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 composition comprising, (a) a Cas polypeptide that comprises a RuvC-like nuclease domain and a zinc finger, but does not comprise an HNH domain, or a polynucleotide encoding the Cas polypeptide, and (b) one or more engineered nucleic acid components lacking a tracr sequence 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 polynucleotide sequence adjacent to a protospacer adjacent motif (PAM) in the genome of a eukaryotic cell, or a polynucleotide encoding the one or more engineered nucleic acid components.
2. The composition of claim 1 , wherein the Cas polypeptide is a Type V Cas polypeptide.
3. The composition of claim 1 , that is capable of directing cleavage at the target sequence of the polynucleotide.
4. The composition of claim 1 , wherein the one or more nucleic acid components comprises a guide RNA.
5. The composition of claim 1 , 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 moieties.
6. The composition of claim 1 , wherein the Cas polypeptide is linked to a heterologous functional domain.
7. The composition of claim 1 , wherein the composition comprises the formed CRISPR-Cas complex.
8. The composition of claim 1 , wherein the composition comprises one or more vectors encoding the Cas polypeptide and the one or more nucleic acid components.
9. The composition of claim 8 , wherein a polynucleotide sequence in the one or more vectors encoding the Cas polypeptide is codon optimized for expression in a eukaryotic cell.
10. The composition of claim 8 , wherein the one or more vectors are viral vectors, optionally an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.
11. The composition of claim 1 , wherein the composition comprises a mRNA encoding the Cas polypeptide.
12. The composition of claim 11 , wherein the mRNA encoding the Cas polypeptide is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.
13. A method of targeting a polynucleotide, comprising contacting a sample comprising the polynucleotide with the composition of claim 1 .
14. The method of claim 13 , wherein the Cas polypeptide is a Type V Cas polypeptide.
15. The method of claim 13 , wherein the target sequence is in a eukaryotic cell, and wherein contacting the eukaryotic cell with the CRISPR-Cas complex results in modification of a gene or gene product, or modification in the expression of a gene product.
16. The method of claim 13 , wherein the target sequence is in a eukaryotic cell, and wherein contacting the eukaryotic cell with the CRISRP-Cas complex results in cleavage of the target sequence.
17. A composition comprising (a) a Cas polypeptide that comprises a RuvC-like nuclease domain, but does not comprise an HNH domain, or a polynucleotide encoding the Cas polypeptide, and (b) one or more engineered nucleic acid components lacking a tracr sequence that form a complex with the Cas polypeptide and that is capable of directing sequence-specific binding of said complex to a target polynucleotide sequence adjacent to a protospacer adjacent motif (PAM) in the genome of a eukaryotic cell, or a polynucleotide encoding the one or more engineered nucleic acid components, wherein the Cas polypeptide comprises at least one mutation in the RuvC-like nuclease domain and is catalytically inactive.
18. The composition of claim 17 , wherein the Cas polypeptide is a Type V Cas polypeptide.
19. The composition of claim 17 , wherein the as polypeptide comprises one or more nuclear localization signals and/or one or more nucleic acid components comprising one or more modified nucleotides or one or more non-nucleotide moieties.
20. The composition of claim 17 , wherein the Cas polypeptide is linked to a heterologous functional domain.
21. The composition of claim 17 , wherein the composition comprises the formed CRISPR-Cas complex.
22. The composition of claim 17 , wherein the composition comprises one or more vectors encoding the Cas polypeptide and the one or more nucleic acid components.
23. The composition of claim 22 , wherein a polynucleotide sequence in the one or more vectors encoding the Cas polypeptide is codon optimized for expression in a eukaryotic cell.
24. The composition of claim 22 , wherein the one or more vectors are viral vectors, optionally an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.
25. The composition of claim 17 , wherein the composition comprises a mRNA encoding the Cas polypeptide.
26. The composition of claim 25 , wherein the mRNA encoding the Cas polypeptide is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.
27. A method of targeting a polynucleotide, comprising contacting a sample comprising the polynucleotide with the composition of claim 17 .
28. The method of claim 27 , wherein the Cas polypeptide is a Type V Cas polypeptide.
29. The method of claim 27 , wherein the target sequence is in a eukaryotic cell.
30. The method of claim 29 , wherein contacting the eukaryotic cell with the CRISPR-Cas complex results in modification of a gene or gene product, or modification in the expression of a gene product.