Recruitment of Cas3 enables DNA cleavage by the type I-A CRISPR-Cas system of Saccharolobus islandicus.

Type I CRISPR-Cas systems constitute the most prevalent prokaryotic adaptive immune pathways and are classified into seven subtypes (I-A to I-G). These antiviral systems typically exploit a Cascade complex for RNA-guided DNA recognition and a Cas3 helicase-nuclease effector for DNA degradation, yet their diverse activation mechanisms remain not fully understood. In this study, we isolate the I-A Cascade from Saccharolobus islandicus, revealing a minimal form of Cascade lacking both Cas3 and the CRISPR-RNA maturase Cas6. Cas3 is recruited to the R-loop structure formed after Cascade binding to target DNA, which activates the effector for both cis- and trans-DNA cleavage. Strikingly, ATP not only enables the processive target degradation by Cas3 but also suppresses the trans-cleavage of the same enzyme. Together, the Sa. islandicus I-A system operates via target-dependent Cas3 recruitment-a mechanism distinct from other characterized I-A systems, thus underscoring the mechanistic diversity within type I CRISPR-Cas immunity.