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A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants.

Haijie Zhang·Bo Chen·Lina Gu·Chenlong Wang·Lei Xu·Xinyuan Ji·Jie Wang·Zhiqiang Wang·Xia Xiao·Yuan Liu
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Abstract

INTRODUCTION: The widespread misuse and overuse of antibiotics have driven the emergence of multidrug-resistant and pan drug-resistant bacteria, constituting a formidable global health threat. Antibiotic adjuvants that potentiate the efficacy of existing antibiotics represent a particularly promising avenue to address this challenge. METHODS: We performed a genome-wide CRISPR interference (CRISPRi) screening to identify potential targets for broad-spectrum antibiotic adjuvants, which highlighted the homologous recombination pathway as a promising candidate. To functionally validate this pathway, we employed three strategies to suppress the expression and function of recA, a key component of homologous recombination, including a CRISPRi system delivered via transconjugation, a RecX-derived peptide (RecX-20) fused to a cell-penetrating motif, and a small-molecule inhibitor cisplatin validated by surface plasmon resonance. RESULTS: Disruption of the homologous recombination pathway not only significantly increased bacterial susceptibility to multiple classes of antibiotics, including quinolones, β-lactams, aminoglycosides, and nitrofurantoin, but also reduced horizontal gene transfer of antibiotic resistance. In addition, recA deficiency resulted in a cascade of physiological disruptions, including membrane damage, efflux pump dysfunction, oxidative stress imbalance and metabolic disruption. All three recA-targeting strategies enhanced the antibacterial activity, with cisplatin exhibiting the most pronounced potentiating effect both in vitro and in vivo. CONCLUSIONS: This study reveals that the homologous recombination pathway, particularly RecA, is a viable target for the development of broad-spectrum antibiotic adjuvant. Our findings provide mechanistic insights and practical strategies to restore the effectiveness of existing antibiotics and address the growing threat of antimicrobial resistance.

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Anti-Bacterial AgentsHomologous RecombinationRec A RecombinasesCRISPR-Cas SystemsEscherichia coliMicrobial Sensitivity TestsEscherichia coli ProteinsCisplatinGenome, BacterialAntibioticBroad-spectrum antibiotic adjuvantCRISPR interferenceHomologous recombination pathwayJournal ArticleResearch Support, Non-U.S. Gov't

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Haijie Zhang, Bo Chen, Lina Gu, Chenlong Wang, Lei Xu, Xinyuan Ji, Jie Wang, Zhiqiang Wang, Xia Xiao, Yuan Liu. 2025-12-21. A genome-wide CRISPRi screen identifies homologous recombination pathway as potential target for broad-spectrum antibiotic adjuvants.. https://doi.org/10.1016/j.jare.2025.12.015

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