Canonically minimal RNA-guided insertion sequences expand into large elements that disseminate antimicrobial resistance

IS110 has emerged as a powerful genome-editing tool because it is the smallest RNA-guided system capable of diverse programmable insertions. Naturally existing elements are conventionally modeled as compact ~1.5-kb systems comprising a single transposase and a bridge RNA (bRNA). Using high-throughput junction mapping together with large-scale comparative genomics, we redefined the in vivo structural boundaries, growth, and mobilization of IS110 elements. We uncovered a previously unrecognized size continuum extending to ~100 kb, driven by progressive additions. Experiments confirmed activity of natural IS110s both well below and above the size range of previously characterized elements. The large systems comprised mostly plasmid-derived DNA, with antimicrobial resistance genes being the most enriched. Boundary configurations at expanded loci and the range of partial excision intermediates they produce both indicate flexible sequence recognition by IS110, with half-matches between the bRNA and complementary DNA sequence as the most enriched configuration. This sequence tolerance allows loci to expand with diverse cargo. Together, these findings redefine IS110 from a compact insertion sequence into a dynamic platform that disseminates adaptive cargos.