Fig. 3. Schematic model of target searching and R-loop formation by Cascade or Cas9 on long stretches of DNA. (A) Model of target searching and discrimination by the Cascade complex. The Cascade–crRNA complex searches for the target sequence through random 3D collisions. When the complex recognizes a bona fide target, R-loop propagation starts from the PAM proximal region; the full R-loop structure is stabilized by DNA-RNA hybridization for target cleavage. In contrast, when Cascade complex binds weakly to a mutated target, Cascade processing the exogenous mutant DNA incorporation to the CRISPR spacer locus called priming. Target and PAM sequence is shown in green and yellow box respectively. DNA sequence mutation is shown in orange circle. (B) Model of target searching and R-loop formation by Cas9. The Cas9-guide RNA complex scans the target sequence through random 3D collisions. Non-specific binding to off-target sites induces the formation of an intermediate R-loop structure; failure to maintain the full R-loop structure generates a quick dissociation from the off-target sequence. This weak binding helps make searching for the on-target sequence efficient. On-target Cas9 binding promotes R-loop formation, similar to the Cascade complex, and stabilization of the R-loop structure allows cleavage of the DNA target.