Abstract
Anti-CRISPR (Acr) proteins are natural inhibitors of clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) systems, providing valuable tools for regulating genome editing. Here, we present the crystal structure of AcrIIA19, a plasmid-encoded Type II-A CRISPR-Cas system inhibitor that targets Cas9. AcrIIA19 adopts a previously uncharacterized fold and forms a stable homodimer. Biochemical assays revealed that AcrIIA19 binds selectively to the wedge (WED) domain of Cas9, a conserved structural interface critical for single guide RNA–DNA duplex stabilization and catalysis. This interaction disrupts Cas9 activity at multiple stages, independent of the order of complex assembly. Notably, AcrIIA19 exhibits broad-spectrum inhibition across divergent Cas9 orthologs, including Streptococcus pyogenes and Staphylococcus aureus Cas9, by exploiting a conserved WED domain vulnerability. Our findings establish AcrIIA19 as a versatile Cas9 inhibitor and highlight the WED domain as a strategic target for developing species-agnostic CRISPR regulatory tools in biotechnology and therapeutic applications.
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Data availability
The coordinate and structural factors have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) under the PDB code 9WA8. Uncropped original gels are provided at the Supplementary Fig. 1. The raw data for quantification analysis for Acr activity and Cas9 binding assays are provided in Supplementary Data 1.
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Acknowledgements
We would like to thank the 5 C beamline staff of the Pohang Accelerator Laboratory (Pohang, Korea) for their assistance during data collection. This study was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2025-02316334 and RS-2025-16065724).
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H.H.P. designed the research and supervised the project. G.E.K. expressed and purified the proteins and performed crystallization and structure determination. G.E.K. and S.Y.L. conducted biochemical and EMSA experiments. S.Y.L. and Y.J.K. performed structural prediction and MALS analysis. H.B.J. assisted with structural determination. H.H.P., G.E.K., and S.Y.L. interpreted the data and wrote the manuscript with contributions from all authors. All authors reviewed and approved the final manuscript.
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Kim, G.E., Lee, S.Y., Kang, Y.J. et al. AcrIIA19 binds to the WED domain and inhibits various Cas9 orthologs at multiple stages. Commun Biol (2025). https://doi.org/10.1038/s42003-025-09417-6
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DOI: https://doi.org/10.1038/s42003-025-09417-6
