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bioRxiv · 10.1101/2021.05.25.445634

Structural basis for target-site selection in RNA-guided DNA transposition systems

Park, J.-U.·Tsai, A.·Mehrotra, E.·Petassi, M. T.·Hsieh, S.-C.·Ke, A.·Peters, J. E.·Kellogg, E. H.
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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.

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Park, J.-U., Tsai, A., Mehrotra, E., Petassi, M. T., Hsieh, S.-C., Ke, A., Peters, J. E., Kellogg, E. H.. 2021-05-25. Structural basis for target-site selection in RNA-guided DNA transposition systems. https://doi.org/10.1101/2021.05.25.445634

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