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移位的 PAMs 会产生 DNA 突出端，并增强 SpCas9 酶切后的复合物解离。

Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation.

机构信息

Institut Jacques Monod, Université de Paris Cité, Paris, France.

Institut de Biologie, Ecole Normale Supérieure, Université PSL, Paris, France.

出版信息

Nat Struct Mol Biol. 2023 Nov;30(11):1707-1718. doi: 10.1038/s41594-023-01104-6. Epub 2023 Oct 12.

DOI:10.1038/s41594-023-01104-6
PMID:37828409
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10643121/
Abstract

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.

摘要

使用桑格测序（Sanger sequencing）和高通量基因组测序技术对 DNA 切割反应进行分析，我们发现化脓性链球菌 SpCas9 复合物通过生成大量突出而非平齐的 DNA 末端来应对内部机械应变。内部机械应变是通过改变（增加或减少）RNA-DNA 杂交体与下游标准 PAM 之间的间距而产生的。通过增加杂交体与 PAM 之间 2 个碱基的距离，可以稳定地产生长达 2 个碱基的 3' 突出。我们还使用单分子实验实时重建 CRISPR-SpCas9 反应的全过程，结构和动力学监测和定量 R 环形成、第一个和第二个 DNA 切口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 SpCas9 被充分去稳定化，以至于在形成断裂的 DNA 末端后迅速解离。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/5e9a470c212e/41594_2023_1104_Fig16_ESM.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/02badedc6e16/41594_2023_1104_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/884b176f4eff/41594_2023_1104_Fig7_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/8873a0d1fd99/41594_2023_1104_Fig8_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/5292720fac2a/41594_2023_1104_Fig9_ESM.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/94441f37bdda/41594_2023_1104_Fig12_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/7cbdf1145639/41594_2023_1104_Fig13_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/4891526fe929/41594_2023_1104_Fig14_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/e85215af0d94/41594_2023_1104_Fig15_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ff26/10643121/5e9a470c212e/41594_2023_1104_Fig16_ESM.jpg

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