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

• US 9,822,370 B2
• Filed: 09/12/2014
• Issued: 11/21/2017
• Est. Priority Date: 04/04/2013
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation, President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Children's Medical Center Corporation, President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Musunuru, Kiran, Cowan, Chad A., Rossi, Derrick J.
• Primary Examiner(s): WILSON, MICHAEL C
• Application Number:
  US14/485,288
• Publication Number:
  US 20150071889A1
• Time in Patent Office: 1,166 Days
• Field of Search: 435455, 435325
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

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

Claims

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)₁₉NGG.

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.