A Staphylococcus phage recombination cassette that enables recombineering across distant bacteria

Modifying bacterial genomes with phage recombinases, a process termed recombineering, has enabled precise genome editing, but its reach is limited by host specificity: each new organism generally requires its own recombinase to be identified and optimized. Using known phage recombination genes as synteny anchors, we mined Staphylococcus genomes and recovered a diverse collection of phage recombination cassettes, spanning the RAD52-like annealase superfamily (Sak, Erf) and the RAD51/RecA-like Sak4 family. Functional testing of representatives in S. aureus identified several highly efficient systems that support recombineering. We engineered the most efficient of these into a single-plasmid tool that yields markerless editing in S. aureus in four days. Notably, where other recombineering systems rely on host-specific factors for nuclease evasion and ssDNA loading, this cassette encodes both activities itself: a predicted broad-spectrum nuclease inhibitor and a dedicated single-stranded DNA-binding protein. We therefore tested the cassette across phylogenetically distant hosts, and observed recombination in Streptococcus pyogenes, Enterococcus faecium, Francisella novicida, and Escherichia coli. These results identify a self-sufficient class of phage recombination systems and provide a portable tool for