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 composition comprising, a Type V Cas polypeptide that comprises a RuvC-like nuclease domain, a Zn finger region with at least one Zn-binding cysteine, but does not comprise an HNH domain, and one or more engineered nucleic acid components comprising a heterologous guide sequence 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 of a polynucleotide, wherein the heterologous guide sequence comprises a tracr sequence.
2 . The composition of claim 1 , that is capable of directing cleavage at the target sequence of the polynucleotide.
3 . The composition of claim 1 , wherein the one or more nucleic acid components are engineered to hybridize with a target sequence adjacent to a protospacer motif (PAM) in a genome of a eukaryotic cell.
4 . The composition of claim 1 , wherein the Cas polypeptide comprises one or more nuclear localization signals.
5 . The composition of claim 1 , wherein 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 composition comprises the formed CRISPR-Cas complex.
7 . 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.
8 . The composition of claim 7 , wherein a polynucleotide sequence in the one or more vectors encoding the Cas polypeptide is codon optimized for expression in a eukaryotic cell.
9 . The composition of claim 7 , wherein the one or more vectors are viral vectors.
10 . The composition of claim 9 , wherein the one or more viral vectors are adenoviral vectors, lentiviral vectors, or adeno-associated viral vectors.
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 a CRISPR-Cas complex comprising, 1) a Type V Cas polypeptide comprising a RuvC nuclease domain, a Zn finger region with at least one Zn-binding cysteine, but not comprising a HNH domain, and 2) one or more nucleotide components comprising a heterologous guide sequence capable of directing sequence-specific binding of the CRISPR-Cas complex to a target sequence of the polynucleotide, wherein the heterologous guide sequence comprises a tracr sequence.
14 . The method of claim 13 , wherein the target sequence is in a eukaryotic cell.
15 . The method of claim 14 , 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 . A method of cleaving a polynucleotide, comprising contacting a sample comprising the polynucleotide with a CRISPR-Cas complex comprising, 1) a Type V Cas polypeptide comprising a RuvC nuclease domain, a Zn finger region with at least one Zn-binding cysteine, but not comprising a HNH domain, and 2) one or more nucleotide components comprising a heterologous guide sequence capable of directing sequence-specific cleavage with said complex at a target sequence of a polynucleotide, wherein the heterologous guide sequence comprises a tracr sequence.
17 . The method of claim 16 , wherein the target sequence is in a eukaryotic cell.
18 . The method of claim 17 , 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.
19 . A composition comprising a Type V Cas polypeptide that comprises a RuvC-like nuclease domain, a Zn finger region with at least one Zn-binding cysteine, but does not comprise an HNH domain, and one or more engineered nucleic acid components comprising a heterologous guide sequence that form a complex with the Type V Cas polypeptide and that is capable of directing sequence-specific binding off said complex to a target sequence of a polynucleotide, wherein the Cas polypeptide comprises at least one mutation in the RuvC-like nuclease domain and is catalytically inactive, wherein the heterologous guide sequence comprises a tracr sequence.
20 . The composition of claim 19 , wherein the Cas polypeptide comprises one or more nuclear localization signals.
21 . The composition of claim 19 , wherein the Cas polypeptide is linked to a heterologous functional domain.
22 . The composition of claim 19 , wherein the one or more nucleic acid components comprise one or more modified nucleotides or one or more non-nucleotide moieties.
23 . The composition of claim 19 , wherein the composition comprises a formed CRISPR-Cas complex.
24 . The composition of claim 19 , wherein the composition comprises one or more vectors encoding the Cas polypeptide and the one or more nucleic acid components.
25 . The composition of claim 24 , wherein a polynucleotide sequence in the one or more vectors encoding the Cas polypeptide is codon optimized for expression in a eukaryotic cell.
26 . The composition of claim 24 , wherein the one or more vectors are viral vectors.
27 . The composition of claim 26 , wherein the one or more viral vectors are adenoviral vectors, lentiviral vectors, or adeno-associated viral vectors.
28 . The composition of claim 24 , wherein the composition comprises a mRNA encoding the Cas polypeptide.
29 . The composition of claim 28 , wherein the mRNA encoding the Cas polypeptide is comprised in a lipid nanoparticle, a liposome, an exosome, or a microvesicle.
30 . A method of targeting a polynucleotide, comprising contacting a sample comprising, 1) a catalytically inactive Type V Cas polypeptide linked to one or more heterologous functional domains, the Type V Cas polypeptide comprising a mutated RuvC-like nuclease domain, a Zn finger region with at least one Zn-binding cysteine, but does not comprise an HNH domain, and 2) one or more nucleic acid components comprising a heterologous guide sequence 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 of a polynucleotide, wherein the heterologous guide sequence comprises a tracr sequence.
31 . The method of claim 30 , wherein the target sequence is in a eukaryotic cell.
32 . The method of claim 31 , wherein contacting the eukaryotic cell with the CRISPR-Cas complex results in modification of a gene or gene product, or modification in expression of a gene product.