Enhanced homologous recombination by the modulation of targeting vector ends

Shinji Hirotsune, Hiroshi Kiyonari, Mingyue Jin, Kanako Kumamoto, Kayo Yoshida, Miki Shinohara, Hitomi Watanabe, Anthony Wynshaw-Boris, Fumio Matsuzaki

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)


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.

Original languageEnglish
Article number2518
JournalScientific reports
Issue number1
Publication statusPublished - 01-12-2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General


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