Methods for increasing the efficiency of homology directed repair (HDR) in the cellular genome

Abstract

In certain embodiments, the disclosure provides a method for increasing the efficiency of homology directed repair (HDR) in the genome of a cell, comprising: (a) introducing into the cell: (i) a nuclease; and (ii) a donor nucleic acid which comprises a modification sequence to be inserted into the genome; and (b) subjecting the cell to a temperature shift from 37 DEG C to a lower temperature; wherein the nuclease cleaves the genome at a cleavage site in the cell, and the donor nucleic acid directs the repair of the genome sequence with the modification sequence through an increased rate of HDR.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of US Provisional Application No. 62 / 437,042, filed December 20, 2016, which application is hereby incorporated in its entirety for all purposes. Background technique [0003] Various methods for DNA targeted cleavage of genomic sequences have been described in the art. Such targeted cleavage events can be used to induce targeted mutagenesis, induce targeted deletion of cellular DNA sequences, and facilitate targeted recombination at predetermined chromosomal loci. These methods typically involve the use of engineered cleavage systems to induce double-strand breaks (DSBs) or nicks in the target DNA sequence such that the DNA is broken through error-generating processes such as non-homologous end joining (NHEJ) or homology-directed repair (HDR). Repair of the break can result in gene inactivation or insertion of an exogenous sequence of interest. Cleavage can occur by using specific nu...

AI Technical Summary

Problems solved by technology

[0004] The efficiency of genome modification at a specific target location by the HDR process is relatively low in cells

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