Research Article

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

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Science  15 Jul 2021:
eabi8976
DOI: 10.1126/science.abi8976

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Abstract

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo 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-electron microscopy (cryo-EM). Polymerization of ATP-bound TnsC helical filaments could explain how polarity information is passed to the transposase. 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, ADPᐧAlF3, 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.

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