Figure 2. Mechanism of crRNA biogenesis and targeting in the three Types of CRISPR-Cas systems.

Black arrowheads, primary processing sites of the crRNA precursor (pre-crRNA) to liberate intermediate crRNAs (int-crRNA). White arrowhead, further processing of the int-crRNA to yield mature crRNAs (mat-crRNA). Green line, target sequence (same sequence as crRNA spacer). Purple line, PAM (Mojica et al., 2009). (A) In Type I systems, primary processing of the pre-crRNA is achieved by the Cas6 endoribonuclease within the Cascade complex (Brouns et al., 2008). Cleavage occurs at the base of the stem-loop formed by the repeat RNA to release mat-crRNAs. The Cascade recruits the Cas3 nuclease to nick the DNA strand complementary to the proto-spacer, immediately downstream of the region of interaction with the crRNA spacer (Sinkunas et al., 2013). (B) In Type II systems primary processing requires the annealing of the tracrRNA to the repeat sequences of the pre-crRNA and the subsequent cleavage of the dsRNA by the host RNase III (Deltcheva et al., 2011). Primary processing occurs in the contex of Cas9 and it is followed by the trimming of the 5′-end repeat and spacer sequences of the int-crRNA to yield mat-crRNAs. Target cleavage requires the crRNA, the tracrRNA and the RuvC and HNH domains of Cas9, each of which cleaves one DNA strand of the proto-spacer region, 3-nt upstream of the PAM (Gasiunas et al., 2012; Jinek et al., 2012). (C) In Type III systems Cas6 cleaves the pre-crRNA to generate int-crRNAs that are incorporated into a Cmr/Cas10 or Csm/Cas10 complex, where further maturation occurs through the trimming of 3′-end sequences (Hale et al., 2012; Hale et al., 2009). While genetic evidence indicates that III-A subTypes cleave target DNA sequences (Hatoum-Aslan et al., 2014), biochemical data suggests that subType III-B cleave RNA molecules (Hale et al., 2009).