Fig. 5: Updated model of Cas9 reaction cycle. | Nature Communications

Fig. 5: Updated model of Cas9 reaction cycle.

From: Visualization of a multi-turnover Cas9 after product release

Fig. 5

The cycle begins with a Cas9-sgRNA binary complex searching for PAM-containing DNA targets. Complementarity of the sgRNA spacer and target sequence initiates R-loop propagation where the distal duplex adopts a linear conformation. When the R-loop is fully formed, the PAM-distal DNA duplex adopts a kinked conformation and initiates nuclease lobe reorganization. The L1 and L2 linker helices precisely position HNH onto the target strand. HNH reorganization exposes the RuvC active site and enables accommodation of the NTS. HNH and RuvC catalyze concerted TS and NTS cleavage, respectively. Following TS cleavage, the HNH domain moves in reverse and adopts the checkpoint conformation again. Coordinated cleavage of the NTS allows for PAM-distal DNA rewinding and triggers the slow release of the PAM-distal product. The PAM-proximal product remains bound to the complex, prevents re-binding of a new target, and leads to product inhibition. Eventual release of the PAM-proximal product resets Cas9 to the binary surveillance complex and restarts the cycle.

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