US2019382800A1PendingUtilityA1

Novel crispr enzymes and systems

Assignee: BROAD INST INCPriority: Jun 18, 2015Filed: Jun 24, 2019Published: Dec 19, 2019

Est. expiryJun 18, 2035(~8.9 yrs left)· nominal 20-yr term from priority

Inventors:Konstantin Severinov Feng Zhang Yuri I. Wolf Sergey Shmakov Ekaterina Semenova Leonid Minakhin Kira S. Makarova Eugene Koonin Silvana Konermann Julia Joung Jonathan Gootenberg Omar Abudayyeh Eric S. Lander

C12N 15/63C12N 15/111C12N 15/907C12N 15/113C12N 9/22C12N 2800/22C12N 15/8201C12N 15/85C12N 2310/111C12N 15/102C12N 2310/20C12N 9/222

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Claims

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.

Claims

exact text as granted — not AI-modified

What is claimed: 
     
         1 . An in vitro method for targeting a target RNA in a sample, comprising contacting said target RNA with a CRISPR-Cas effector protein and a guide RNA, wherein the effector protein forms a complex with the guide RNA, and wherein the guide RNA directs the complex to bind the target RNA. 
     
     
         2 . The method of  claim 1 , wherein the CRISPR-Cas effector protein is a type VI effector protein. 
     
     
         3 . The method of  claim 1 , wherein the CRISPR-Cas effector protein comprises at least one HEPN domain. 
     
     
         4 . The method of  claim 1 , wherein the CRISPR-Cas effector protein comprises two HEPN domains. 
     
     
         5 . The method of  claim 1 , wherein the CRISPR-Cas effector protein is a C2c2 protein. 
     
     
         6 . The method of  claim 5 , 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, Campylobacter, Leptotrichia, Rhodobacter, Lachnospiraceae, Carnobacterium , and  Paludibacter.    
     
     
         7 . The method of  claim 6 , 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 ½b (Ls);  Listeria weihenstephanensis  FSL R9-0317 (Liw); and  Listeria bacterium  FSL M6-0635 (Lib). 
     
     
         8 . The method of  claim 7 , 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);  Leptotrichia wadei  F0279 (Lew);  Carnobacterium gallinarum  DSM 4847 (Cg);  Carnobacterium gallinarum  DSM 4847 (Cg);  Paludibacter propionicigenes  WB4 (Pp);  Listeria seeligeri  serovar ½b (Ls);  Listeria weihenstephanensis  FSL R9-0317 (Liw); and  Listeria bacterium  FSL M6-0635 (Lib). 
     
     
         9 . The method of  claim 5 , wherein the C2c2 effector protein cleaves the target RNA and cleaves bystander RNAs in said sample. 
     
     
         10 . The method of  claim 9 , which comprises detecting the degradation of said bystander RNAs in said sample. 
     
     
         11 . The method of  claim 9 , wherein said method comprises comparing the cleavage of said bystander RNAs in the presence and absence of the target RNA. 
     
     
         12 . The method of  claim 10 , wherein said method comprises comparing the cleavage of said bystander RNAs in the presence and absence of the target RNA. 
     
     
         13 . The method of  claim 9 , wherein the C2c2 effector protein is LshC2c2. 
     
     
         14 . The method of  claim 1 , wherein the guide RNA comprises a spacer sequence or guide sequence and a direct repeat sequence. 
     
     
         15 . The method of  claim 1 , wherein the guide RNA does not comprise a tracr sequence. 
     
     
         16 . The method of  claim 1 , wherein the target RNA is a disease associated RNA. 
     
     
         17 . The method of  claim 1 , wherein said method is a diagnostic method by sensing of disease-specific RNAs.

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