Fig. 2.

Schematic illustration of CRISPR/Cas9 mechanism. A The Cas9 protein complex contains six domains (Recognition lobe (REC I), REC II, Arginine-rich bridge helix, PAM Interacting, HNH, and RuvC). REC I is the major domain responsible for binding with the gRNA, the REC II function is not studied. The arginine-rich bridge helix initiates cleavage activity upon binding to targeted sequences. The interaction with PAM confers PAM specificity, which is responsible for binding with the target sequence. The HNH and RuvC are nuclease domains to chop the target sequence. The Cas9 protein remains inactive due to the absence of gRNA. B The programmed gRNA binds to the Cas9 and generates changes in the protein, which leads the inactive Cas9 protein into its active form. Once triggered, it searches the target sequence by binding with a sequence that matches the PAM sequence (5′-NGG-3′). Then Cas9 generates DSBs at 3 bp upstream of the PAM using its HNH and RuvC domains