DNA remodeling couples target recognition to directional transposition in a Tn7-like CAST

Abstract

CRISPR-associated transposons (CASTs) couple target recognition to the insertion of large DNA cargoes, but how distinct targeting pathways are converted into productive and directional integration remains poorly understood1. Here we define the assembly pathway of a type I-B1 CAST from Anabaena variabilis, a system closely related to prototypical Tn7 that retains TnsD-mediated glmS recognition while incorporating CRISPR-based RNA-guided targeting2. Cryo-electron microscopy structures across multiple intermediates define a structural trajectory from two-step TnsD-mediated target recognition and stepwise assembly of the AAA+ ATPase TnsC to recruitment and activation of the split TnsA/TnsB transposase module. This trajectory culminates in an asymmetric strand-transfer complex that provides a structural basis for insertion orientation and supports a role for ATP hydrolysis in productive transpososome assembly. In parallel, RNA-guided targeting structures refine the functional PAM to ATG, define TniQ recruitment by Cascade, and show how CRISPR-based recognition converges on the shared TnsABC machinery. Together, these findings establish DNA remodeling and minor-groove positioning of TnsC as a common structural signal that converts protein- and RNA-guided target recognition into directional DNA insertion.