BPS2026 – Biophysical principles for expanding PAM compatibility in CRISPR-Cas9 variants

CRISPR-Cas9 has revolutionized genome editing, yet its activity remains constrained by strict recognition of a guanine-rich protospacer adjacent motif (PAM), limiting targetable DNA sites. Expanding PAM compatibility is crucial for broadening therapeutic and biotechnological applications, but the structural basis enabling Cas9 variants to recognize non-canonical PAMs remains incompletely understood. Here, we combined extensive molecular dynamics (MD) simulations with novel causality inference analysis, graph theory-based network models and centrality analyses to investigate how SpCas9 variants—VQR, VRER, and EQR—achieve altered PAM specificity compared with wild-type Cas9 and two inactive constructs carrying R1333Q/R1335Q substitutions. Our results show that efficient PAM recognition arises from three interdependent principles: stabilization of the PAM-interacting (PI) domain, preservation of long-range allosteric communication between PI domain and the distal REC3, and entropic tuning of DNA engagement. Functional VQR, VRER, and EQR variants maintain stable contacts between PAM-interacting residues and DNA while distal mutations such as D1135V/E promote PI domain stabilization and enable productive coupling to REC3 and the catalytic HNH nuclease. In contrast, inactive engineered constructs disrupt this stabilization, fragment the REC3 dynamic network, and impair the HNH-REC3 allosteric pathway essential for nuclease activation. These findings establish a unified mechanistic framework linking local PAM binding to distal catalytic activation and provide generalizable design principles for rationally engineering next-generation Cas9 variants with expanded PAM compatibility and improved genome editing performance.