Novel crispr enzymes and systems

Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

What is claimed: 
     
         1 . A non-naturally occurring or engineered composition comprising a CRISPR-Cas effector protein comprising at least one HEPN domain and a nucleic acid component, wherein the effector protein forms a complex with the nucleic acid component, said nucleic acid component capable of binding with a target nucleic acid sequence and direct sequence-specific binding of said complex to the target nucleic acid sequence. 
     
     
         2 . The composition of  claim 1 , wherein the CRISPR-Cas protein is a Type VI CRISPR-Cas protein. 
     
     
         3 . The composition of  claim 1 , wherein the CRISPR-Cas protein is a C2c2 protein. 
     
     
         4 . The composition of  claim 3 , wherein the C2c2 protein is from a bacteria belonging to a genus selected from the group consisting of:  Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifactor, Mycoplasma, Camplyobacter, Leptotrichia , Rhodobacter, Lachnospiraceae,  Carnobacterium, Paludibacter.    
     
     
         5 . The composition of  claim 4 , wherein the C2c2 protein is an orthologue comprising 80% sequence identity to an orthologue selected from the group consisting of;  Leptotrichia shahii  DSM 19757 C2c2 : Rhodobacter capsulatus  SB 1003 (RcS);  Rhodobacter capsulatus  R121 (RcR);  Rhodobacter capsulatus  DE442 (RcD); Lachnospiraceae bacterium MA2020 (Lb(X));  Lachnospiraceae bacterium  NK4A179 (Lb(X); [ Clostridium ]  aminophilum  DSM_10710 (CaC);  Lachnospiraceae bacterium  NK4A144 (Lb(X);  Leptotrichia wadei  F0279 (Lew);  Leptotrichia wadei  F0279 (Lew);  Carnobacterium gallinarum  DSM 4847 (Cg);  Carnobacterium gallinarum  DSM 4847 (Cg);  Paludibacter propionicigenes  WB4 (Pp);  Listeria seeligeri  serovar 1/2b (Ls);  Listeria weihenstephanensis  FSL R9-0317 (Liw); and  Listeria bacterium  FSL M6-0635 (Lib). 
     
     
         6 . The composition of  claim 5 , wherein the C2c2 protein is an orthologue selected from the group consisting of;  Leptotrichia shahii  DSM 19757 C2c2 , Rhodobacter capsulatus  SB 1003 (RcS),  Rhodobacter capsulatus  R121 (RcR),  Rhodobacter capsulatus  DE442 (RcD), [Lachnospiraceae bacterium MA2020 (Lb(X)), Lachnospiraceae bacterium NK4A179 (Lb(X)), [ Clostridium ]  aminophilum  DSM_10710 (CaC), Lachnospiraceae bacterium NK4A144 (Lb(X)),  Leptotrichia wadei  F0279 (Lew),  Carnobacterium gallinarum  DSM 4847 (Cg),  Paludibacter propionicigenes  WB4 (Pp),  Listeria seeligeri  serovar 1/2b (Ls),  Listeria weihenstephanensis  FSL R9-0317 (Liw), and  Listeria bacterium  FSL M6-0635 (Lib). 
     
     
         7 . The composition according to  claim 1 , wherein the CRISPR-Cas effector protein comprises at least one or more nuclear localization signals. 
     
     
         8 . The composition according to  claim 3 , wherein the C2c2 effector protein 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 C2c2 effector protein lacking said one or more mutations. 
     
     
         9 . The composition according to  claim 3 , wherein the C2c2 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 operably configured to express the polypeptides 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 11 , wherein the polynucleotide molecule encoding the CRISPR-Cas effector protein is codon optimized for expression in a eukaryotic cell. 
     
     
         13 . An in vitro or ex vivo method of targeting a target nucleic acid in a eukaryotic or prokaryotic cell, the method comprising contacting a sample with a non-naturally occurring or engineered composition according to  claim 1 . 
     
     
         14 . The method of  claim 13 , wherein the effector protein and nucleic acid component(s) are provided via one or more polynucleotide molecules encoding the polypeptides and/or the nucleic acid component(s), and wherein the one or more polynucleotide molecules are operably configured to express the polypeptides and/or the nucleic acid component(s). 
     
     
         15 . The method of  claim 13 , wherein the non-naturally occurring or engineered composition is delivered via liposomes, nanoparticles, exosomes, microvesicles, a gene-gun or one or more viral vectors. 
     
     
         16 . The method of  claim 13 , wherein the method comprises modifying said target locus of interest. 
     
     
         17 . The method of  claim 13 , wherein modifying is or comprises cleaving the target nucleic acid sequence. 
     
     
         18 . The method of  claim 13 , wherein the target nucleic acid sequence comprises RNA. 
     
     
         19 . The method of  claim 13 , wherein the target nucleic acid sequence is complementary to at least a portion of the nucleic acid component. 
     
     
         20 . A cell transiently transfected or non-transiently transfected with the vector of  claim 10 .