小柯机器人

瑞士苏黎世大学Martin Jinek研究组揭示Cas9的R环形成和构象激活机制。相关论文于2022年8月24日在线发表在《自然》杂志上。

研究人员报告了化脓性链球菌Cas9的一系列冷冻电镜结构，并捕捉到靶标DNA杂交的定向过程。在R环形成的早期阶段，Cas9的REC2和REC3结构域形成一个带正电的裂缝，容纳了靶标DNA双链的远端。向导-靶标杂交经过种子区域后，诱导REC2和REC3结构域重新排列，并重新定位HNH核酸酶结构域，使其呈现无法催化的检查点构象。

导向-靶标异源双链的完成触发了HNH核酸酶结构域的构象激活，通过导向-靶标异源双链的扭曲以及互补的REC2和REC3结构域的重排而实现。这些结果共同建立了一个Cas9的靶向DNA依赖性激活的结构框架，揭示了其构象检查机制，并可能促进具有增强特异性和活性的新型Cas9变体和向导RNA设计的发展。

据了解，Cas9是一种CRISPR相关的内切酶，能够在RNA引导下对DNA进行定点切割。Cas9的可编程活性已被广泛用于基因组编辑应用，然而它的靶标DNA结合和非靶标识别的精确机制仍未被完全理解。

附：英文原文

Title: R-loop formation and conformational activation mechanisms of Cas9

Author: Pacesa, Martin, Loeff, Luuk, Querques, Irma, Muckenfuss, Lena M., Sawicka, Marta, Jinek, Martin

Issue&Volume: 2022-08-24

Abstract: Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1,2,3. The programmable activity of Cas9 has been widely utilized for genome editing applications4,5,6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide–target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide–target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide–target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

DOI: 10.1038/s41586-022-05114-0

Source: https://www.nature.com/articles/s41586-022-05114-0

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/
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