Prokaryotic RNAi-like system and methods of use
View Patent ↗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. An isolated polynucleotide comprising at least 23 nucleotides, wherein the isolated polynucleotide comprises a psiRNA-tag and a guide sequence, wherein the psiRNA-tag is the first 5 to 10 nucleotides of the isolated polynucleotide 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 isolated polynucleotide, and wherein the guide sequence is complementary to, and hybridizes to, a target polynucleotide which is cleaved.
2. A vector encoding the isolated polynucleotide of claim 1 .
3. The isolated polynucleotide of claim 1 wherein the isolated polynucleotide is RNA.
4. The isolated polynucleotide of claim 1 wherein the psiRNA-tag has at least 80% sequence similarity with 5′-ATTGAAAS, wherein S is G or C.
5. The isolated polynucleotide of claim 1 wherein the guide sequence comprises at least 31 nucleotides or at least 37 nucleotides.
6. The isolated polynucleotide of claim 1 wherein the guide sequence is 31 nucleotides or is 37 nucleotides.
7. The isolated polynucleotide of claim 1 wherein the psiRNA-tag is the first 8 nucleotides of the isolated polynucleotide.
8. The isolated polynucleotide of claim 1 wherein the target polynucleotide is RNA.
9. The isolated polynucleotide of claim 1 wherein the isolated polynucleotide is associated with CRISPR-associated (Cas) polypeptides to form a complex having endonuclease activity, and wherein the Cas polypeptides are encoded by the microbe.
10. The isolated polynucleotide of claim 9 wherein the Cas polypeptides comprise a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, and a Cmr6 polypeptide, and have endoribonuclease activity.