Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus.
Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus.
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DOI：
10.1038/ncomms13905
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发表时间：
2017-01-09
影响因子：
16.6
通讯作者：
Cotta-Ramusino C
中科院分区：
文献类型：
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作者：
Bothmer A;Phadke T;Barrera LA;Margulies CM;Lee CS;Buquicchio F;Moss S;Abdulkerim HS;Selleck W;Jayaram H;Myer VE;Cotta-Ramusino C
CRISPR-Cas9系统为基因组工程提供了一个通用的工具包，可以在特定的基因组位置引入各种DNA损伤。然而，更好地了解这些病变的性质和修复途径对于实现这项技术的全部潜力至关重要。在这里，我们描述了由每个Cas9变体引起的不同病变以及由此产生的修复途径接合。我们证明了悬垂结构的存在和极性是双链断裂修复途径选择的关键决定因素。类似地，源自不同Cas9变体的单个切口差异性地激活修复：通过同源重组修复D10 A而不是N863 A诱导的切口。最后，我们证明了同源重组是修复病变所需的双链，而不是单链DNA作为模板。响应于CRISPR-Cas9的修复途径选择的这种详细表征为设计研究和治疗性基因组工程策略提供了更确定性的方法。CRISPR-Cas9已迅速成为修饰基因组的常用分子生物学工具，并已被修饰以产生单链切口以及双链断裂。在这里，作者探索了由Cas9的不同变体激活的DNA修复途径。
The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies. CRISPR-Cas9 has rapidly become a common molecular biology tool for modifying genomes and has been modified to generate single-strand nicks as well as double-strand breaks. Here the authors explore the DNA repair pathways activated by the different variants of Cas9.