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 . (canceled)
2 . An engineered CRISPR-Cas system comprising:
(a) a Type-V Cas protein or a polynucleotide encoding the Type-V Cas protein, wherein the Type-V Cas protein is fused with one or more nuclear localization signals (NLSs) and comprises a RuvC domain but not a HNH domain;
(b) a guide RNA or a polynucleotide encoding the guide RNA, wherein the guide RNA comprises a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of a eukaryotic cell;
wherein the CRISPR-Cas system does not comprise a tracrRNA, wherein the guide RNA is capable of forming a CRISPR complex with the Type-V Cas protein in the eukaryotic cell, and wherein the guide sequence is capable of directing sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the genomic locus of interest of the eukaryotic cell.
3 . The engineered CRISPR-Cas system of claim 2 , wherein the engineered CRISPR-Cas system is comprised or encoded in a viral vector for delivery.
4 . The engineered CRISPR-Cas system of claim 3 , wherein the viral vector is an adenoviral vector, a lentiviral vector, or an adeno-associated viral vector.
5 . The engineered CRISPR-Cas system of claim 2 , wherein the engineered CRISPR-Cas system is comprised in a lipid particle for delivery, wherein the lipid particle comprises the guide RNA and an mRNA encoding the Type-V Cas protein.
6 . The engineered CRISPR-Cas system of claim 5 , wherein the lipid particle is a lipid nanoparticle, a liposome, an exosome, or a microvesicle.
7 . The engineered CRISPR-Cas system of claim 2 , wherein the engineered CRISPR-Cas system comprises a ribonucleoprotein comprising the guide RNA in complex with the Type-V Cas protein.
8 . The engineered CRISPR-Cas system of claim 7 , wherein the ribonucleoprotein is formulated for delivery by electroporation or microinjection.
9 . The engineered CRISPR-Cas system of claim 2 , wherein the Type-V Cas protein comprises RuvC-I, RuvC-II, and RuvC-III domains.
10 . The engineered CRISPR-Cas system of claim 2 , wherein the Type-V Cas protein comprises at least one mutation in a catalytic domain and has reduced catalytic activity.
11 . The engineered CRISPR-Cas system of claim 10 , wherein the Type-V Cas protein is fused with at least one heterologous protein domain.
12 . The engineered CRISPR-Cas system of claim 11 , wherein the heterologous protein domain has one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, or nucleic acid binding activity; or wherein the heterologous protein domain comprises a transposase domain, an integrase domain, a recombinase domain, a resolvase domain, an invertase domain, a protease domain, a DNA methyltransferase domain, a DNA hydroxylmethylase domain, a DNA demethylase domain, a histone acetylase domain, a histone deacetylases domain, a nuclease domain, a transcriptional repressor domain, a transcriptional activator domain, a deaminase domain, a transcription-regulatory protein domain, a cellular uptake activity associated domain, a nucleic acid binding domain, an antibody presentation domain, a histone modifying enzyme, a recruiter of histone modifying enzyme, an inhibitor of histone modifying enzyme, a histone methyltransferase, a histone demethylase, a histone kinase, a histone phosphatase, a histone ribosylase, a histone deribosylase, a histone ubiquitinase, a histone deubiquitinase, a histone biotinase, or a histone tail protease.
13 . The engineered CRISPR-Cas system of claim 2 , wherein the guide RNA comprises a direct repeat sequence linked to the guide sequence.
14 . The engineered CRISPR-Cas system of claim 2 , wherein the guide RNA comprises at least one chemical modification.
15 . The engineered CRISPR-Cas system of claim 14 , wherein the chemical modification comprises 2′-O-methyl, 2′-O-methyl 3′ phosphorothioate, or 2′-O-methyl 3′ thioPACE.
16 . The engineered CRISPR-Cas system of claim 2 , wherein the PAM comprises 5′-TTN or 5′-TTTV.
17 . The engineered CRISPR-Cas system of claim 2 , wherein the polynucleotide encoding the Type-V Cas protein is codon-optimized for expression in the eukaryotic cell.
18 . The engineered CRISPR-Cas system of claim 2 , further comprising an exogenous polynucleotide for targeted integration into a DNA break introduced by the CRISPR complex in the genomic locus of interest by non-homologous end joining or by homology directed repair.
19 . The engineered CRISPR-Cas system of claim 2 , wherein the CRISPR complex being directed by the guide sequence is capable of cleaving the genomic locus of interest, thereby modifying the eukaryotic cell with an insertion, deletion, or substitution of one or more nucleotides in the genomic locus of interest.
20 . An engineered CRISPR-Cas system comprising:
(a) a Type-V Cas protein, wherein the Type-V Cas protein is fused with one or more nuclear localization signals (NLSs) and comprises a RuvC domain but not a HNH domain;
(b) a guide RNA, wherein the guide RNA comprises a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of a mammalian cell;
wherein the CRISPR-Cas system does not comprise a tracrRNA, wherein the guide RNA is capable of forming a CRISPR complex with the Type-V Cas protein in the mammalian cell, and wherein the guide sequence is capable of directing sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the genomic locus of interest of the mammalian cell, wherein the CRISPR complex being directed by the guide sequence is capable of cleaving the genomic locus of interest in the mammalian cell.
21 . An engineered CRISPR-Cas system comprising:
(a) an mRNA encoding a Type-V Cas protein, wherein the Type-V Cas protein is fused with one or more nuclear localization signals (NLSs) and comprises a RuvC domain but not a HNH domain;
(b) a guide RNA, wherein the guide RNA comprises a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of a mammalian cell;
wherein the CRISPR-Cas system does not comprise a tracrRNA, wherein the guide RNA is capable of forming a CRISPR complex with the Type-V Cas protein in the mammalian cell, and wherein the guide sequence is capable of directing sequence-specific binding of the CRISPR complex to the target sequence adjacent to the PAM in the genomic locus of interest of the mammalian cell, wherein the CRISPR complex being directed by the guide sequence is capable of cleaving the genomic locus of interest in the mammalian cell.