Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.
Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.
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DOI：
10.1038/s41467-017-01466-8
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发表时间：
2017-11-10
影响因子：
16.6
通讯作者：
Nureki O
中科院分区：
文献类型：
--
作者：
Shibata M;Nishimasu H;Kodera N;Hirano S;Ando T;Uchihashi T;Nureki O
CRISPR相关的核酸内切酶Cas9与向导RNA结合并切割具有与RNA向导互补的序列的双链DNA。Cas9-RNA系统已被用于许多应用，例如基因组编辑。在这里，我们使用高速原子力显微镜（HS-AFM）来可视化CRISPR-Cas9的实时空间和实时动态。HS-AFM电影表明，尽管apo-Cas9采用了意想不到的灵活构象，但Cas9-RNA形成了稳定的双叶结构，并通过三维扩散询问DNA上的靶位点。这些电影还提供了Cas9介导的DNA切割过程的实时可视化。值得注意的是，Cas9 HNH核酸酶结构域在DNA结合时波动，随后采用活性构象，其中HNH活性位点对接在靶DNA中的切割位点处。总的来说，我们的HS-AFM数据扩展了我们对CRISPR-Cas9作用机制的理解。CRISPR RNA引导的核酸内切酶Cas9识别并切割与RNA引导物互补的双链DNA。在这里，作者使用高速原子力显微镜（HS-AFM）来可视化Cas9在其DNA靶向和切割过程中的构象动力学。
The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9–RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9. CRISPR RNA-guided endonuclease Cas9 recognizes and cleaves the double-stranded DNA complementary to the RNA guide. Here the authors use high-speed atomic force micropcopy (HS-AFM) to visualize the conformational dynamics of Cas9 during its DNA targeting and cleavage processes.
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