PROKARYOTIC RNAi-LIKE SYSTEM AND METHODS OF USE

Abstract

Provided herein are methods for inactivating a target polynucleotide. The methods use a psiRNA having a 5′ region and a 3′ region. The 5′ region includes, but is not limited to, 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. The 3′ region is substantially complementary to a portion of the target polynucleotide. The methods may be practiced in a prokaryotic microbe or in vitro. Also provided are polypeptides that have endonuclease activity in the presence of a psiRNA and a target polynucleotide, and methods for using the polypeptides.

1 - 56 . (canceled) 
     
     
         57 . A method for inactivating a target polynucleotide in a cell comprising:
 introducing into a cell a psiRNA comprising at least 23 nucleotides, wherein the psiRNA comprises a psiRNA-tag and a guide sequence, wherein the psiRNA-tag is the first 5 to 10 nucleotides of the psiRNA and comprises a nucleotide sequence chosen from nucleotides of a repeat from a CRISPR locus that are immediately upstream of a spacer present in a microbe comprising the CRISPR locus, wherein the guide sequence is located immediately downstream of the psiRNA-tag and comprises the remaining nucleotides of the psiRNA, wherein the guide sequence is (i) substantially complementary to a target polynucleotide or (ii) selectively hybridizes under stringent hybridization conditions to a target polynucleotide, and wherein the target polynucleotide is cleaved.   
     
     
         58 . The method of  claim 57  wherein the guide sequence comprises at least 31 nucleotides or at least 37 nucleotides. 
     
     
         59 . The method of  claim 57  wherein the guide sequence is 31 nucleotides or is 37 nucleotides. 
     
     
         60 . The method of  claim 57  wherein the psiRNA-tag is the first 8 nucleotides of the psiRNA. 
     
     
         61 . The method of  claim 57  wherein the target polynucleotide is RNA. 
     
     
         62 . The method of  claim 57  wherein the psiRNA associates in the cell with CRISPR-associated (Cas) polypeptides to form a complex having endonuclease activity, and wherein the Cas polypeptides are encoded by the cell. 
     
     
         63 . The method of  claim 62  wherein the Cas polypeptides comprise a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, a Cmr5 polypeptide, and a Cmr6 polypeptide, and have endoribonuclease activity. 
     
     
         64 . The method of  claim 57  wherein the psiRNA is introduced into the cell as an RNA polynucleotide. 
     
     
         65 . The method of  claim 57  wherein the psiRNA introduced into the cell as a DNA polynucleotide encoding the psiRNA. 
     
     
         66 . The method of  claim 57  wherein the target polynucleotide is a polynucleotide endogenous to the cell. 
     
     
         67 . The method of  claim 57  wherein the cell is a bacterial cell or an archaeal cell. 
     
     
         68 . The method of  claim 57  wherein the cell is a eukaryotic cell. 
     
     
         69 . The method of  claim 57  wherein the cell is a genetically modified microbe. 
     
     
         70 . The method of  claim 57  wherein the target polynucleotide is DNA. 
     
     
         71 . A method for cleaving a target polynucleotide comprising:
 incubating under suitable conditions a composition comprising:
 a target polynucleotide; 
 a psiRNA comprising at least 23 nucleotides, wherein the psiRNA comprises a psiRNA-tag and a guide sequence, wherein the psiRNA-tag is the first 5 to 10 nucleotides of the psiRNA and comprises a nucleotide sequence chosen from nucleotides of a repeat from a CRISPR locus that are immediately upstream of a spacer present in a microbe comprising the CRISPR locus, wherein the guide sequence is located immediately downstream of the psiRNA-tag and comprises the remaining nucleotides of the psiRNA, and wherein the guide sequence is (i) substantially complementary to a target polynucleotide or (ii) selectively hybridizes under stringent hybridization conditions to a target polynucleotide; and a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, a Cmr5 polypeptide, and a Cmr6 polypeptide; 
   wherein the target polynucleotide is cleaved.   
     
     
         72 . The method of  claim 71  wherein the guide sequence comprises at least 31 nucleotides or at least 37 nucleotides. 
     
     
         73 . The method of  claim 71  wherein the guide sequence is 31 nucleotides or is 37 nucleotides. 
     
     
         74 . The method of  claim 71  wherein the psiRNA-tag is the first 8 nucleotides of the psiRNA. 
     
     
         75 . The method of  claim 71  wherein the method is in vivo. 
     
     
         76 . The method of  claim 71  wherein the method is in vitro. 
     
     
         77 . The method of  claim 71  wherein the target polynucleotide is DNA.