Dynamics and thermostability of HNH nuclease in divergent Cas9 species

CRISPR-Cas9 is a powerful technology that has rapidly transformed genome editing. The Cas9 from Streptococcus pyogenes (SpCas9) is susceptible to degradation and unsuitable for applications requiring cleavage at elevated temperatures. A recently discovered Cas9 homolog from the thermophilic bacterium Geobacillus strearothermophillus (GeoCas9) showed stability and enzymatic activity at a wider range of temperatures than that of SpCas9. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, we investigated the GeoHNH using extensive molecular dynamics (MD) simulations and solution NMR to assess its structure dynamics in comparison to the canonical mesophilic SpHNH. We show that the mesophilic SpHNH undergoes extensive μs-ms motion while its thermophilic counterpart, GeoHNH, was flexible on a faster timescale (ps-ns). While specific intradomain pathways were found to drive SpHNH function, a non-specific and poorly formed signaling was found in GeoHNH. We also examined residue mutations in GeoHNH that have shown to increase the specificity in the SpCas9. The K597A mutation (corresponding to K855A in SpCas9) reduces the thermostability of GeoCas9, disrupting a network of critical salt-bridge interactions. These findings show that K597A alters dynamics and structure of GeoCas9, analogously to the specificity-enhancing K855A mutation in SpCas9, suggesting that lysine-to-alanine mutations might be critical for improving the specificity of GeoHNH and other Cas9 HNH domains. Our results provide the mechanistic differences between mesophilic and thermophilic Cas9 species which can aid in the development of alternate gene editing tools in mammalian cells.