Genome editing using targeting endonucleases and single-stranded nucleic acids

Patent metadata

Field	Value
Publication number	US-9512444-B2
Application number	US-201113811884-A
Country	US
Kind code	B2
Filing date	Jul 22, 2011
Priority date	Jul 23, 2010
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

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.

First claim

What is claimed is:

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.

Assignees

• Chen Fuqiang
• Pruett-Miller Shondra M
• Davis Gregory D
• Sigma Aldrich Co Llc

Inventors

• Chen Fuqiang
• Pruett-Miller Shondra M
• Davis Gregory D

Classifications

• C12Q2521/301

  Endonuclease · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N15/00

  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 title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title