DNA-targeting CRISPR/Cas systems. DNA-targeting CRISPR/Cas systems operate in three stages, based on distinct targeting mechanisms. (A) Type I involves Cascade, which identifies PAM and recruits Cas3 for DNA nicking along a single strand. (B) In Type II, Cas9 pairs with crRNA, locates PAM, and induces blunt dsDNA breaks by aligning its HNH and RuvC domains. (C) Type V uses Cas12 with a crRNA guide to recognize PAM, cleaving the non-target strand first, followed by the target strand through the RuvC domain, which after a staggered dsDNA break is formed, remains active for further ssDNA cleavage.
