Structural basis of RNA-guided DNA integration by type I CRISPR-associated transposases

ABSTRACT 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 complex1,2. Understanding the detailed mechanisms of this elaborate process is paramount to engineering CAST systems into programmable genetic tools3–6. The type I-F Pseudoalteromonas CAST (PseCAST) displays the highest activity in mammalian cells to date7 and has been the subject of extensive directed evolution8, but efforts to rationally engineer further improvements have been hampered by critical gaps in our understanding of transpososome assembly and activation9. Here we use cryo-EM structural analysis, validated by DNA transposition assays, to visualize the PseCAST 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 PseCAST 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. Competing Interest Statement G.D.L. and S.H.S. are inventors on patent applications related to CAST systems and uses thereof. All remaining authors declare no conflicts of interest.