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

Abstract

Target site selection in CAST systems Exciting genomic engineering possibilities exist for natural integration systems called transposons, which have coopted CRISPR/Cas systems. An unexplained feature of these systems involves how they direct insertions in a single orientation at a precise distance from the programmed target sequence. Park et al. show that orientation information is communicated to the transposase TnsB using the unidirectional growth of a helical filament made up of an AAA+ protein, TnsC. ATP hydrolysis trims the filament to a minimal unit that is marked by TniQ and defined by the Cas12k protein to provide spacing information. This finding may help future engineering of these systems for therapeutic applications. Science, abi8976, this issue p. 768 Cryo-EM studies reveals a role for polymerization of a transposition regulator, TnsC, in CRISPR-associated transposition systems. 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 used cryo–electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate–bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing 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, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.