切割后靶点驻留决定了cas12a诱导的DNA双链断裂非同源端连接的不对称性

IF 10.1 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Ruo-Dan Chen, Yi Yang, Kun-Ming Liu, Jing-Zhen Hu, Yi-Li Feng, Chun-Yi Yang, Rui-Rui Jiang, Si-Cheng Liu, Yue Wang, Ping-An Han, Ru-Gang Tian, Yu-Long Wang, Shi-Ming Xu, An-Yong Xie
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引用次数: 0

摘要

在Cas12a切割其DNA靶后,它产生具有两个兼容的5 '交错末端的DNA双链断裂(DSB)。Cas12a-gRNA复合物在释放PAM-远端(PDE)的同时保持在原间隔器邻近基序(PAM)-近端(PPE)。这种不对称保留对DSB修复的影响目前尚不清楚。LbCas12a在PDEs处的切割后保留抑制了经典非同源末端连接(c-NHEJ)核心因子的招募,导致PDEs处的缺失时间比PDEs长。这种c-NHEJ接合的不对称性导致配对LbCas12a诱导的两个兼容pde之间的连接比涉及兼容PPE的连接精确大约十倍。此外,连接到spcas9诱导的dsb的给定末端表明,与cas12a诱导的dsb的PDE连接比与PPE连接更有效。在lbcas12a诱导的nhej介导的靶向整合中,只有来自lbcas12a诱导的dsbs的两个兼容的pde -一个来自供体模板,另一个来自靶点-促进准确和定向的连接。基于这些发现,我们开发了一种名为cas12a诱导PDE连接(CIPDEL)的策略,用于nhej介导的高效和精确的基因校正和插入。CRISPR-LbCas12a在DSB末端的不对称保留抑制PPEs处的c-NHEJ,而不是PDEs处的c-NHEJ。这种独特的修复机制可以用于CIPDEL策略,为同源定向定向集成提供了更好的选择。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Post-cleavage target residence determines asymmetry in non-homologous end joining of Cas12a-induced DNA double strand breaks
After 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. Post-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. The 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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来源期刊
Genome Biology
Genome Biology Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
21.00
自引率
3.30%
发文量
241
审稿时长
2 months
期刊介绍: Genome Biology stands as a premier platform for exceptional research across all domains of biology and biomedicine, explored through a genomic and post-genomic lens. With an impressive impact factor of 12.3 (2022),* the journal secures its position as the 3rd-ranked research journal in the Genetics and Heredity category and the 2nd-ranked research journal in the Biotechnology and Applied Microbiology category by Thomson Reuters. Notably, Genome Biology holds the distinction of being the highest-ranked open-access journal in this category. Our dedicated team of highly trained in-house Editors collaborates closely with our esteemed Editorial Board of international experts, ensuring the journal remains on the forefront of scientific advances and community standards. Regular engagement with researchers at conferences and institute visits underscores our commitment to staying abreast of the latest developments in the field.
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