文献类型： 外文期刊

作者： Chen, Ruo-Dan ¹ ; Yang, Yi ¹ ; Liu, Kun-Ming ¹ ; Hu, Jing-Zhen ¹ ; Feng, Yi-Li ¹ ; Yang, Chun-Yi ¹ ; Jiang, Rui-Rui ³ ; Liu, Si-Cheng ¹ ; Wang, Yue ¹ ; Han, Ping-An ⁶ ; Tian, Ru-Gang ⁶ ; Wang, Yu-Long ⁵ ; Xu, Shi-Ming ¹ ; Xie, An-Yong ¹ ;

作者机构： 1.Zhejiang Univ, Sir Run Run Shaw Hosp, Dept Gen Surg, Key Lab Laparoscop Technol Zhejiang Prov,Sch Med, Hangzhou 310016, Zhejiang, Peoples R China

2.Hangzhou Qiantang Hosp, Hangzhou 310018, Zhejiang, Peoples R China

3.Zhejiang Univ, Inst Translat Med, Sch Med, Hangzhou 310029, Zhejiang, Peoples R China

4.Zhejiang Univ, Canc Ctr, Hangzhou 310029, Zhejiang, Peoples R China

5.Tsinghua Univ, Zhejiang Key Lab Multi & Mol Enzymol, Yangtze Delta Reg Inst, Jiaxing 314006, Zhejiang, Peoples R China

6.Inner Mongolia Acad Agr & Anim Husb Sci, Inst Anim Husb, Hohhot 010031, Inner Mongolia, Peoples R China

7.Inner Mongolia Acad Agr & Anim Husb Sci, Inner Mongolia Key Lab Sugarbeet Genet & Germplasm, Hohhot 010031, Inner Mongolia, Peoples R China

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

页码：

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.