Cas9-nucleic acid complexes and uses related thereto

Patent metadata

Field	Value
Publication number	US-10544405-B2
Application number	US-201414760113-A
Country	US
Kind code	B2
Filing date	Jan 15, 2014
Priority date	Jan 16, 2013
Publication date	Jan 28, 2020
Grant date	Jan 28, 2020

Abstract

This disclosure relates to Cas9-nucleic acid complexes and uses related thereto. In certain embodiments, the disclosure contemplates transgenic plants and animals genetically engineered to express Cas9-nucleic acid complexes disclosed herein. In certain embodiments, the disclosure relates to methods of treating or preventing, diseases, conditions, cancer, viral infections or other pathogenic infection using vectors configured to express a Cas9-nucleic acid complex disclosed herein.

First claim

What we claim:

1. A nucleic acid composition comprising:
a first nucleic acid sequence comprising a Cas9 gene encoding a Cas9 protein,
a second nucleic acid sequence comprising
a first RNA segment that binds, in a eukaryotic cell, to the Cas9 protein, wherein the first RNA segment comprises a double-stranded region and
a second single-stranded RNA segment that binds to a target nucleic acid,

wherein the target nucleic acid sequence is genomic RNA or an mRNA transcript from an RNA virus with no DNA stage or is microRNA associated with an oncogene.

2. The nucleic acid composition of claim 1 , wherein the Cas9 protein comprises SEQ ID NO: 1 or a conserved variant thereof.

3. The nucleic acid composition of claim 2 , wherein the Cas9 protein has an arginine-rich motif, a RuvC-III motif, and a RuvC-IV motif.

4. The nucleic acid composition of claim 1 , wherein the Cas9 protein comprises a sequence with at least 40% identity to SEQ ID NO: 1.

5. The nucleic acid composition of claim 1 , wherein the first segment forms a hairpin structure.

6. The nucleic acid composition of claim 1 , wherein the second segment comprises more than 50 continuous nucleotides configured to hybridize to the target nucleic acid sequence.

7. The nucleic acid composition of claim 1 , wherein the target nucleic acid sequence is a genomic RNA from a virus.

8. The nucleic acid composition of claim 1 wherein the target nucleic acid sequence is mRNA or microRNA associated with an oncogene.

9. The nucleic acid composition of claim 1 , wherein the Cas9 gene comprises a sequence with 60% or more identity to SEQ ID NO: 9.

10. The nucleic acid composition of claim 7 , wherein the virus is influenza A virus including subtype H1N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus, coxsackie A virus, norovirus, Rubella virus, lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, California encephalitis virus, hantavirus, rabies virus, ebola virus, marburg virus, hepatitis A virus (HAV), hepatitis C virus (HCV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

11. The nucleic acid composition of claim 1 wherein the Cas9 protein does not include a nuclear localization signal.

12. A recombinant vector comprising the nucleic acid composition of claim 1 .

13. A eukaryotic cell comprising
a mRNA encoding a Cas9 protein, and
a RNA comprising
a first segment that binds to the Cas9 protein, wherein the first RNA segment comprises a double-stranded region and
a second single-stranded segment that binds to a target nucleic acid sequence,

wherein the target nucleic acid sequence is a genomic RNA or mRNA sequence from a virus, wherein the virus is influenza A virus including subtype H1N1, influenza B virus, influenza C virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, SARS coronavirus, coxsackie A virus, norovirus, Rubella virus, lymphocytic choriomeningitis virus (LCMV), yellow fever virus, measles virus, mumps virus, respiratory syncytial virus, rinderpest virus, California encephalitis virus, hantavirus, rabies virus, ebola virus, marburg virus, hepatitis A virus (HAV), hepatitis C virus (HCV), hepatitis D virus (HDV), or hepatitis E virus (HEV).

14. The cell of claim 13 comprising the Cas9 protein complexed with the first segment.

15. The cell of claim 13 that is a mammalian cell.

16. The cell of claim 13 wherein the Cas9 protein does not include a nuclear localization signal.

17. The cell of claim 13 , wherein the Cas9 protein is encoded by a gene comprising a sequence with 60% or more identity to SEQ ID NO: 9.

Assignees

• Univ Emory

Inventors

• Weiss David S
• Grakoui Arash
• Sampson Timothy R
• Price Aryn Alaine

Classifications

• C12N15/8213

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/902

  using homologous recombination · CPC title

• C07K2319/85

  containing an RNA binding domain · CPC title

• C07K2319/42

  containing a HA(hemagglutinin)-tag · CPC title