Comparative Structural and Dynamics Study of Free and gRNA-bound FnCas9 and SpCas9 Proteins

The introduction of CRISPR/Cas9 based gene editing has greatly accelerated therapeutic genome editing. However, the off-target DNA cleavage by CRISPR/Cas9 protein hampers its clinical translation, thereby hindering its widespread use as a programmable genome editing tool. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. Here, we have compared the dynamics of a more specific naturally occurring Cas9 from Francisella novicida (FnCas9) to the most widely used, SpCas9 protein. Long-scale atomistic MD simulations of free and gRNA-bound forms of both the Cas9 proteins were performed, and their domain rearrangements and binding affinity with gRNA were compared to decipher the possible reason behind the enhanced specificity of FnCas9 protein. The greater binding affinity with gRNA, high domain electrostatics, and more volatility of FnCas9 than SpCas9 may explain its increased specificity and lower tolerance for mismatches. Highlights The gRNA binding led to the opening of the structure of Cas9 endonuclease and exposure of hydrophobic residues to accommodate it. Concerted domain movement was observed in both SpCas9 (HNH-REC2 domains, CTD-Topo domains, and REC3-RuvC) and FnCas9 (HNH-REC3-REC1 and PI-WED-RuvC). Both Cas9s have shown a decrease in percentage helicity and an increase in the percentage of structurally dynamic residues (more secondary structural transitions) post gRNA binding. In the gRNA-bound form of FnCas9, the PAM-interacting domain was found to have a larger structural transition and increased coil%. Electrostatic interactions have a major contribution to the high binding affinity of FnCas9 with gRNA. Arginine-helix has shown a pivotal role in the binding of gRNA in FnCas9 protein.