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Type I CRISPR-Cas systems utilize the RNA-guided Cascade complex to identify matching DNA targets and the nuclease-helicase Cas3 to degrade them. Among the seven subtypes, type I-C is compact in size and highly active in creating large-sized genome deletions in human cells. Here, we use four cryoelectron microscopy snapshots to define its RNA-guided DNA binding and cleavage mechanisms in high resolution. The non-target DNA strand (NTS) is accommodated by I-C Cascade in a continuous binding groove along the juxtaposed Cas11 subunits. Binding of Cas3 further traps a flexible bulge in NTS, enabling NTS nicking. We identified two anti-CRISPR proteins AcrIC8 and AcrIC9 that strongly inhibit Neisseria lactamica I-C function. Structural analysis showed that AcrIC8 inhibits PAM recognition through allosteric inhibition, whereas AcrIC9 achieves so through direct competition. Both Acrs potently inhibit I-C-mediated genome editing and transcriptional modulation in human cells, providing the first off-switches for type I CRISPR eukaryotic genome engineering. [Display omitted] • Four cryo-EM snapshots reveal DNA capture and Cas3 activation for type I-C CRISPR • Cas3 is recruited via "handshaking" with Cascade to the full R-loop • AcrIC8 and AcrIC9 inhibit PAM recognition by I-C Cascade via distinct mechanisms • AcrIC8 and AcrIC9 provide off-switches for type I CRISPR gene editing in human cells Hu et al. elucidate the PAM recognition, R-loop formation, and Cas3 recruitment processes for type I-C CRISPR activation. Two anti-CRISPR proteins, AcrIC8 and AcrIC9, block PAM recognition by I-C Cascade via distinct mechanisms and can serve as off-switches for type I-C genome deletion and transcriptional activation applications in human cells. [ABSTRACT FROM AUTHOR]