Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

Mikihiro Shibata (), Hiroshi Nishimasu (), Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi and Osamu Nureki ()
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Mikihiro Shibata: Kanazawa University
Hiroshi Nishimasu: The University of Tokyo
Noriyuki Kodera: Kanazawa University
Seiichi Hirano: The University of Tokyo
Toshio Ando: Kanazawa University
Takayuki Uchihashi: Kanazawa University
Osamu Nureki: The University of Tokyo

Nature Communications, 2017, vol. 8, issue 1, 1-9

Abstract: Abstract 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.

Date: 2017
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DOI: 10.1038/s41467-017-01466-8

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