Abstact

CRISPR-associated transposases (CASTs) achieve site-specific DNA integration by coupling the RNA-guided targeting action of a nuclease-deficient CRISPR-Cas system with the assembly of a Tn7-like transpososome complex (1,2) . Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools (3-6) . The type I-F Pseudoalteromonas CAST ( Pse CAST) displays the highest activity in mammalian cells to date (7) and has been the subject of extensive directed evolution (8) , but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation (9) . Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the Pse CAST system in a series of functional states that define the stepwise mechanism of RNA-guided DNA integration. The structure of a target DNA-bound Cascade-TniQ-TnsC complex reveals that conformational changes induced by R-loop formation are coupled to target DNA stabilization and TnsC heptamerization, which in turn recruits the TnsAB transposase via conserved interactions with its C-terminal tail. Finally, the structure of the 1.2 MDa Pse CAST transpososome holocomplex reveals specific TnsC-TnsB and TnsB-target DNA interactions that drive allosteric remodelling of the TnsB catalytic site to activate donor DNA integration. Together, these findings establish a unified structural and mechanistic blueprint for RNA-guided DNA integration and lay the foundation for engineering next-generation DNA insertion systems for genome editing applications.