ABSTRACT
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.