The crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA (sgRNA) and target DNA at 2.5 Å resolution reveals its bilobed architecture, composed of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe binds the sgRNA:DNA heteroduplex in a positively-charged groove, while the NUC lobe contains the HNH and RuvC nuclease domains, which cleave complementary and non-complementary DNA strands. The NUC lobe also includes a carboxyl-terminal domain that interacts with the protospacer adjacent motif (PAM). The structure elucidates the molecular mechanism of RNA-guided DNA targeting by Cas9, providing insights into its function and paving the way for the rational design of new genome-editing technologies. The REC lobe recognizes the repeat:anti-repeat duplex, while the PAM-interacting (PI) domain confers PAM specificity. The RuvC domain has an RNase H fold and cleaves the non-complementary strand via a two-metal mechanism. The HNH domain has a ββα-metal fold and cleaves the complementary strand via a single-metal mechanism. The sgRNA:DNA complex adopts a T-shaped architecture, with the guide:target heteroduplex, repeat:anti-repeat duplex, and stem loops 1–3. The conserved arginine cluster on the Bridge helix is critical for sgRNA:DNA recognition. The REC1 and RuvC domains facilitate RNA-guided DNA targeting. The repeat:anti-repeat duplex is recognized by the REC and NUC lobes in a sequence-dependent manner. The structural flexibility of Cas9 and sgRNA allows for conformational changes necessary for DNA cleavage. The study provides a critical step towards understanding the molecular mechanism of RNA-guided DNA targeting by Cas9 and highlights the importance of structural and functional studies for the development of Cas9-based genome engineering technologies.The crystal structure of Streptococcus pyogenes Cas9 in complex with guide RNA (sgRNA) and target DNA at 2.5 Å resolution reveals its bilobed architecture, composed of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe binds the sgRNA:DNA heteroduplex in a positively-charged groove, while the NUC lobe contains the HNH and RuvC nuclease domains, which cleave complementary and non-complementary DNA strands. The NUC lobe also includes a carboxyl-terminal domain that interacts with the protospacer adjacent motif (PAM). The structure elucidates the molecular mechanism of RNA-guided DNA targeting by Cas9, providing insights into its function and paving the way for the rational design of new genome-editing technologies. The REC lobe recognizes the repeat:anti-repeat duplex, while the PAM-interacting (PI) domain confers PAM specificity. The RuvC domain has an RNase H fold and cleaves the non-complementary strand via a two-metal mechanism. The HNH domain has a ββα-metal fold and cleaves the complementary strand via a single-metal mechanism. The sgRNA:DNA complex adopts a T-shaped architecture, with the guide:target heteroduplex, repeat:anti-repeat duplex, and stem loops 1–3. The conserved arginine cluster on the Bridge helix is critical for sgRNA:DNA recognition. The REC1 and RuvC domains facilitate RNA-guided DNA targeting. The repeat:anti-repeat duplex is recognized by the REC and NUC lobes in a sequence-dependent manner. The structural flexibility of Cas9 and sgRNA allows for conformational changes necessary for DNA cleavage. The study provides a critical step towards understanding the molecular mechanism of RNA-guided DNA targeting by Cas9 and highlights the importance of structural and functional studies for the development of Cas9-based genome engineering technologies.