Method of making a deletion in a target sequence in isolated primary cells using Cas9 and two guide RNAs

Patent metadata

Field	Value
Publication number	US-9822370-B2
Application number	US-201414485288-A
Country	US
Kind code	B2
Filing date	Sep 12, 2014
Priority date	Apr 4, 2013
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells.

First claim

What is claimed is:

1. A method of making a deletion in a target polynucleotide sequence in an isolated mammalian primary cell comprising contacting the mammalian cell with a nucleic acid sequence encoding a clustered regularly interspersed short palindromic repeats-associated 9 (Cas9) protein and two guide ribonucleic acid sequences that hybridize to target sites in the target polynucleotide sequence such that a deletion in the target polynucleotide sequence occurs, and wherein the efficiency of making the deletion in the target polynucleotide sequence is at least 18%.

2. The method according to claim 1 , wherein the Cas9 protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof.

3. The method according to claim 2 , wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

4. The method according to claim 1 , wherein the nucleic acid sequence encoding the Cas9 protein comprises a modified nucleic acid.

5. The method according to claim 4 , wherein the modified nucleic acid comprises a ribonucleic acid containing at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate.

6. The method according to claim 1 , wherein each target site is a 20-nucleotide DNA sequence.

7. The method according to claim 1 , wherein each target site is a 20-nucleotide DNA sequence beginning with G and immediately precedes an NGG motif recognized by the Cas protein.

8. The method according to claim 1 , wherein each target site is G(N) 19 NGG.

9. The method according to claim 1 , wherein the target polynucleotide sequence encodes CCR5.

10. The method according to claim 1 , wherein the target polynucleotide sequence encodes CXCR4.

11. The method according to claim 1 , wherein the Cas9 protein is from any bacterial species or a functional portion thereof.

12. The method according to according to claim 11 , wherein the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

Assignees

• Harvard College
• The Children'S Medical Center Corp

Inventors

• Musunuru Kiran
• Cowan Chad A
• Rossi Derrick J

Classifications

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• A61P31/18

  for HIV · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P35/00

  Antineoplastic agents · CPC title