Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications

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.