Allen A. Cheng, Huiming Ding, and Timothy K. Lu
New therapeutic strategies are needed to treat infections caused by drug-resistant bacteria, which constitute a major growing threat to human health. Here, we use a high-throughput technology to identify combinatorial genetic perturbations that can enhance the killing of drug-resistant bacteria with antibiotic treatment. This strategy, Combinatorial Genetics En Masse (CombiGEM), enables the rapid generation of high-order barcoded combinations of genetic elements for high-throughput multiplexed characterization based on next-generation sequencing. We created ∼34,000 pairwise combinations of Escherichia coli transcription factor (TF) overexpression constructs. Using Illumina sequencing, we identified diverse perturbations in antibiotic-resistance phenotypes against carbapenem-resistant Enterobacteriaceae. Specifically, we found multiple TF combinations that potentiated antibiotic killing by up to 106-fold and delivered these combinations via phagemids to increase the killing of highly drug-resistant E. coli harboring New Delhi metallo-beta-lactamase‑1. Moreover, we constructed libraries of three-wise combinations of transcription factors with >4 million unique members and demonstrated that these could be tracked via next-generation sequencing. We envision that CombiGEM could be extended to other model organisms, disease models, and phenotypes, where it could accelerate massively parallel combinatorial genetics studies for a broad range of biomedical and biotechnology applications, including the treatment of antibiotic-resistant infections.