Structure of the Type I-F3 CAST holo integration complex reveals licensing mechanisms during RNA-guided DNA integration

Precise genomic integration of large DNA cargo remains a central challenge in genome engineering. In this context, CRISPR-associated transposases (CASTs) offer a promising solution by coupling RNA-guided DNA recognition to transposon-mediated integration. Type I-F3 CASTs exhibit exceptional targeting fidelity, making them a leading focus for biotechnology and gene therapy applications. Yet, despite extensive characterization, the structural basis for RNA-guided DNA integration of type I-F3 CASTs has remained elusive. Here, using cryo-EM and functional assays, we report the structural licensing mechanism of the Vibrio cholerae CAST (VchCAST) holo integration complex (HIC). Our structures reveal that sequential conformational licensing events govern selective activation of the integration machinery at the on-target site. Different components, including Cascade-TniQ, TnsC, and TnsA/B, undergo conformational changes upon assembly into the HIC, and the catalytically competent state is stabilized only within the fully assembled complex. Finally, comparison with existing CAST integration complex structures reveals a set of conserved architectural features, suggesting a common molecular basis for RNA-guided transposition across CAST diversity.