Structure of Geobacillus stearothermophilus Cas9: insights into the catalytic process and thermostability of CRISPR-Cas9

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