Structure of the minimal type I-F2 CRISPR-Cas DNA-interference complex

CRISPR–Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the type I-F2 system represents the most compact type I CRISPR–Cas system, distinguished by the absence of small and large subunits. These subunits are otherwise essential for target recognition and nuclease recruitment. To elucidate how this minimal system mediates DNA interference, we determined the cryo-electron microscopy (cryo-EM) structure of the I-F2 Cascade complex bound to its target DNA and the effector helicase-nuclease Cas3. Our structure reveals a unique mechanism of Cas3 recruitment, predominantly mediated by the Cas7 subunit. We show how the helicase and C-terminal domains of Cas3 engage the single-stranded DNA to initiate directional DNA-unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference in the absence of canonical subunits and emphasize the mechanistic diversity among closely related type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the minimal I-F2 system for genome editing and biotechnological applications.