@article {Park2021.05.25.445634, author = {Jung-Un Park and Amy Tsai and Eshan Mehrotra and Michael T. Petassi and Shan-Chi Hsieh and Ailong Ke and Joseph E. Peters and Elizabeth H. Kellogg}, title = {Structural basis for target-site selection in RNA-guided DNA transposition systems}, elocation-id = {2021.05.25.445634}, year = {2021}, doi = {10.1101/2021.05.25.445634}, publisher = {Cold Spring Harbor Laboratory}, 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{\textperiodcentered}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 Summary Cryo-EM studies reveals the role of the AAA+ regulator TnsC for target-site selection in CRISPR-associated transposition systems.Competing Interest StatementThe Peters lab has corporate funding for research that is not directly related to the work in this publication. Cornell University has filed patent applications with J.E.P. as inventor involving CRISPR-Cas systems associated with transposons that is not directly related to this work.}, URL = {https://www.biorxiv.org/content/early/2021/05/25/2021.05.25.445634}, eprint = {https://www.biorxiv.org/content/early/2021/05/25/2021.05.25.445634.full.pdf}, journal = {bioRxiv} }