PT  - JOURNAL ARTICLE
AU  - Saki Osuka
AU  - Kazushi Isomura
AU  - Shohei Kajimoto
AU  - Tomotaka Komori
AU  - Hiroshi Nishimasu
AU  - Tomohiro Shima
AU  - Osamu Nureki
AU  - Sotaro Uemura
TI  - Real-time observation of flexible domain movements in Cas9
AID  - 10.1101/122069
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 122069
4099  - http://biorxiv.org/content/early/2017/03/29/122069.short
4100  - http://biorxiv.org/content/early/2017/03/29/122069.full
AB  - The CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to sgRNA and the target DNA. These studies also have hinted at flexibility between the domains, but whether these flexible movements take place under dynamic and physiological conditions is unclear. Here, we directly observed dynamic fluctuations by multiple Cas9 domains using single-molecule FRET. The flexible domain movements allow Cas9 to take transient conformations beyond those revealed by crystal structures. Importantly, one Cas9 nuclease domain accessed to the DNA cleavage position only during such flexible movement, suggesting the importance of this flexibility in the DNA cleavage process. Moreover, changes in domain flexibility in the presence of nucleic acids indicated that flexible Cas9 domain movements are involved in the sgRNA and the target DNA binding processes. Collectively, our results highlight the potential role of fluctuations in driving Cas9 catalytic processes.