Prokaryotic RNAi-like system and methods of use
First Claim
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.
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Accused Products
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.
178 Citations
10 Claims
- 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.
Specification