Cas3 Mediated Target DNA Recognition and Cleavage is Independent of the Composition and Architecture of Cascade Surveillance Complex

In type I CRISPR-Cas system, Cas3 -a nuclease cum helicase- in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organisation of Cascade subunits in type I-C influences the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues -that are unique to type I-C- located in the uncharacterised C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the nontarget strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.