Structural Basis of PAM-Induced Conformational Changes in SpCas9: A Molecular Dynamics Study

As the most widely utilized CRISPR gene-editing enzyme,
SpCas9
has been extensively studied and applied. However, its strict dependence
on the canonical NGG PAM sequence significantly restricts its targeting
scope. Although recent research has successfully engineered SpCas9
variants capable of recognizing noncanonical (non-NGG) PAMs, these
variants still exhibit limitations when binding noncanonical PAMs,
including substantially reduced cleavage efficiency. To elucidate
the molecular mechanisms underlying noncanonical PAM recognition by
SpCas9, we employed molecular dynamics simulations to compare the
structural differences within the Cas9-gRNA-DNA ternary complex when
bound to various PAM sequences. Our analysis revealed significant
conformational changes within SpCas9 upon engagement with noncanonical
PAMs and uncovered the regulatory mechanisms underpinning these changes.
We further identified key dynamic determinants governing the extensive
conformational transitions occurring during the noncanonical PAM binding
process. These findings provide insights into the dynamic landscape
of noncanonical PAM recognition, offering crucial mechanistic guidance
for designing efficient, PAM-compatible Cas9 variants.