Research On The Molecular Mechanism Of AcrIF11 Inhibiting Type I-F CRISPR-Cas System

CRISPR-Cas systems are part of the prokaryotic adaptive immune system against foreign genetic elements,including invading viruses.In order to effectively evade CRISPR systems,viruses such as bacteriophages have evolved protein-based inhibitors,called anti-CRISPR(Acrs),to inactivate the CRISPR-Cas immune systems.So far,studies have identified a large number of Acrs,targeting different types of CRISPR-Cas systems.Despite the growing number of identified Acr proteins,the working mechanisms of only a few of them have been characterized in detail.The type I-F CRISPR-Cas system is the first subtype to discovery Acrs,and it is also the subtype with the most families of Acrs identified at present.The traditional type I-F Acrs inhibit CRISPR-Cas system through direct interactions with either the crRNA-guided surveillance(Csy)complex or the Cas3 helicase-nucleas.Here,we reveal a novel mechanism by which AcrIF 11 inhibits the type I-F CRISPR system.By crystallizing AcrIF 11 protein,we determine its protein crystal structure and find NAD+located within the structure.Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase(mART)to modify N250 of the Cas8f subunit,a residue required for recognition of the invading DNA,within the Csy complex from Pseudomonas aeruginosa.The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA(dsDNA)binding activity.Biochemical studies also show that AcrIF11 requires,besides Cas8f,the Cas7f subunit for binding to and modifying the Csy complex.Therefore,our results not only reveal an unprecedented mechanism for inacticating the type I CRISPR-Cas system,but also provide an approach for designing efficient regulatory tools in the future applications of type I CRISPR-Cas systems.