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<p>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 <i>Streptococcus pyogenes</i> and <i>Staphylococcus aureus</i> 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.</p>

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AcrIIA19 binds to the WED domain and inhibits various Cas9 orthologs at multiple stages

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月23日

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  • Gi Eob Kim,
  • So Yeon Lee,
  • Yong Jun Kang,
  • Hyo Bin Jin,
  • Hyun Ho Park

摘要

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.