Figure 3.

DNA targeting by the Cas9 family of RNA-guided endonucleases. (A) Schematic of dsDNA target (dark blue) recognition by Cas9s. Cas9 protein (grey) associates with two RNA partners, the crRNA (red) and the tracrRNA (purple), that are partially duplexed together. The HNH and RuvC nuclease motifs of Cas9 cleave the targeted DNA strand and the non-complementary DNA strand, respectively. Arrows denotes the cleavage sites. Recognition and cleavage of dsDNA targets require the complementarity between the DNA target and the crRNA spacer, and a short flanking protospacer-adjacent motif (PAM, yellow). (B) Schematic of NmeCas9's in vitro DNase H activity. NmeCas9 generates site-specific cuts (denoted by arrows, highlighted by green circle) in the DNA strand of a RNA-DNA hybrid substrate. Dark blue, DNA strand; red, RNA strand; light blue oval, NmeCas9.