Cas9-nucleic acid complexes and uses related thereto

• US 10,544,405 B2
• Filed: 01/15/2014
• Issued: 01/28/2020
• Est. Priority Date: 01/16/2013
• Status: Active Grant

• Current Assignee: Emory University
• Original Assignee: Emory University
• Inventors: Weiss, David S., Grakoui, Arash, Sampson, Timothy R., Price, Aryn Alaine
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US14/760,113
• Publication Number:
  US 20150353905A1
• Time in Patent Office: 2,204 Days
• Field of Search: None
• CPC Class Codes: A61K 2035/11 Medicinal preparations comprising living procariotic cells
• CPC Class Codes: A61K 2039/5156 expressing foreign proteins
• CPC Class Codes: A61K 2039/523 expressing foreign proteins
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/42 containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/85 containing an RNA binding domain
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2800/22 Vectors comprising a coding region that has been codon optimised for expression in a respective host
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

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.