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.

What is claimed is: 
     
         1 - 149 . (canceled) 
     
     
         150 . A composition comprising:
 a. a nucleic acid sequence that encodes a protein at least 95% identical to SEQ ID NO: 120; and   b. a DNA molecule encoding a guide RNA, or a guide RNA thereof, wherein the guide RNA comprises:
 i. a nucleotide sequence that is complementary to a target sequence of a target nucleic acid; and 
 ii. a region that associates with a polypeptide to form a guide RNA-polypeptide complex wherein (i) is heterologous to (ii). 
   
     
     
         151 . The composition of  claim 150 , wherein the DNA molecule comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 139. 
     
     
         152 . The composition of  claim 150 , wherein the DNA molecule encodes a sequence that transcribes into, or the guide RNA is, a sequence that is 80% similar to SEQ ID NO: 181 and is in a 5′ orientation with respect to the nucleotide sequence that is complementary to the target sequence. 
     
     
         153 . The composition of  claim 150 , wherein the composition further comprises a sequence that encodes a nuclear localization signal (NLS) at the 5′ terminus, the 3′ terminus, or both termini of the nucleic acid sequence that encodes SEQ ID NO: 120. 
     
     
         154 . The composition of  claim 153 , wherein the NLS encoded is the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 50. 
     
     
         155 . The composition of  claim 150 , comprising a lipid, a liposome, a vector, or a particle. 
     
     
         156 . The composition of  claim 150 , comprising one or more of: a buffer, a nuclease inhibitor, and a protease inhibitor. 
     
     
         157 . The composition of  claim 150 , comprising an additional recombinant guide RNA, wherein said additional recombinant guide RNA comprises a region that is complementary to a second target sequence. 
     
     
         158 . The composition of  claim 150 , comprising the target nucleic acid, wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) adjacent to said target sequence, wherein the PAM comprises the sequence 5′ -NTTN-3′, wherein N is any nucleotide and T is thymine. 
     
     
         159 . The composition of  claim 158 , wherein the target nucleic acid comprises:
 (i) a target strand (TS) and (ii) a non-target strand (NTS) comprising the PAM wherein the PAM is 5′ to the target sequence.   
     
     
         160 . The composition of  claim 150 , wherein the nucleic acid sequence encodes a heterologous polynucleotide that is fused to the protein. 
     
     
         161 . The composition of  claim 150 , comprising a pharmaceutically acceptable excipient. 
     
     
         162 . The composition of  claim 150 , wherein the nucleic acid sequence is a messenger RNA. 
     
     
         163 . The composition of  claim 150 , wherein the nucleic acid sequence is an expression vector. 
     
     
         164 . The composition of  claim 163 , wherein the expression vector is an adeno associated viral vector. 
     
     
         165 . The composition of  claim 150 , wherein the target sequence is a eukaryotic sequence. 
     
     
         166 . The composition of  claim 150 , wherein the recombinant guide RNA comprises one or more of a base modification, a sugar modification, and a backbone modification. 
     
     
         167 . A population of cells comprising the composition of  claim 150 . 
     
     
         168 . A cell modified by the composition of  claim 150 . 
     
     
         169 . A method of editing a gene comprising contacting a cell comprising the gene with the composition of  claim 150 .