Differential Allosteric Modulation of Cas9 Specificity

Both
RNA- and protein-based strategies have been developed to mitigate
off-target cleavage by CRISPR–Cas9, yielding noncanonical guide
RNAs (gRNAs) and Cas9 variants with enhanced gene-editing precision.
However, the molecular mechanisms by which such PAM-distal alterationsremote
from the nuclease centersmodulate Cas9 activity and specificity
remain incompletely understood. Here, we performed near-millisecond all-atom molecular dynamics simulations
to elucidate how diverse PAM-distal perturbationsincluding
gRNA truncation, base mismatching, and evolved mutationsreshape
the conformational dynamics and allosteric regulation of Cas9. Despite
their distinct origins, all perturbations ultimately modulate Cas9
function by altering HNH dynamics that impede the transition from
the checkpoint to the catalytically active state, yet they do so through
distinct allosteric routes. The 16-nt gRNA induces a pronounced REC3
reorientation toward the L2 linker and HNH domain, while PAM-distal
mismatches with the 18-nt gRNA promote engagement of the unwound target
DNA strand with L2both effectively restraining HNH rotation.
In contrast, evolved mutations remodel the global motional modes so
that REC2 swivels inward, constraining the HNH flexibility. These
perturbations delineate multiple structural paths converging on a
shared allosteric outcomeHNH immobilization and catalytic
suppressionthereby unifying RNA-, DNA-, and protein-level
effects within a single dynamic framework linking distal structural
perturbations to activity control. This work provides mechanistic
insight into the regulation of Cas9 fidelity and offers principles
for the design of next-generation genome editors.