Abstract

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, structures with the transition state mimic, ADP{middle dot}AlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications. One Sentence SummaryCryo-EM studies reveals the role of the AAA+ regulator TnsC for target-site selection in CRISPR-associated transposition systems.