Effector proteins and methods of use

Abstract

Provided herein are compositions, systems, and methods comprising effector proteins and uses thereof. These effector proteins are shown to be active with guide RNAs and may be characterized as CRISPR-associated (Cas) proteins. Various compositions, systems, and methods of the present disclosure leverage the activities of these effector proteins for the modification, detection, and engineering of nucleic acids.

1 .- 54 . (canceled) 
     
     
         55 . A system comprising:
 a) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 796 (MEME_4); and   b) an engineered guide nucleic acid or a DNA molecule encoding the engineered guide nucleic acid.   
     
     
         56 . The system of  claim 55 , wherein the length of the effector protein is 400 amino acids to 700 amino acids. 
     
     
         57 . The system of  claim 55 , wherein the effector protein comprises an additional amino acid sequence that is at least 90% identical to at least one of SEQ ID NO: 793 (MEME_1), SEQ ID NO: 795 (MEME_3), and SEQ ID NO: 798 (MEME_6). 
     
     
         58 . The system of  claim 55 , wherein the effector protein comprises an additional amino acid sequence that is at least 90% identical to at least one of SEQ ID NO: 793 (MEME_1), SEQ ID NO: 794 (MEME_2), SEQ ID NO: 795 (MEME_3), SEQ ID NO: 797 (MEME_5), SEQ ID NO: 798 (MEME_6), and SEQ ID NO: 799 (MEME_7). 
     
     
         59 . The system of  claim 55 , wherein the effector protein comprises an amino acid sequence that is at least 70% identical to each of SEQ ID NO: 793 (MEME_1), SEQ ID NO: 794 (MEME_2), SEQ ID NO: 795 (MEME_3), SEQ ID NO: 797 (MEME_5), SEQ ID NO: 798 (MEME_6), and SEQ ID NO: 799 (MEME_7). 
     
     
         60 . The system of  claim 55 , wherein the amino acid sequence of the effector protein is at least 70% identical to any one of SEQ ID NOs: 23-34. 
     
     
         61 . The system of  claim 55 , wherein the amino acid sequence of the effector protein is at least 80% identical to any one of SEQ ID NOs: 23-34. 
     
     
         62 . The system of  claim 55 , wherein the engineered guide nucleic acid comprises a sequence that is at least 95% complementary to a eukaryotic sequence. 
     
     
         63 . The system of  claim 55 , wherein the engineered guide nucleic acid comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID NOs: 624, 628, 630, 634, 638, 641, 643, 645, 646, and 827-929. 
     
     
         64 . The system of  claim 55 , wherein the engineered guide nucleic acid is a single guide RNA (sgRNA). 
     
     
         65 . The system of  claim 55 , wherein the engineered guide nucleic acid comprises one or more modifications selected from a 2′-OMe modification, a 2′-Fluoro modification, and a phosphorothioate linkage. 
     
     
         66 . The system of  claim 55 , wherein the effector protein comprises a RuvC domain. 
     
     
         67 . The system of  claim 55 , comprising an expression vector, wherein the expression vector comprises the nucleic acid encoding the effector protein, and optionally the DNA molecule encoding the engineered guide nucleic acid. 
     
     
         68 . The system of  claim 67 , wherein the expression vector is a viral vector. 
     
     
         69 . The system of  claim 68 , wherein the viral vector is an adeno associated viral (AAV) vector. 
     
     
         70 . The system of  claim 67 , wherein the viral vector is a self-complementary AAV (scAAV) vector 
     
     
         71 . The system of  claim 55 , wherein the nucleic acid encoding the effector protein is a messenger RNA (mRNA). 
     
     
         72 . The system of  claim 71 , comprising a lipid or lipid nanoparticle. 
     
     
         73 . The system of  claim 55 , comprising a fusion partner protein. 
     
     
         74 . The system of  claim 73 , wherein the fusion partner protein is fused or linked to the effector protein. 
     
     
         75 . The system of  claim 73 , wherein the fusion partner protein is selected from: a transcriptional activator, a transcriptional repressor, a deaminase, a reverse transcriptase, a methyltransferase, a demethylase, a histone acetyltransferase, a histone deacetylase, a deaminase, a polymerase, and a reverse transcriptase. 
     
     
         76 . The system of  claim 55 , wherein the effector protein comprises a nuclear localization signal. 
     
     
         77 . A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with the system of  claim 55 . 
     
     
         78 . The method of  claim 76 , comprising contacting a cell with the system of  claim 55 . 
     
     
         79 . The method of  claim 77 , wherein the cell is a mammalian cell. 
     
     
         80 . The method of  claim 76 , wherein the target nucleic acid comprises double stranded DNA (dsDNA). 
     
     
         81 . The method of  claim 76 , comprising modifying a nucleobase of the target nucleic acid to a different nucleobase. 
     
     
         82 . The method of  claim 76 , comprising modifying a target sequence of the target nucleic acid. 
     
     
         83 . One or more nucleic acid vectors encoding at least one of:
 a) an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 796 (MEME_4); and   b) an engineered guide nucleic acid.   
     
     
         84 . A method of detecting a target nucleic acid in a cell, a subject or a sample, the method comprising contacting the cell, subject or sample with:
 a) an effector protein, or a nucleic acid encoding an effector protein, wherein the effector protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 796 (MEME_4); and   b) an engineered guide nucleic acid or a DNA molecule encoding the engineered guide nucleic acid.