A diverse single-stranded DNA–annealing protein library enables efficient genome editing across bacterial phyla

Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA-annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia (<i>Mycobacterium smegmatis</i> and <i>Corynebacterium glutamicum</i>), Alphaproteobacteria (<i>Agrobacterium tumefaciens</i> and <i>Caulobacter crescentus</i>), and Bacilli (<i>Lactococcus lactis</i> and <i>Staphylococcus aureus</i>). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in <i>C. crescentus</i> to introduce single amino acid mutations with >70% efficiency. Second, we adapt SSAPs for dsDNA editing in <i>C. glutamicum</i> and <i>S. aureus</i>, enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in <i>S. aureus</i> to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.