DNA seauencing technologies have vastly outpaced our rate of protein functional characterization, leaving over 100,000 bacterial protein families of unknown function across all metagenomic datasets. This knowledge gap, known as the "genetic dark matter" comprises many uncharacterized genesequired for survival in diverse environments, such as those underlying virulence or maintenance in the microbiome. Functional genomics techniaues promise high-throughput characterization of genes across conditions, but are generally limited to a handful of methods that can only dentify single-mutants. Since genes with overlapping function often compensate for each other, the development of double-mutant phenotyping approaches is necessary to identify redundant systems and map geneunction en masse. Here, we developed dual transposon sequencing (dual Tn.seq), the first unbiased high-throughput double mutant phenotyping platform described in bacteria. Dual Tn-seg combines random barcode (RB)Tn-seq with the Cre-lox system to recombine distant insertion identifiers invivo to track the fate of all randomly generated double-mutants in a pooled population. We designed and validated dual Tn-seg in Streptococcus pneumoniae, identifying hundreds of genetic interactions to direct downstream functional studies. Due to its relative simplicity, dual Tn-seq is amenable to highly diverse bacteria and conditions and will be a powerful tool for unraveling unknown gene function and revealing elusive redundant systems ensuring cell survival.
