Modes of inhibition used by phage anti-CRISPRs to evade type I-C Cascade

Cascades are RNA-guided multi-subunit CRISPR-Cas surveillances complexes that target foreign nucleic acids for destruction. Here, we present a 2.9-[A] resolution cryo-electron (cryo-EM) structure of the D. vulgaris type I-C Cascade bound to a double-stranded (ds)DNA target. Our data shows how the 5-TTC-3 protospacer adjacent motif (PAM) sequence is recognized, and provides a unique mechanism through which the displaced, single-stranded non-target strand (NTS) is stabilized via stacking interactions with protein subunits in order to favor R-loop formation and prevent dsDNA re-annealing. Additionally, we provide structural insights into how diverse anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking initial DNA binding. These observations provide a structural basis for directional R-loop formation and reveal how divergent Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.