Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics
Abstract 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 encouragin...
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Nature Portfolio
2025-01-01
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| Online Access: | https://doi.org/10.1038/s41598-024-81989-5 |
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| author | Angelica Frusteri Chiacchiera Michela Casanova Massimo Bellato Aurora Piazza Roberta Migliavacca Gregory Batt Paolo Magni Lorenzo Pasotti |
| author_facet | Angelica Frusteri Chiacchiera Michela Casanova Massimo Bellato Aurora Piazza Roberta Migliavacca Gregory Batt Paolo Magni Lorenzo Pasotti |
| author_sort | Angelica Frusteri Chiacchiera |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-1c88d28c2cde4edeb8540db2f11fdafa |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
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| spelling | doaj-art-1c88d28c2cde4edeb8540db2f11fdafa2025-01-05T12:17:57ZengNature PortfolioScientific Reports2045-23222025-01-0115111910.1038/s41598-024-81989-5Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibioticsAngelica Frusteri Chiacchiera0Michela Casanova1Massimo Bellato2Aurora Piazza3Roberta Migliavacca4Gregory Batt5Paolo Magni6Lorenzo Pasotti7Department of Electrical, Computer and Biomedical Engineering, University of PaviaDepartment of Electrical, Computer and Biomedical Engineering, University of PaviaDepartment of Molecular Medicine, Department of Information Engineering, University of PaduaDepartment of Clinical-Surgical, Diagnostic and Pediatric Sciences, University of PaviaDepartment of Clinical-Surgical, Diagnostic and Pediatric Sciences, University of PaviaInstitut Pasteur, Inria, Université Paris CitéDepartment of Electrical, Computer and Biomedical Engineering, University of PaviaDepartment of Electrical, Computer and Biomedical Engineering, University of PaviaAbstract 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.https://doi.org/10.1038/s41598-024-81989-5Antimicrobial resistanceAntibiotic re-sensitizationCRISPR arrayEscherichia coli clinical isolatesbla NDM-typebla ctx−M-type |
| spellingShingle | Angelica Frusteri Chiacchiera Michela Casanova Massimo Bellato Aurora Piazza Roberta Migliavacca Gregory Batt Paolo Magni Lorenzo Pasotti Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics Scientific Reports Antimicrobial resistance Antibiotic re-sensitization CRISPR array Escherichia coli clinical isolates bla NDM-type bla ctx−M-type |
| title | Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics |
| title_full | Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics |
| title_fullStr | Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics |
| title_full_unstemmed | Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics |
| title_short | Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics |
| title_sort | harnessing crispr interference to resensitize laboratory strains and clinical isolates to last resort antibiotics |
| topic | Antimicrobial resistance Antibiotic re-sensitization CRISPR array Escherichia coli clinical isolates bla NDM-type bla ctx−M-type |
| url | https://doi.org/10.1038/s41598-024-81989-5 |
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