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

Author

Listed:
  • Mikihiro Shibata

    (Kanazawa University
    Kanazawa University)

  • Hiroshi Nishimasu

    (The University of Tokyo
    JST, PRESTO)

  • Noriyuki Kodera

    (Kanazawa University
    JST, PRESTO)

  • Seiichi Hirano

    (The University of Tokyo)

  • Toshio Ando

    (Kanazawa University
    CREST/JST)

  • Takayuki Uchihashi

    (Kanazawa University
    CREST/JST
    Kanazawa University
    Department of Physics, Nagoya University)

  • Osamu Nureki

    (The University of Tokyo)

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.

Suggested Citation

  • Mikihiro Shibata & Hiroshi Nishimasu & Noriyuki Kodera & Seiichi Hirano & Toshio Ando & Takayuki Uchihashi & Osamu Nureki, 2017. "Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy," Nature Communications, Nature, vol. 8(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01466-8
    DOI: 10.1038/s41467-017-01466-8
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    Cited by:

    1. Kazuki Kato & Sae Okazaki & Soumya Kannan & Han Altae-Tran & F. Esra Demircioglu & Yukari Isayama & Junichiro Ishikawa & Masahiro Fukuda & Rhiannon K. Macrae & Tomohiro Nishizawa & Kira S. Makarova & , 2022. "Structure of the IscB–ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    2. Aldo S. Bader & Martin Bushell, 2023. "iMUT-seq: high-resolution DSB-induced mutation profiling reveals prevalent homologous-recombination dependent mutagenesis," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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