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Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9

Haruka Narita, Hiroshi Ebata, Karibu Sakai, Katsuhiko Minami, Sotaro Uemura, View ORCID ProfileTomohiro Shima
doi: https://doi.org/10.1101/2020.04.13.039537
Haruka Narita
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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Hiroshi Ebata
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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Karibu Sakai
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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Katsuhiko Minami
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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Sotaro Uemura
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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Tomohiro Shima
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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  • ORCID record for Tomohiro Shima
  • For correspondence: tomohiro.shima@bs.s.u-tokyo.ac.jp
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Abstract

SHORT ABSTRACT This paper summarizes how to visualize the flexible inter-domain movements of CRISPR-associated protein Cas9 using single molecule FRET

LONG ABSTRACT The CRISPR-associated protein Cas9 is widely used as a genome editing tool because of its ability to be programmed to cleave any DNA sequence that is followed by a protospacer adjacent motif. The continuing expansion of Cas9 technologies has stimulated studies regarding the molecular basis of the Cas9 catalytic process. Here we summarize methods for single molecule FRET (smFRET) to visualize the inter-domain movements of Cas9 protein. Our measurements and analysis demonstrate flexible and reversible movements of the Cas9 domains. Such flexible movements allow Cas9 to adopt transient conformations beyond those solved by crystal structures and play important roles in the Cas9 catalytic process. In addition to the smFRET measurement itself, to obtain precise results, it is necessary to validate Cas9 catalytic activity. Also, fluorescence anisotropy data are required to interpret smFRET data properly. Thus, in this paper, we describe the details of these important additional experiments for smFRET measurements.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted April 14, 2020.
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Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9
Haruka Narita, Hiroshi Ebata, Karibu Sakai, Katsuhiko Minami, Sotaro Uemura, Tomohiro Shima
bioRxiv 2020.04.13.039537; doi: https://doi.org/10.1101/2020.04.13.039537
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Implementation of single molecule FRET for visualizing intramolecular movement in CRISPR-Cas9
Haruka Narita, Hiroshi Ebata, Karibu Sakai, Katsuhiko Minami, Sotaro Uemura, Tomohiro Shima
bioRxiv 2020.04.13.039537; doi: https://doi.org/10.1101/2020.04.13.039537

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