Year: 2025
Type: article
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. Using a combination of X-ray crystallography and biochemical approaches, the authors have characterized a plasmid-encoded Type II-A CRISPR-Cas system inhibitor, namely AcrIIA19, that exhibits broad-spectrum inhibition across divergent Cas9 orthologs. This work also identifies the WED domain as a strategic target for developing species-agnostic CRISPR regulatory tools for biotechnology and therapeutic applications.
Source: Communications Biology
Institution: Chung-Ang University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $4,190
Awards: G1856817905, G2722238760, G3677610472
OpenAlex ID: W7116982676