Structure of Geobacillus stearothermophilus Cas9: Insights into\nthe Catalytic Process and Thermostability of\nCRISPR-Cas9

CRISPR-Cas9 has been developed as a powerful gene editing\ntool,\nbut the mechanism that governs its intricate catalytic process remains\nincompletely resolved. Here, the cryo-electron microscopy structures\nof thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) in a complex with sgRNA and target DNA are reported. The\nstructure of GeoCas9 in a complex with sgRNA reveals a slit termed\nan L1 crevice comprising HNH, RuvC, and an L1 helix as a transient\nstorage site of the 5′ spacer fragment of sgRNA. When the loosely\nbound 5′ spacer segment is extracted to pair with the target\nDNA, the L1 crevice collapses to trigger subsequent HNH domain translocation.\nIn addition, structural and biochemical analyses suggest that the\nresilience of GeoCas9 at elevated temperatures is related to the unique\nPI domain conformation and a higher proline content of the WED domain.\nThese results advance our understanding of the catalytic process of\nCas9 and unveil the molecular mechanism that accounts for the superior\nthermal profile of GeoCas9.