Structure of <i>Geobacillus stearothermophilus</i> Cas9: Insights into the Catalytic Process and Thermostability of CRISPR-Cas9

CRISPR-Cas9 has been developed as a powerful gene editing tool, but the mechanism that governs its intricate catalytic process remains incompletely resolved. Here, the cryo-electron microscopy structures of thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) in a complex with sgRNA and target DNA are reported. The structure of GeoCas9 in a complex with sgRNA reveals a slit termed an L1 crevice comprising HNH, RuvC, and an L1 helix as a transient storage site of the 5′ spacer fragment of sgRNA. When the loosely bound 5′ spacer segment is extracted to pair with the target DNA, the L1 crevice collapses to trigger subsequent HNH domain translocation. In addition, structural and biochemical analyses suggest that the resilience of GeoCas9 at elevated temperatures is related to the unique PI domain conformation and a higher proline content of the WED domain. These results advance our understanding of the catalytic process of Cas9 and unveil the molecular mechanism that accounts for the superior thermal profile of GeoCas9.