小柯机器人

美国德克萨斯大学David W. Taylor和Kenneth A. Johnson研究小组合作揭示了CRISPR-Cas9错配监测的结构基础。2022年3月2日，国际学术期刊《自然》发表了这一成果。
研究人员使用动力学引导的冷冻电镜来确定Cas9在错配切割不同阶段的结构。研究人员观察到在错配存在情况下，引导RNA-DNA双链体形成的独特线性结构，可防止Cas9激活。尽管典型的扭结引导RNA-DNA双链体构象有助于DNA切割，但研究人员观察到含有远离原型间隔区相邻基序的错配底物通过重组RuvC结构域中的环而得到稳定。错配稳定残基诱变减少了脱靶DNA切割，但保证了快速的靶向DNA切割。通过靶向仅涉及错配区域，研究人员为下一代高保真Cas9变体的设计提供了新思路。

据介绍，CRISPR-Cas9是基因组编辑的一种工具但受限于脱靶DNA切割，并且对Cas9识别错配位点的潜在机制知之甚少。尽管已经设计了对错配具有更大辨别力的Cas9变体，但这些变体对目标DNA的切割率大大降低。

附：英文原文

Title: Structural basis for mismatch surveillance by CRISPR–Cas9

Author: Bravo, Jack P. K., Liu, Mu-Sen, Hibshman, Grace N., Dangerfield, Tyler L., Jung, Kyungseok, McCool, Ryan S., Johnson, Kenneth A., Taylor, David W.

Issue&Volume: 2022-03-02

Abstract: CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1,2,3,4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5,6,7. Although Cas9 variants with greater discrimination against mismatches have been designed8,9,10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

DOI: 10.1038/s41586-022-04470-1

Source: https://www.nature.com/articles/s41586-022-04470-1

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
官方网址：http://www.nature.com/
投稿链接：http://www.nature.com/authors/submit_manuscript.html