arginine rich cluster, but which does not bind Zinc.
3. The composition of claim 1 , wherein the Type V Cas polypeptide is a C2c1 or C2c3 polypeptide.
4. The composition of claim 3 , wherein the C2c1 polypeptide is obtained from a bacterial species selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Desulfovibrio inopinatus, Desulfonatronum thiodismutans, Opitutaceae bacterium TAV5 , Tuberibacillus calidus, Bacillus thermoamylovorans, Brevibacillus sp. CF112, Bacillus sp. NSP2.1 , Desulfatirhabdium butyrativorans, Alicyclobacillus herbarius, Citrobacter freundii, Brevibacillus agri , and Methylobacterium nodulans.
5. The composition of claim 3 , wherein the C2c1 polypeptide comprises at least 95% sequence identity to a C2c1 polypeptide selected from the group consisting of: SEQ ID NOs: 548-567.
6. The composition of claim 3 , wherein the Type V Cas polypeptide of a) is C2c1 polypeptide, or the Type V Cas polypeptide of b) is C2c3 polypeptide.
7. The composition according to claim 1 , wherein the Type V Cas polypeptide comprises one or more nuclear localization signals.
8. The composition according to claim 1 , wherein the Type V Cas polypeptide comprises a mutation of one or more amino acid residues in a catalytically active domain of the effector protein and has reduced or abolished nuclease activity compared with a Type V Cas polypeptide lacking said one or more mutations.
9. The composition according to claim 1 , wherein the Type V Cas polypeptide is linked to a heterologous functional domain.
10. A vector system comprising one or more vectors, the one or more vectors comprising one or more polynucleotide molecules encoding components of the non-naturally occurring or engineered composition of claim 1 .
11. The vector system according to claim 10 , wherein the one or more polynucleotide molecules comprise one or more regulatory elements capable of expressing the polypeptide(s) and/or the nucleic acid component(s), optionally wherein the one or more regulatory elements comprise inducible promotors.
12. The vector system according to claim 10 , wherein the Type V Cas polypeptide is codon optimized for expression in a eukaryotic cell.
13. The method of claim 12 , wherein modifying comprises cleaving the sequence.
14. The method of claim 12 , wherein the target sequence comprises DNA.
15. An isolated eukaryotic or prokaryotic cell transiently or non-transiently transfected with the vector of claim 10 .
16. An in vitro, in vivo, or ex vivo method of targeting a target sequence in a eukaryotic or prokaryotic cell, the method comprising: contacting a sample with the engineered composition of claim 1 .
17. The method of claim 16 , wherein the sample comprises a polynucleotide comprising the target sequence in a eukaryotic cell.
18. The method of claim 16 , wherein the Type V Cas polypeptide and nucleic acid component(s) are provided via one or more polynucleotide molecules encoding the polypeptide(s) and/or the nucleic acid component(s), and wherein the one or more polynucleotide molecules are capable of expressing the polypeptide(s) and/or the nucleic acid component(s).
19. The method of claim 16 , wherein the engineered composition is delivered via liposomes, nanoparticles, exosomes, microvesicles, a gene-gun or one or more viral vectors.
20. The method of claim 16 , further comprising modifying said target sequence.