CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids using an RNA-mediated interference mechanism. Interference in Type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Cas3 bound to ss-DNA substrate and show that it is an obligated 3′-to-5′ ss-DNase preferentially accepting substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ss-DNA cleavage. ATP coordination and conformational flexibility are revealed for the SF2-type helicase moiety. Cas3 is specifically guided towards Cascade-bound target DNA with a correct PAM sequence, through physical interactions to both the non-target substrate strand and the CasA protein. The cascade of recognition events ensures a well-controlled DNA targeting and degradation of alien DNA by Cascade and Cas3.