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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
He X,Chen W,Liu Z,Yu G,Chen Y,Cai YJ,Sun L,Xu W,Zhong L,Gao C,Chen J,Zhang M,Yang S,Yao Y,Zhang Z,Ma F,Zhang CC,Lu HP,Yu B,Cheng TL,Qiu J,Sheng Q,Zhou HM,Lv ZR,Yan J,Zhou Y,Qiu Z,Cui Z,Zhang X,Medoi
10.1093/nar/gkaa195subject
Has Abstractpub_date
2020-06-04 00:00:00pages
e57issue
10eissn
0305-1048issn
1362-4962pii
5813803journal_volume
48pub_type
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