Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.
Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.
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DOI：
10.1038/s41594-023-01104-6
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发表时间：
2023-11
影响因子：
16.8
通讯作者：
Strick, Terence R.
中科院分区：
文献类型：
--
作者：
Wang, Jinglong;Le Gall, Julien;Frock, Richard L.;Strick, Terence R.
使用DNA切割反应的桑格测序和高通量基因组测序，我们发现化脓链球菌SpCas 9复合物通过稳健地产生悬垂而不是钝的DNA末端的分布来响应内部机械应变。内部机械应变是通过移动（增加或减少）RNA-DNA杂交体和下游典型PAM之间的间距产生的。通过杂交体和PAM之间的距离增加2个碱基，可以稳健地产生多达2个碱基的3 '突出端。我们还使用单分子实验来实时重建CRISPR-SpCas 9反应的整个过程，在结构和动力学上监测和量化R环形成，第一和第二次DNA切割事件以及催化后复合物的解离。复合物解离和断裂DNA末端的释放是反应的限速步骤，并且移位的SpCas 9被充分去稳定化，以便在形成断裂DNA末端后快速解离。在这里，使用单分子实验，作者表明SpCas 9通过诱导突出的DNA末端来响应移动的PAM。这种移位-PAM靶向通过生理水平的DNA超螺旋增强，并且反过来促进催化后复合物的解离。
Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3′ overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR–SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends. Here, using single-molecule experiments, the authors show that SpCas9 responds to shifted PAMs by inducing overhanging DNA ends. Such shift-PAM targeting is enhanced by physiological levels of DNA supercoiling and in turn promotes dissociation of the complex after catalysis.
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影响因子：
14.8
作者：
Hu J;Meyers RM;Dong J;Panchakshari RA;Alt FW;Frock RL
通讯作者：
Frock RL
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
DOI：
10.1073/pnas.2103630118
发表时间：
2021-05-25
影响因子：
11.1
作者：
Liang, Zhuoyi;Kumar, Vipul;Frock, Richard L.
通讯作者：
Frock, Richard L.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle