Enhanced homologous recombination by the modulation of targeting vector ends

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

doi:10.1038/s41598-020-58893-9

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.

Highlights

After the digestion of an eGFP empty vector by various restriction enzymes, SYBR Green I was intercalated into the eGFP empty vector, and the vector was treated with Exonuclease III (ExoIII) or T7
We first examined the frequency of homology-directed repair (HDR)-mediated gene targeting to the Hprt gene and other error-prone repair systems, including C-nonhomologous end joining (NHEJ) and MMEJ, by selection with G418 for one week; double selection was performed using G418 and 6-TG (HDR-mediated gene targeting of the Hprt gene) for an additional week
We demonstrated that the use of targeting constructs carrying long 3′ overhangs facilitates the frequency of HDR, suggesting that the creation of 3′ single-stranded DNA (3′ ssDNA) overhangs is a rate-limiting step in HDR

Summary

Introduction

The repair process is followed by overhang removal, gap filling DNA repair and ligation These systems often generate small sequence changes near the DSB site and promote random integration during targeted mutation processes[13,14]. HDR promotes genetic changes by using an uninjured homologous region as a template to restore correct information that was lost during a DSB. This allows HDR to be mostly error-free[15]. The three subpathways of HDR are distinct, they share the same initial steps, in which a 3′ overhanging tail is generated by the resection of DSB ends. We discovered that treatment with T7 exonuclease, which catalyzes the removal of 5′ mononucleotides from duplex DNA in the 5′ to 3′ direction, provides a 3′ overhanging tail on the targeting vector that increases the efficiency of HDR

Methods

Results

Conclusion