Movements in CRISPR-Cas9, a genome-editing tool, during the DNA cleavage process
Overview of the press release
The CRISPR-associated protein Cas9 is widely used as a powerful genome editing tool because it can be programmed to cleave specific DNA sequences. In the present study, a research team led by Assistant Professor Tomohiro Shima and Professor Osamu Nureki (Department of Biological Sciences) has clarified conformational dynamics of the Cas9 molecule. While crystal structures have provided a static view of Cas9 bound to target DNA, the research team used single-molecule FRET (Fluorescent Resonance Energy Transfer) to illustrate how inter-domain fluctuations help drive Cas9-catalyzed DNA cleavage. The measurements revealed Cas9 to exist in both static and fluctuating phases. Interestingly, the catalytic domain in Cas9 (HNH domain) accessed the DNA cleavage competent position only during the fluctuating phase.
This study highlights the importance of conformational fluctuations in Cas9 and provides useful information for the future improvement of this genome editing tool.
Figure: Movements of the HNH catalytic domain in CRISPR-Cas9.
Cas9 molecules showed both static and fluctuating states. The HNH catalytic domain accessed the DNA cleavage competent (D) position only during the fluctuating state. During the fluctuating state, the HNH domain fluctuated between the intermediate (I) position and D position or newly revealed semi-docked (D*) position. The distance between the HNH domain and the DNA cleavage site was monitored by the single-molecule FRET technique.
Journal The EMBO Journal Title Real-time observation of flexible domain movements in CRISPR-Cas9 Authors Saki Osuka, Kazushi Isomura, Shohei Kajimoto, Tomotaka Komori, Hiroshi Nishimasu, Tomohiro Shima*, Osamu Nureki*, Sotaro Uemura DOI 10.15252/embj.201796941 Paper link http://emboj.embopress.org/cgi/doi/10.15252/embj.201796941
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