LAUSR

E. coli maintain CRISPR-Cas adaptive immune systems to protect the cell against invading genetic elements. Immunity relies on the RNA guided surveillance complex Cascade (CRISPR-associated complex for antiviral defense) and the trans-acting Cas3 protein with helicase and nuclease activities. We recently showed that Cas3 generates degradation products ranging from 30 to 150 nt that act as pre-cursors for primed spacer acquisition. However, it remains unclear which mechanism drives the generation of these fragments with a specific size. Here we employed single-molecule FRET to probe the molecular dynamics of Cas3 in real-time. Our data shows that Cas3 repeatedly generates DNA loops in the target strand whilst remaining in tight contact with Cascade. DNA loops are generated by breaking open the dsDNA helix in distinctive steps of 3 bp, arising from the RecA like folds of the helicase domain. Repetitive unwinding achieved by slipping of the helicase domain, which limits the average translocation distance to ∼90 nt. Taken together, our data suggest that the inherent helicase properties of Cas3 drive the generation precursors of adequate size for primed spacer integration.