Catalytic-state structure and engineering of Streptococcus thermophilus Cas9

Cas9 nucleases recognize and cleave their target DNA through base pairing of a guide RNA with a spacer adjacent to a protospacer adjacent motif (PAM).Streptococcus thermophilusCas9 (St1Cas9), a smaller Cas9 orthologue thanStreptococcus pyogenesCas9, enables robust genome editing in diverse organisms. Here we report high-resolution structures of St1Cas9 in complex with a single-guide RNA and different PAM-containing DNAs. All of the structures represent an HNH catalytic state that is rarely observed in other Cas9 structures, clearly depicting the active conformation. A unique wing region in the REC domain forms intensive interactions with the HNH domain, playing a key role in regulating St1Cas9 DNA cleavage activity and probably stabilizing the active conformation. Furthermore, St1Cas9 applies a strategy distinct from those of other Cas9 orthologues for PAM recognition. Structure-guided engineering of St1Cas9 substantially expanded its targeting scope. These molecular-level characterizations will facilitate the rational engineering of St1Cas9.