Discovery of human gut phage-encoded anti-CRISPR proteins unveils diverse mechanisms for phages to evade type II...

Phages encode diverse anti-CRISPR (Acr) proteins to counteract bacterial CRISPR-Cas systems. However, gut phage Acrs remain poorly characterized. Using an integrated bioinformatics and high-throughput functional screening approach, we identify 651 phage-encoded positive Acr candidates that target type II CRISPR systems, which predominate in the human gut. Among these, a subset of Acrs is verified through plasmid interference assays, with plaque assays confirming CRISPR-Cas inhibitory activity for 36 Acr candidates. Mechanistic characterization of five Acrs, including the Acr against subtype II-B systems (AcrIIB-1), reveals distinct inhibition strategies. Remarkably, 213 positive Acr candidates, designated here as GutAcr_aca, exhibit structural convergence by adopting similar folds and exhibit dual functionality: transcription regulation to support their production and inhibition of CRISPR-Cas systems. These GutAcr_aca are widely distributed across microbial species (detected in 26% of species). Our work uncovers the extensive diversity of phage-encoded Acrs in the human gut and highlights their potential as biotechnology tools.