Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species. Issue 10 (30th March 2020)

• Record Type: Journal Article
• Authors: He, Xiaozhen; Chen, Wenfeng; Liu, Zhen; Yu, Guirong; Chen, Youbang; Cai, Yi-Jun; Sun, Ling; Xu, Wanli; Zhong, Lili; Gao, Caixi; Chen, Jishen; Zhang, Minjie; Yang, Shengxi; Yao, Yizhou; Zhang, Zhiping; Ma, Fujun; Zhang, Chen-Chen; Lu, Hui-Ping; Yu, Bin; Cheng, Tian-Lin; Qiu, Juhui; Sheng, Qing; Zhou, Hai-Meng; Lv, Zhi-Rong; Yan, Junjun; Zhou, Yongjian; Qiu, Zilong; Cui, Zongbin; Zhang, Xi; Meng, Anming; Sun, Qiang; Yang, Yufeng;
• Is Part Of: Nucleic acids research. Volume 48:Issue 10(2020)
• Page Start: e57
• Page End: e57
• Publication Date: 2020-03-30
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkaa195
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.