Journal Article

Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics

Casanova, Michela, Pasotti, Lorenzo, Magni, Paolo, Batt, Gregory, Frusteri Chiacchiera, Angelica, Piazza, Aurora, Bellato, Massimo, Migliavacca, Roberta

2025

Overview

The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli . Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/326/22/1434 / 631/553/2691 / 631/553/552 / Anti-Bacterial Agents - pharmacology / Anti-Bacterial Agents - therapeutic use / Antibiotic re-sensitization / Antibiotic resistance / Antibiotics / Antimicrobial agents / Antimicrobial resistance / bla ctx−M-type / bla NDM-type / Cefotaxime / Clinical isolates / Colistin / CRISPR / CRISPR array / CRISPR-Cas Systems / DNA damage / Drug delivery / Drug resistance / Drug Resistance, Bacterial - genetics / E coli / Escherichia coli - drug effects / Escherichia coli - genetics / Escherichia coli clinical isolates / Gene targeting / Growth conditions / Humanities and Social Sciences / Humans / Life Sciences / Meropenem / Microbial Sensitivity Tests / multidisciplinary / Mutation / Nucleotide sequence / Science / Science (multidisciplinary)