Secondary Conformational Checkpoint in CRISPR-Cas9.

A specific checkpoint between target DNA binding and cleavage primarily governs the precision of Cas9 gene editing. Although various CRISPR-Cas9 variants have been developed to improve DNA cleavage accuracy, we still lack a comprehensive understanding of how they work at the molecular level. Herein, we have focused on studying the late-stage conformational transitions of Cas9 and an evolved Cas9 mutant (evoCas9) that start from the precleavage state. Our submilliseconds of dynamic simulations reveal that the presence of base mismatches leads the HNH nuclease domain of Cas9 to alter its principal functional modes of motion, thereby impairing its conformational activation. This observation suggests the existence of a secondary conformational checkpoint that fine-tunes the final DNA cleavage activation. Remarkably, evoCas9 is prone to deviating from the normal activation pathway with base mismatches. This is characterized by a noticeable shift in the positioning of the HNH domain and a significantly perturbed allosteric communication network within the enzyme. Therefore, the mutations evolved in evoCas9 also reinforce the secondary checkpoint in addition to the previously identified primary checkpoint, collectively ensuring this variant's high gene-editing accuracy. This mechanism should also apply to other Cas9-guide RNA variants with enhanced fidelity.