Genome editing using targeting endonucleases and single-stranded nucleic acids

• US 9,512,444 B2
• Filed: 07/22/2011
• Issued: 12/06/2016
• Est. Priority Date: 07/23/2010
• Status: Active Grant

• Current Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.)
• Original Assignee: Sigma-Aldrich Co. LLC (Merck & Co., Inc.)
• Inventors: Chen, Fuqiang, Pruett-Miller, Shondra M., Davis, Gregory D.
• Primary Examiner(s): Dunston, Jennifer
• Application Number:
  US13/811,884
• Publication Number:
  US 20130137180A1
• Time in Patent Office: 1,964 Days
• Field of Search: None
• US Class Current: 1/1
• 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: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides methods and kits for editing specific chromosomal sequences in cells. In particular, targeting endonucleases and single-stranded nucleic acids are used to edit the chromosomal sequence.

Claims

1. A method for integrating at least one exogenous sequence into at least one chromosomal sequence in a cell, the method comprising:

a) introducing into the cell (i) at least one targeting endonuclease or nucleic acid encoding a targeting endonuclease, the targeting endonuclease being able to introduce a double-stranded break at a targeted cleavage site in the chromosomal sequence, (ii) at least one first single-stranded nucleic acid comprising a first region having substantial sequence identity to one side of the targeted cleavage site, (iii) at least one second single-stranded nucleic acid comprising a first region having substantial sequence identity to the other side of the targeted cleavage site, and (iv) at least one donor polynucleotide comprising the exogenous sequence that is flanked by a first sequence having substantial sequence identity to a second region of the first single-stranded nucleic acid and a second sequence having substantial sequence identity to a second region of the second single-stranded nucleic acid; and

b) maintaining the cell under conditions such that exogenous sequence is integrated into the chromosomal sequence during repair of the double-stranded break introduced by the targeting endonuclease.

2. The method of claim 1, wherein the targeting endonuclease is a pair of zinc finger nucleases;

and each of the first and second single-stranded nucleic acids is a deoxyribonucleic acid that is at least 30 nucleotides in length.

3. The method of claim 1, wherein the donor polynucleotide is a plasmid vector.