Prokaryotes have a wide range of defense mechanisms against invading nucleic acids. CRISPR-Cas is by far the most extensively studied and successfully applied anti-phage system. The vast diversity of CRISPR-Cas systems is divided into two classes and six types with different modes of action. The focus of this study is the type I-F CRISPR-Cas defense system found in A. actinomycetemcomitans bacteria[1]. This system encodes four different proteins (Cas5, Cas6, Cas7, Cas8), which together with crRNA form the ribonucleoprotein effector complex called Cascade (Csy), as well as Cas2/3 nuclease-helicase[2]. Cascade binds to the invading DNA site that is complementary to the crRNA, forming the R-loop structure. The R-loop triggers the Cas2/3 to degrade the foreign DNA eliminating the infection[3].
We have recently solved the cryo-EM structure of Cas2/3 bound to the Cascade-DNA complex and determined the interaction surfaces of Cas2/3 with Cascade. Here, using in vitro biochemical methods we analyse the importance of these surfaces for the DNA interference in the type I-F system.