Prokaryotic RNAi-like system and methods of use

• US 8,546,553 B2
• Filed: 07/24/2009
• Issued: 10/01/2013
• Est. Priority Date: 07/25/2008
• Status: Active Grant

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Terns, Rebecca, Terns, Michael, Hale, Caryn
• Primary Examiner(s): SHIN, DANA H
• Application Number:
  US13/055,769
• Publication Number:
  US 20110189776A1
• Time in Patent Office: 1,530 Days
• Field of Search: None
• US Class Current: 536/24.5
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid

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.

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.

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.