A Metabolism-Wide E. coli CRISPRi Library to Screen Antibiotic Susceptibility and Metabolome Responses

Abstract

Understanding gene function and stress response mechanisms in bacteria requires tools that enable precise and scalable genetic perturbations, coupled with comprehensive phenotypic readouts. In this work, an arrayed CRISPR interference (CRISPRi) library targeting the metabolic genes of Escherichia coli was developed and validated to facilitate high-resolution functional genomics. The library was constructed from a pooled resource and quality-controlled to ensure accurate and reproducible gene knockdowns, enabling systematic phenotypic analysis under defined experimental conditions. The arrayed library was used to explore the impact of metabolic gene knockdowns on antibiotic susceptibility. A focused investigation of fosfomycin responses revealed that repression of specific metabolic genes, including atpH and pykF, modulates tolerance phenotypes through transcriptional and metabolic shifts. This work was extended to a broader antibiotic stress screen involving ciprofloxacin, gentamicin, rifampicin, and meropenem. Comparative analysis identified both antibiotic-specific and shared tolerance phenotypes, highlighting non-inherited survival strategies. To support metabolomic applications, a high-throughput sampling workflow was established and optimized for reproducibility, throughput, and compatibility with untargeted mass spectrometry. This protocol was applied in a genome-wide screen using the arrayed CRISPRi library, resulting in the identification of metabolic bottlenecks, buffering mechanisms, and phenotypic consequences of gene repression. Targeted metabolite profiling data integration were used to validate and refine these observations. Taken together, these findings demonstrate a platform that integrates gene perturbation with multi-omics profiling to investigate bacterial physiology. The arrayed CRISPRi library and associated workflows provide tools to dissect genotype–phenotype relationships and open avenues for applications in antibiotic development, metabolic engineering, and systems biology.