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-targeting systems comprising a novel DNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. Methods for making and using and uses of such systems, methods, and compositions and products from such methods and uses are also disclosed and claimed.
1. A CRISPR-Cas system comprising, a Type V Cas polypeptide and one or more engineered nucleic acid components that form a CRISPR-Cas complex with the Type V Cas polypeptide and that is capable of directing sequence-specific binding of said complex to a target sequence present in a eukaryotic cell, wherein the complex lacks a tracr sequence.
2. The CRISPR-Cas system of claim 1 , wherein the Type V Cas polypeptide comprises a RuvC-like nuclease domain, but does not comprise an HNH domain.
3. The CRISPR-Cas system of claim 1 , wherein the Type V Cas polypeptide is a Type V-A Cas polypeptide.
4. The CRISPR-Cas system of claim 1 , wherein the Type V Cas polypeptide comprises at least one mutation in the RuvC-like nuclease domain and is catalytically inactive.
5. The CRISPR-Cas system of claim 1 , wherein the Type V Cas polypeptide is catalytically active.
6. The CRISPR-Cas system of claim 1 , wherein the Type V Cas polypeptide is configured to cleave only one strand of the target sequence.
7. The CRISPR-Cas system of claim 1 , wherein the Type V Cas polypeptide comprises a heterologous functional domain.
8. A composition comprising a CRISPR-Cas complex, which complex comprises a Type V Cas polypeptide and one or more engineered nucleic acid components that is capable of directing sequence-specific binding of said complex to a target sequence present in a eukaryotic cell, wherein the Type V Cas polypeptide and the one more engineered nucleic acid components do not naturally occur together, and wherein the complex lacks a tracr sequence.
9. The composition of claim 8 , wherein the complex comprises a Type V Cas polypeptide comprising one or more catalytic motifs of a RuvC nuclease domain but not comprising a HNH domain.
10. The composition of claim 8 , wherein the complex comprises (a) a Type V Cas polypeptide comprising one or more catalytic motifs of the RuvC nuclease domain and a Zn finger region, but not comprising a HNH domain, and (b) one or more nucleic acid components.
11. The composition of claim 10 , wherein the Zn finger region is inactivated.
12. The composition of claim 8 , wherein the Type V Cas polypeptide is a Type V-A Cas polypeptide.
13. The composition of claim 8 , wherein the Type V Cas polypeptide comprises at least one mutation in the RuvC-like nuclease domain and is catalytically inactive.
14. The composition of claim 8 , wherein the Type V Cas polypeptide is catalytically active.
15. The composition of claim 8 , wherein the Type V Cas polypeptide is configured to cleave only one strand at the location of the target sequence.
16. The composition of claim 8 , wherein the Type V Cas polypeptide comprises a heterologous functional domain.
17. An isolated eukaryotic cell comprising: a Cas polypeptide that comprises a RuvC-like nuclease domain, but does not comprise an HNH domain; and one or more engineered nucleic acid components that form a CRISPR-Cas complex with the Cas polypeptide and that is capable of directing sequence-specific binding of said complex to a target sequence present in the eukaryotic cell, wherein the complex lacks a tracr sequence.
18. The isolated eukaryotic cell of claim 17 , wherein the Cas polypeptide is a Type V Cas polypeptide.
19. The isolated eukaryotic cell of claim 17 , in which the target sequence is cleaved.
20. The isolated eukaryotic cell of claim 17 , wherein the one or more nucleic acid components are engineered to hybridize with the target sequence adjacent to a protospacer motif (PAM) in the genome of the eukaryotic cell.
21. The isolated eukaryotic cell of claim 17 , wherein the Cas polypeptide comprises one or more nuclear localization signals and/or the one or more nucleic acid components comprise one or more modified nucleotides or one or more non-nucleotide components.
22. The isolated eukaryotic cell of claim 17 , which comprises the formed CRISPR-Cas complex.
23. A method of targeting a polynucleotide, comprising contacting a eukaryotic cell comprising the polynucleotide with the composition of claim 8 , wherein: 1) the CRISPR-Cas complex comprises a Cas polypeptide comprising a RuvC nuclease domain, but not comprising a HNH domain, 2) the one or more nucleotide components directs sequence-specific binding of the CRISPR-Cas complex to a target sequence of the polynucleotide, and 3) wherein the complex lacks a tracr sequence.
24. The method of claim 23 , wherein the Cas polypeptide is a Type V-A Cas polypeptide.
25. The method of claim 23 , wherein the Cas polypeptide comprises at least one mutation in the RuvC-like nuclease domain and is catalytically inactive.
26. The method of claim 23 , wherein the Cas polypeptide is catalytically active.
27. The method of claim 23 , wherein the Cas polypeptide is capable of cleaving only one strand of the target sequence.
28. The method of claim 23 , wherein the Cas polypeptide comprises a heterologous functional domain.
29. The method of claim 23 , which comprises contacting the eukaryotic cell with the CRISPR-Cas complex and results in modification of a gene or gene product, or modification in the expression of a gene product.
30. The method of claim 23 , which comprises contacting the eukaryotic cell with the CRISPR-Cas complex and results in cleavage of the target sequence.