Figure 4. The proposed model of bimodal Cas9–RNA binding along with the kinetics of Cas9–RNA DNA targeting as a function of mismatches.

Cas9-RNA targeting occurs in predominantly two steps. The first step, that is, initial Cas9-RNA binding to DNA target is a transient PAM surveillance step, independent of the DNA sequence. In the second step, following the PAM detection, Cas9-RNA proceeds to form RNA-DNA heteroduplex in a unidirectional manner, that is, from PAM-proximal to PAM-distal end. Rate of transition between various Cas9-RNA targeting steps for different DNA targets is indicated by the size of arrows.