Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus

Abstract

A Type III-A CRISPR-Cas (StCsm) complex of Streptococcus thermophilus comprising crRNA, Csm4, and Csm3 and use for cleavage of RNA bearing a nucleotide sequence complementary to the crRNA, in vitro or in vivo. Methods for site-specific cleavage/shredding of a target RNA molecule using an RNA-guided RNA endonuclease comprising a minimal complex of crRNA, Csm4, and Csm3, and methods of RNA knock-down and RNA knock-out are disclosed.

What is claimed is: 
     
       1. A method of programmable RNA shredding comprising:
 providing a Type III-A CRISPR-Cas (Csm) complex comprising at least crRNA, Csm4, and Csm3, or at least crRNA, Csm4, Csm3, and Cas10, and any other subunits; 
 identifying target RNA; 
 exposing the complex to the target RNA, thereby generating a plurality of cleaved fragments of the target RNA; 
 wherein the cleavage sites are at 6 nucleotide intervals. 
 
     
     
       2. The method of  claim 1  wherein 1 to 10 Csm3 subunits are present in the complex. 
     
     
       3. The method of  claim 1  wherein the crRNA comprises a 5′ handle and a spacer, and optionally a 3′ handle, wherein the spacer is complementary or substantially complementary to a region of a target RNA. 
     
     
       4. The method of  claim 1  wherein the programmable RNA shredding occurs in vitro. 
     
     
       5. The method of  claim 1  wherein the programmable RNA shredding occurs in vivo. 
     
     
       6. A method of programmable RNA knock-out or knock-down comprising:
 identifying a target RNA; 
 exposing the target RNA to a Type III-A Csm (III-A) (Csm) complex comprising at least crRNA, Csm4, and Csm3 and any other subunits; 
 providing conditions facilitating the cutting of the target RNA by the complex; 
 wherein the expression of a gene complementary to the target RNA is knocked down or knocked out. 
 
     
     
       7. The method of  claim 6  wherein the Csm3 contains a mutation which inactivates the endoribonuclease activity, and the method results in RNA knock-down. 
     
     
       8. The method of  claim 6  wherein the complex further comprises Cas 10. 
     
     
       9. The method of  claim 6  wherein the programmable RNA knock-out or knock-down occurs in vitro. 
     
     
       10. The method of  claim 6  wherein the programmable RNA knock-out or knock-down occurs in vivo. 
     
     
       11. A composition comprising an engineered Csm complex comprising only crRNA, Csm4, and Csm3, or only crRNA, Csm4, Csm3, and Cas10, where the crRNA of the engineered complex is configured for complementary binding to a selected site in a target RNA molecule, and wherein the Csm complex is capable of cutting the target RNA molecule under suitable conditions. 
     
     
       12. The composition of  claim 11  wherein 1 to 10 Csm3 subunits are present in the complex. 
     
     
       13. The composition of  claim 11  wherein the RNA cutting occurs in vitro. 
     
     
       14. The composition of  claim 11  wherein the RNA cutting occurs in vivo.