(A) Arrangement of the Pyrococcus furiosus Type I-A CRISPR-cas operon. (B) Agarose EMSA showing that Pfu Cas3 significantly improved the DNA target binding behavior of Pfu Cascade. (C) Agarose EMSA using differentially labeled binding partners to reveal that I-A Pfu Cas3 bound constitutively to Pfu Cascade. In contrast, I-E Tfu Cas3 only interacted with Tfu Cascade upon full R-loop formation. Cas3, Cascade, and dsDNA were labeled with Cy3 (rendered in green), Cy5 (red), and FAM (blue), respectively. Overlapping colors indicate complex formation that survived electrophoresis.