In silico identification and experimental validation of long-range allosteric inhibition of Staphylococcus aureus Cas9 catalytic activity by an anti-CRISPR protein AcrIIA14

Effective temporal and spatial regulation of CRISPR-Cas9 catalytic activity remains a key challenge, limiting the clinical application of CRISPR-Cas9 gene-editing. Here, we investigated the long-range allosteric inhibition of Staphylococcus aureus Cas9 (SauCas9) catalytic activity by its anti-CRISPR (Acr) protein, AcrIIA14, aiming to uncover remote allosteric mechanisms in large protein complexes and identify potential allosteric sites for the design of SauCas9 inhibitors. Through a combined computational-experimental framework integrating extensive molecular dynamics simulations, Markov state models, network community modeling, and site-directed mutagenesis, we identified canonical and non-canonical inhibitory states of SauCas9 regulated by AcrIIA14. Key domains, including REC, L1, HNH, L2, and PI, play crucial roles in transmitting the AcrIIA14-meidated inhibitory signal. Introducing point mutations on the routes of allosteric communication and analyzing these mutants using in vitro DNA cleavage assays and surface plasmon resonance analysis revealed that SauCas9 escaped AcrIIA14's inhibition owing to the disruption of AcrIIA14-meidated allosteric communication. Moreover, two cryptic allosteric sites on SauCas9 were identified as mutations of these sites prevented inhibition of SauCas9 by AcrIIA14. Overall, our results provide a dynamic understanding of CRISPR-Cas9 regulation and an avenue to design SauCas9 inhibitors with a broad range of applications in Cas9 enzyme catalysis, biophysics, and gene-editing.