Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks

文献类型： 外文期刊

第一作者： Chen, Ruo-Dan

作者： Chen, Ruo-Dan;Yang, Yi;Liu, Kun-Ming;Hu, Jing-Zhen;Feng, Yi-Li;Yang, Chun-Yi;Liu, Si-Cheng;Wang, Yue;Xu, Shi-Ming;Xie, An-Yong;Chen, Ruo-Dan;Feng, Yi-Li;Liu, Si-Cheng;Xie, An-Yong;Chen, Ruo-Dan;Yang, Yi;Liu, Kun-Ming;Hu, Jing-Zhen;Feng, Yi-Li;Yang, Chun-Yi;Jiang, Rui-Rui;Wang, Yue;Xu, Shi-Ming;Xie, An-Yong;Chen, Ruo-Dan;Yang, Yi;Liu, Kun-Ming;Hu, Jing-Zhen;Feng, Yi-Li;Yang, Chun-Yi;Jiang, Rui-Rui;Wang, Yue;Xu, Shi-Ming;Xie, An-Yong;Jiang, Rui-Rui;Wang, Yu-Long;Han, Ping-An;Tian, Ru-Gang;Han, Ping-An;Tian, Ru-Gang

作者机构：

关键词： CRISPR-Cas12a; Asymmetric retention; PAM-distal end; Classical NHEJ; Targeted integration

期刊名称：GENOME BIOLOGY （ 影响因子：9.4； 五年影响因子：16.3 ）

ISSN： 1474-760X

年卷期： 2025 年 26 卷 1 期

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收录情况： SCI

摘要： BackgroundAfter Cas12a cleaves its DNA target, it generates a DNA double strand break (DSB) with two compatible 5 '-staggered ends. The Cas12a-gRNA complex remains at the protospacer adjacent motif (PAM)-proximal end (PPE) while releasing the PAM-distal end (PDE). The effects of this asymmetric retention on DSB repair are currently unknown.ResultsPost-cleavage retention of LbCas12a at PPEs suppresses the recruitment of classical non-homologous end joining (c-NHEJ) core factors, leading to longer deletions at PPEs compared to PDEs. This asymmetry in c-NHEJ engagement results in approximately tenfold more accurate ligation between two compatible PDEs induced by paired LbCas12a than ligation involving a compatible PPE. Moreover, ligation to a given end of SpCas9-induced DSBs demonstrates more efficient ligation with a PDE from Cas12a-induced DSBs than with a PPE. In LbCas12a-induced NHEJ-mediated targeted integration, only two compatible PDEs from LbCas12a-induced DSBs-one from donor templates and the other from target sites-promote accurate and directional ligation. Based on these findings, we developed a strategy called Cas12a-induced PDE ligation (CIPDEL) for NHEJ-mediated efficient and precise gene correction and insertion.ConclusionsThe asymmetric retention of CRISPR-LbCas12a at DSB ends suppresses c-NHEJ at PPEs, not at PDEs. This unique repair mechanism can be utilized in the CIPDEL strategy, offering a potentially better alternative for homology-directed targeted integration.

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