Novel cas13b orthologues 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 Cas13b effector protein and at least one targeting nucleic acid component like a guide RNA or crRNA.

1 - 50 . (canceled) 
     
     
         51 . A non-naturally occurring or engineered composition for modifying a target RNA sequence, said composition comprising:
 (a) a Cas13b effector protein comprising any one of amino acid sequences of SEQ ID NO: 31-38,   (b) an engineered guide molecule, and   (c) one or more heterologous functional domains associated with the Cas13b effector protein,   wherein the engineered guide molecule is capable of forming a CRISPR-Cas complex with the Cas13b effector protein and comprises (i) a guide sequence that directs sequence-specific binding to a target RNA sequence other than the naturally occurring Cas13b protospacer and that reprograms the CRISPR-Cas complex to bind said target RNA sequence, and (b) a direct repeat sequence, and   wherein the one or more heterologous functional domains modifies the target RNA sequence.   
     
     
         52 . The composition of  claim 51 , wherein the Cas13b effector protein is catalytically inactive. 
     
     
         53 . The composition of  claim 51 , wherein the Cas13b effector protein comprises one or more mutations to the two HEPN domains. 
     
     
         54 . The composition of  claim 51 , wherein the Cas13b effector protein comprises a mutation in one or more of positions R116A, H121A, R1177A, and H1182A of Cas13b effector protein originating from  Bergeyella zoohelcum  ATCC 43767 or amino acid positions corresponding thereto of a Cas13b ortholog. 
     
     
         55 . The composition of  claim 51 , wherein the Cas13b effector protein is truncated. 
     
     
         56 . The composition of  claim 55 , wherein the Cas13b effector protein is a truncated functional variant of the corresponding wildtype Cas13b. 
     
     
         57 . The composition of  claim 56 , wherein the Cas13b effector protein corresponds to nucleotides 1-984 of  Prevotella  sp. P5-125 Cas13b. 
     
     
         58 . The composition of  claim 55 , wherein the Cas13b effector protein is catalytically inactive. 
     
     
         59 . The composition of  claim 51 , wherein the guide sequence hybridizes to a target RNA sequence comprising an adenine to form an RNA duplex, wherein the guide sequence comprises a non-pairing cytosine at a position corresponding to said adenine, resulting in an A-C mismatch in the RNA duplex formed. 
     
     
         60 . The composition of  claim 51 , wherein the guide sequence comprises more than one mismatch corresponding to different adenosine sites in the target RNA sequence or wherein two guide molecules are used, each comprising a mismatch corresponding to a different adenosine site in the target RNA sequence. 
     
     
         61 . The composition of  claim 51 , wherein the one or more heterologous functional domains modifies the target RNA sequence by converting adenosine to inosine. 
     
     
         62 . The composition of  claim 61 , wherein the one or more heterologous functional domains comprises an adenosine deaminase. 
     
     
         63 . The composition of  claim 62 , wherein the adenosine deaminase is fused to a N- or C-terminus of the Cas 13b effector protein. 
     
     
         64 . The composition of  claim 63 , wherein the adenosine deaminase is fused by a linker. 
     
     
         65 . The composition of  claim 64 , wherein the linker is (GGGGS) 3-11 , GSG 5 , or LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR, or wherein the linker is an XTEN linker. 
     
     
         66 . The composition of  claim 62 , wherein the adenosine deaminase is linked to an adaptor protein and the guide molecule. 
     
     
         67 . The composition of  claim 62 , wherein the catalytically inactive Cas13b effector protein comprises an aptamer sequence capable of binding to an adaptor protein, wherein the adaptor protein is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, and PRR1. 
     
     
         68 . The composition of  claim 62 , wherein the adenosine deaminase is an RNA-specific adenosine deaminase or catalytic domain thereof. 
     
     
         69 . The composition of  claim 68 , wherein the RNA-specific adenosine deaminase is ADAR. 
     
     
         70 . The composition of  claim 69 , wherein the ADAR is human ADAR (huADAR) and/or ADAR1 or ADAR2, or a catalytic domain thereof. 
     
     
         71 . The composition of  claim 70 , wherein the ADAR is huADAR or a catalytic domain thereof. 
     
     
         72 . The composition of  claim 69 , wherein the ADAR is a mutated hADAR2d comprising mutation E488Q or a mutated hADAR1d comprising mutation E1008Q. 
     
     
         73 . The composition of  claim 51 , wherein the Cas13b effector protein comprises one or more heterologous nuclear export signals (NES) or nuclear localization signals (NLS). 
     
     
         74 . The composition of  claim 73 , wherein the NES is an HIV Rev NES or MAPK NES. 
     
     
         75 . The composition of  claim 73 , wherein the heterologous NES or NLS is fused at the C-terminal of the Cas13b effector protein. 
     
     
         76 . The composition of  claim 51 , wherein the target RNA sequence is within a cell. 
     
     
         77 . A cell comprising the composition of  claim 51 . 
     
     
         78 . The cell of  claim 77 , wherein the cell is a prokaryotic cell. 
     
     
         79 . The cell of  claim 77 , wherein the cell is a eukaryotic cell. 
     
     
         80 . The eukaryotic cell of  claim 79 , wherein the cell is a mammalian cell, a human cell, a non-human animal cell, or a plant cell. 
     
     
         81 . A cell line comprising the cell of  claim 79 , or progeny thereof. 
     
     
         82 . A non-human animal comprising the cell of any one of  claim 79 . 
     
     
         83 . A plant comprising the cell of  claim 79 . 
     
     
         84 . A method of prophylactically or therapeutically treating a subject in need thereof, comprising the composition of  claim 51 . 
     
     
         85 . A method of prophylactically or therapeutically treating a subject in need thereof, comprising the cell of  claim 79 . 
     
     
         86 . A method of modifying a target RNA sequence, said method comprising delivering to said target RNA sequence a composition comprising:
 (a) a Cas13b effector protein comprising any one of amino acid sequences of SEQ ID NO: 31-38,   (b) an engineered guide molecule, and   (c) one or more heterologous functional domains associated with the Cas13b effector protein,   wherein the engineered guide molecule is capable of forming a CRISPR-Cas complex with the Cas13b effector protein and comprises (i) a guide sequence that directs sequence-specific binding to a target RNA sequence other than the naturally occurring Cas13b protospacer and that reprograms the CRISPR-Cas complex to bind said target RNA sequence, and (b) a direct repeat sequence,   wherein the one or more heterologous functional domains modifies the target RNA sequence, and   wherein the target RNA sequence is in a prokaryotic cell or a eukaryotic cell.   
     
     
         87 . The method of  claim 86 , wherein the one or more heterologous functional domains modifies the target RNA sequence by converting adenosine to inosine. 
     
     
         88 . The method of  claim 87 , wherein the one or more heterologous functional domains comprises an adenosine deaminase. 
     
     
         89 . The method of  claim 88 , wherein the Cas13b effector protein, engineered guide molecule, and a coding sequence of the adenosine deaminase, are delivered as one or more polynucleotide molecules or as a ribonucleoprotein complex. 
     
     
         90 . The method of  claim 89 , wherein the Cas13b effector protein, engineered guide molecule, and coding sequence of the adenosine deaminase are delivered by means of particles, vesicles, or one or more viral vectors.