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. 2022 Mar 2;603(7900):343–347. doi: 10.1038/s41586-022-04470-1

Fig. 5. Model for Cas9 activation.

Fig. 5

During R-loop propagation (step 1), the gRNA–TS duplex adopts a linear conformation. After R-loop completion, the PAM-distal end of the linear duplex is captured by REC3 (steps 2 and 3). Mismatches in the PAM-distal region appear to prevent REC3 docking and thereby block subsequent steps of Cas9 activation. Once the kinked R-loop conformation has been formed, L1 and L2 linkers use the gRNA–TS duplex as a scaffold to position the HNH domain at the scissile phosphate of the target strand and to position the NTS in the RuvC site (step 4), which enables Cas9 to make a double-strand break (step 5). According to this model, mutations in the RuvC loop (corresponding to SuperFi-Cas9) inhibit formation of the kinked conformation and subsequent cleavage of the gRNA–TS duplex with mismatches at the PAM-distal end.