IS110 transposon utilizes two mechanistically distinct RNA-guided transposition pathways

The IS110 elements employ RNA-guided transposases using bridge-RNA to coordinate donor and target DNA recognition and integration via donor- and target-binding loops, which offers promise for DSB-free genome editing. However, their transposition mechanism remains unclear, with prior inconsistent observations. Using an IS110 element from Caloranaerobacter azorensis, we show that IS110 transposons utilize two independent transposition pathways. The first depends on bridge-RNA (bRNA) and excises the element via top-strand excision and rejoining, which corresponds to the initial step of the canonical copy-out model and revisits the earlier cut-out model. Integration occurs predominantly through top-strand insertion, which suggests an alternative to the Holliday junction-mediated dual-strand mechanism. The second involves cooperation between a truncated target-binding loop RNA and bRNA, which enables direct top-strand transfer to a new target via two sequential reactions. Both pathways preserve the original transposon copy. These findings redefine the mechanistic understanding of IS110 transposition and reconcile prior discrepancies, thus offering an insight into programmable, RNA-guided genome modification.