Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species

作者：Xiaozhen He, Wenfeng Chen, Zhen Liu, Guirong Yu, Youbang Chen, Yijun Cai, Ling Sun, Wanli Xu, Lili Zhong, Caixi Gao, Jishen Chen, Minjie Zhang, Shengxi Yang, Yizhou Yao, Zhiping Zhang, Fujun Ma, Chenchen Zhang, Huiping Lu, Bin Yu, Tian‐Lin Cheng, Juhui Qiu, Qing Sheng, Hai‐Meng Zhou, Zhi-Rong Lv, Junjun Yan, Yongjian Zhou, Zilong Qiu, Zongbin Cui, Xi Zhang, Anming Meng, Qiang Sun, Yufeng Yang · 发表于：Nucleic Acids Research · 年份：2020 · DOI：10.1093/nar/gkaa195 · 被引用次数：6 · 研究领域：CRISPR and Genetic Engineering、Cytomegalovirus and herpesvirus research、RNA and protein synthesis mechanisms

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.