Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9

The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of Geo Cas9, a thermophilic Cas9 from Geobacillus stearothermophilus . Our results provide new atomic details about the Geo Rec subdomains ( Geo Rec1, Geo Rec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout Geo Rec, and NMR studies of the interaction between Geo Rec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of Geo Cas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Streptococcus pyogenes Cas9. A K267E/R332A double mutant did also did not enhance Geo Cas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of Geo Cas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of Geo Cas9, setting the stage for future studies of Geo Cas9 variants and their effect on its allosteric mechanism.