Cascade-crRNA complexes mediate the identification of the invading foreign DNA and initiate its neutralization by formation of an R-loop (RNA-induced DNA-loop) at the crRNA-complementary sequence (protospacer). After initial unspecific binding to the double-stranded DNA, Cascade-crRNA complex slides along the DNA to find the protospacer. Once the target site is detected, the crRNA hybridizes to the complementary strand with subsequent displacement of the non-complementary strand to form an R-loop structure. Here, we describe how Cascade-DNA complexes and the Cascade-induced strand separation can be characterized in detail by combining chemical and enzymatic footprint analyses. Selective modification of unpaired thymines by permanganate (KMnO4) and the specific cleavage of single-stranded DNA by Nuclease P1 can be used to probe an R-loop formation by Cascade. Localization of the Cascade-crRNA complex on the DNA can be achieved by an Exonuclease III protection assay.