Enhanced homologous recombination by the modulation of targeting vector ends.

Abstract:

:The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Hirotsune S,Kiyonari H,Jin M,Kumamoto K,Yoshida K,Shinohara M,Watanabe H,Wynshaw-Boris A,Matsuzaki F

doi

10.1038/s41598-020-58893-9

subject

Has Abstract

pub_date

2020-02-13 00:00:00

pages

2518

issue

1

issn

2045-2322

pii

10.1038/s41598-020-58893-9

journal_volume

10

pub_type

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