AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid.

Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems, allowing phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors. While enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present a nuclear magnetic resonance (NMR) structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein-level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in . Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that a human macrodomain can indeed remove the modification from Csy1 in lysate. Together, these experiments connect the observations of AcrIF11's enzymatic activity to its potent and specific effects , clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.IMPORTANCEBacteria have evolved diverse immune systems to prevent phage infection, and, consequently, phages have evolved diverse methods of evading bacterial immune systems. To evade the bacterial CRISPR-Cas immune system, phages encode anti-CRISPR proteins (Acrs). Acrs disable CRISPR-Cas by either stably binding to the CRISPR-Cas complex or by enzymatic modification. However, Acr enzymes have not been characterized during lytic infection or lysogenic maintenance. Here, we report the benefits and drawbacks of enzymatic inhibition with AcrIF11, an ADP-ribosyltransferase. Under "high pressure" scenarios such as high CRISPR targeting or CRISPR-Cas overexpression, AcrIF11 outperforms a strong, stable binding Acr by very specifically modifying the Cas8 protein, but nothing else in the cell. AcrIF11 additionally stabilizes lysogeny effectively, but the ADP-ribose modification can potentially be removed by macrodomains, which are ADP-ribose eraser enzymes. AcrIF11 is therefore a potent and widespread plasmid/phage-encoded inhibitor of Type I-F CRISPR-Cas systems with catalytic activity.