Modules of native crRNA:tracrRNA duplexes and engineered single guide RNAs interact with protein domains of Cas9. The six functional modules formed in the crRNA:tracrRNA duplex that allow it to bind Cas9 (light gray) are shown in A; these include the spacer (black), lower stem (dark gray), bulge (green), upper stem (yellow), nexus (blue), and terminal hairpins (red). The protein domains within Cas9 that bind and interact with the guide RNA are shown in B, including the arginine bridge helix (green), PAM interacting motif (blue), HNH nuclease domain (red), and RuvC nuclease domain (yellow). Additionally, the recognition (REC) lobe and nuclease (NUC) lobe of Cas9 are labeled. The full native dual crRNA (blue) and tracrRNA (red) duplex is shown for S. pyogenes, S. aureus, and N. meningitidis in C, E, and G, respectively. The single guide RNA is shown for the same organisms in complex with target DNA (black) flanked by the PAM (green), for S. pyogenes (Jinek et al. 2012), S. aureus (Ran et al. 2015), and N. meningitidis (Hou et al. 2013) in D, F, and H, respectively. The sgRNA combines the crRNA and tracrRNA by a 4-nt tetraloop (dark gray).