Genome editing using targeting endonucleases and single-stranded nucleic acids
View Patent ↗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.
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.