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Nat Commun. 2017 Nov 10;8(1):1430. doi: 10.1038/s41467-017-01466-8.

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

Author information

1
High-speed AFM for Biological Application Unit, Institute for Frontier Science Initiative, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan. msshibata@staff.kanazawa-u.ac.jp.
2
Bio-AFM Frontier Research Center, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan. msshibata@staff.kanazawa-u.ac.jp.
3
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan. nisimasu@bs.s.u-tokyo.ac.jp.
4
JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan. nisimasu@bs.s.u-tokyo.ac.jp.
5
Bio-AFM Frontier Research Center, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan.
6
JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
7
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.
8
CREST/JST, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan.
9
Department of Physics, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan.
10
Department of Physics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
11
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan. nureki@bs.s.u-tokyo.ac.jp.

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.

PMID:
29127285
PMCID:
PMC5681550
DOI:
10.1038/s41467-017-01466-8
[Indexed for MEDLINE]
Free PMC Article

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