Crispr-cas effector polypeptides and methods of use thereof

Abstract

The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.

1 .- 149 . (canceled) 
     
     
         150 . A composition comprising:
 a) a nuclease comprising a single RuvC active site capable of both cleaving DNA and binding crRNA and wherein the amino terminus (N terminus) of the nuclease does not begin with the amino acid sequence “MIS”; and   b) a recombinant guide RNA.   
     
     
         151 . A composition comprising:
 a) a nuclease or a nucleic acid encoding the nuclease, wherein the nuclease comprises a RuvC active site capable of both pre-crRNA processing and DNA cleavage; and   b) a recombinant guide RNA (gRNA) or a nucleic acid encoding the recombinant gRNA, wherein the recombinant gRNA comprises a repeat sequence and a spacer sequence,   wherein the spacer sequence hybridizes to a target sequence of the target nucleic acid   wherein the nuclease binds the repeat sequence, and   wherein the nuclease is capable of cleaving a target nucleic acid without a tracrRNA.   
     
     
         152 . The composition of  claim 151 , wherein the nuclease is fused to a nuclear localization signal. 
     
     
         153 . The composition of  claim 151 , comprising a DNA donor template. 
     
     
         154 . The composition of  claim 151 , comprising a pharmaceutical excipient. 
     
     
         155 . The composition of  claim 151 , wherein the target sequence is a eukaryotic sequence. 
     
     
         156 . The composition of  claim 151 , wherein the nuclease comprises an amino acid sequence that can be encoded by a nucleotide sequence that is at least 90% identical to a nucleotide sequence found in a phage with a genome of at least 120 kbp in length. 
     
     
         157 . The composition of  claim 151 , wherein the target nucleic acid comprises double stranded DNA (dsDNA). 
     
     
         158 . The composition of  claim 157 , wherein the guide sequence hybridizes to a complementary strand of the dsDNA, and wherein there is a proto spacer adjacent motif (PAM) 5′ of the target sequence on a non-complementary strand of the dsDNA. 
     
     
         159 . The composition of  claim 158 , wherein the PAM is 5′-NTTN-3′, wherein T is thymine and N is any nucleotide. 
     
     
         160 . The composition of  claim 151 , wherein the nuclease comprises nickase activity. 
     
     
         161 . The composition of  claim 151 , wherein the nucleic acid encoding the nuclease comprises messenger RNA. 
     
     
         162 . The composition of  claim 161 , comprising a lipid nanoparticle. 
     
     
         163 . The composition of  claim 151 , wherein the composition does not comprise a tracrRNA. 
     
     
         164 . The composition of  claim 151 , wherein at least one of the nucleic acid encoding the nuclease and the nucleic acid encoding the recombinant gRNA are present in an adeno-associated viral vector. 
     
     
         165 . The composition of  claim 151 , wherein the nuclease has been engineered to have reduced nuclease activity relative to a naturally occurring nuclease. 
     
     
         166 . The composition of  claim 165 , wherein the nuclease comprises one to three amino acid substitutions relative to the naturally occurring nuclease that results in the reduced nuclease activity. 
     
     
         167 . The composition of  claim 165 , wherein the nuclease is fused to a heterologous polypeptide. 
     
     
         168 . The composition of  claim 167 , wherein the heterologous polypeptide is selected from a deaminase, a reverse transcriptase, a methyltransferase. 
     
     
         169 . A kit comprising:
 a) a nuclease or a nucleic acid encoding the nuclease, wherein the nuclease comprises a RuvC active site capable of both pre-crRNA processing and DNA cleavage; and   b) a recombinant guide RNA (gRNA) or a nucleic acid encoding the recombinant gRNA, wherein the recombinant gRNA comprises a repeat sequence and a spacer sequence,   wherein the spacer sequence hybridizes to a target sequence of the target nucleic acid   wherein the nuclease binds the repeat sequence, and   wherein the nuclease is capable of cleaving a target nucleic acid without a tracrRNA.