Engineered FnCas12a with enhanced activity through directional evolution in human cells

Clustered regularly interspaced short palindromic repeat–Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and scr...

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Bibliographic Details
Published in:The Journal of biological chemistry 2021-01, Vol.296, p.100394, Article 100394
Main Authors: Liu, Xiexie, Liu, Xiaoyu, Zhou, Chenchen, Lv, Jineng, He, Xiubin, Liu, Yuanyuan, Xie, Haihua, Wang, Bang, Lv, Xiujuan, Tang, Lianchao, Li, Mingchun, Liu, Changbao, Zhao, Junzhao, Liu, Yong, Song, Zongming, Gu, Feng
Format: Article
Language:English
Subjects:
activity
FnCas12a
PAM
RS1
variant
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Summary:Clustered regularly interspaced short palindromic repeat–Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.
ISSN:0021-9258
1083-351X
DOI:10.1016/j.jbc.2021.100394