IS110 transposon utilizes two mechanistically distinct RNA-guided transposition pathways

The recently discovered IS110 elements employ RNA-guided transposases that use bridge-RNA to coordinate donor and target DNA recognition and integration via donor- and target-binding loops, offering promise for precise, DSB-free genome editing. However, their transposition mechanism remains unclear, with prior inconsistent observations. Using a IS110 element from Caloranaerobacter azorensis, we show that IS110 transposons utilize two independent transposition pathways. The first depends on bridge-RNA and excises the element via copy-out to form a circular intermediate, revisiting the earlier cut-out model. Integration occurs predominantly through top-strand insertion, suggesting an alternative to the earlier Holliday junction-mediated dual-strand mechanism. The second involves cooperation between a truncated target-binding loop RNA and bridge-RNA, enabling direct top-strand transfer to a new target via two sequential reactions, bypassing circular intermediates. Both pathways preserve the original transposon copy. These findings redefine the mechanistic understanding of IS110 transposition and reconcile prior discrepancies, offering insight into programmable, RNA-guided genome modification.