researcher Johns Hopkins University, United States
Introduction/Rationale: CRISPR-Cas systems provide prokaryotes with adaptive immunity against phages, and the RNA-guided Cas9 from type II-A systems has become a transformative genome editing tool. However, therapeutic applications requiring precise insertions remain constrained by off-target effects, as prolonged Cas9 activity correlates with diminished specificity. Anti-CRISPR (Acr) proteins, phage-encoded inhibitors of CRISPR-Cas immunity, offer strategies for Cas9 regulation and potential antimicrobial applications. While Acrs hold significant promise for research and clinical use, many lack the structural characterization required to fully realize their capabilities.
Methods: I determined the 3.0 Å cryo-electron microscopy structure of AcrIIA26 bound to Cas9–sgRNA and characterized its mechanism of DNA binding inhibition.
Results: AcrIIA26 adopts a novel fold distinct from known protein architectures, featuring a bifurcated shape comprising two α-helical bundles connected by a central β-sheet. It has a highly acidic surface mimicking duplex DNA, allowing AcrIIA26 to effectively compete for the Cas9 binding interface.
I showed that AcrIIA26 functions via a dual-inhibition mechanism. First, the protein occupies the DNA-binding channel, preventing the PI domain from recognizing PAM sequences and precluding DNA engagement. The 5-helix bundle occupies the PAM recognition site and directly engages key residues (R1333, R1335) within the PI domain, blocking PAM interaction. Second, the 4-helix bundle locks the REC2 domain in a conformation that hinders HNH domain activation and prevents the rearrangement required for catalysis.
Mutagenesis confirmed that residues in both bundles are essential for inhibition, establishing that AcrIIA26 simultaneously disrupts DNA engagement and Cas9 activation.
Conclusion: Our findings expand the mechanistic diversity of anti-CRISPRs, providing a structural framework to engineer AcrIIA26 as a precision “off-switch” for novel antimicrobial strategies and controllable CRISPR therapies.
