A dynamically frustrated allosteric checkpoint consistent with conformational proofreading in CRISPR–Cas9

Abstract

Abstract CRISPR–Cas9 DNA cleavage requires R-loop extension to activate the histidine–asparagine–histidine (HNH) nuclease domain, yet how heteroduplex maturation gates catalytic commitment remains unclear. Using extensive molecular dynamics simulations across seven experimentally trapped heteroduplex states (6–18 nt), we define a three-stage activation pathway: unlocking, preorganization, and precatalytic gating. At the 18-nt checkpoint, HNH and REC2 regain mobility but do not adopt a fully cleavage-competent orientation; this dynamically frustrated metastate is consistent with a conformational-proofreading model. Here, dynamic frustration is used operationally to describe renewed mobility and weakened directional coupling without commitment to the cleavage-competent state, rather than a formal energetic frustration calculation. Two sensors, Y450 at the sgRNA: DNA hybrid interface and K1200 in the PI domain, respond to distinct maturation milestones, encoding heteroduplex length into domain-specific conformational outputs. Integrating these insights with two deep-learning analyses and deep mutational scanning recovers known high-fidelity positions and prioritizes three unannotated residues for testing. In a cellular cleavage reporter assay, R1210D reproduces the attenuated low-completion phenotype of eSpCas9, identifying it as a candidate for direct specificity testing, while destabilizing L1-linker substitutions increase cleavage efficiency. These results connect atomic-level dynamics to experimentally testable, structure-mechanism-informed engineering hypotheses; direct matched-versus mismatched validation is required to establish effects on discrimination fidelity.