By establishing bridge recombinase as a programmable, cross-phylum genome editing platform capable of megabase-scale rearrangements and directional horizontal gene transfer, this work transforms microbiome engineering from organism-specific tinkering into controlled, ecosystem-level rewriting.
When bridge recombinase is directed by two bridge RNAs that each specify one of two recombination junctions, it can programmably swap two distinct, large DNA segments in a single step, a genuine genomic "search-and-replace" we call TRADE editing. To achieve high-fidelity TRADE editing, we engineered orthogonal bRNAs that act like color-coded wires, so the left and right junctions each connect only to their matching ends rather than cross-wiring. This effectively makes it possible to pick up any piece of DNA and move it to any location in any other DNA molecule. Harnessing this, we achieved Programmable Horizontal Gene Transfer: relocation of large, defined regions of chromosomal DNA between diverse bacteria to confer new functional traits on demand.
Bridge recombinase enables programmable, versatile genome editing across diverse bacterial phyla and intact microbial communities, performing insertions up to 142 kb, inversions up to 2.3 Mb, and pathway-scale excisions with high efficiency in single steps, while optimized dual-bRNA systems achieve search-and-replace editing and programmable horizontal gene transfer of functional biosynthetic pathways between phyla without reliance on homologous recombination or host-specific factors.
Bacteria drive crucial processes across ecosystems and profoundly impact human health, yet tools to rewrite microbiomes remain limited. Here, we show that bridge recombinase enables versatile and programmable genome editing across the bacterial tree of life. In Escherichia coli, we achieved 142 kb insertions at >90% efficiency, megabase-scale inversions (2.3 Mb), and pathway-scale 50 kb excisions. With a single ortholog and bridge RNA (bRNA), we edited bacterial isolates spanning five phyla and performed metagenomic editing in human gut microbiomes. We overcame cross-reactivity between co-expressed bRNAs to establish single-step search-and-replace editing, and demonstrated capture and interphylum transfer of functional chromosomal pathways, enabling programmable horizontal gene transfer. These advances establish bridge recombinase as a foundation for orchestrating controlled gene flow in complex microbial ecosystems.