ArticleMicrobial cell factories2017
A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.
Article in Microbial cell factories, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed.
- Sensitizing carbapenem-resistant Klebsiella pneumoniae using a compact Cascade-Cas3 system.International microbiology : the official journal of the Spanish Society for Microbiology · 2026Article
- Metabolic engineering enablesSynthetic and systems biotechnology · 2026Article
- Anaerobic metabolic evolution for homotypic L-valine fermentation.Nature communications · 2026Article
- Genome minimization of a Burkholderia bacterial host.Metabolic engineering · 2026Article
- Targeted elimination of Staphylococcus aureus mastitis infections with synthetic phage-based CRISPR-Cas delivery systems.NPJ biofilms and microbiomes · 2026Article
- Functional characterization of a type I-F1 CRISPR-cas system from the clinical isolate Shewanella xiamenensis Sh95 reveals constitutive activity and plasmid-curing capability.Scientific reports · 2026Article
- Optimization of Plasmid Curing from Genetically EngineeredACS synthetic biology · 2025Article
- CRISPR-Cas9 Gene Therapy: Non-Viral Delivery and Stimuli-Responsive Nanoformulations.Molecules (Basel, Switzerland) · 2025Review
- A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440.Microbial cell factories · 2024Article
- Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.Microbial biotechnology · 2024Article
- Comparison of Plasmid Curing Efficiency across Five Lactic Acid Bacterial Species.Journal of microbiology and biotechnology · 2024Article
- A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.Plant physiology · 2024Article
- In vivo cloning of PCR product via site-specific recombination in Escherichia coli.Applied microbiology and biotechnology · 2024Article
- Resensitizing tigecycline- and colistin-resistantMicrobiology spectrum · 2024Article
- Article
- Plasmids, a molecular cornerstone of antimicrobial resistance in the One Health era.Nature reviews. Microbiology · 2024Review
- Article
- CRISPR-Cas-Based Antimicrobials: Design, Challenges, and Bacterial Mechanisms of Resistance.ACS infectious diseases · 2023Review
- Inhibition of Plasmid Conjugation inInternational journal of molecular sciences · 2023Article
- Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.PloS one · 2023Article
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundPlasmids are widely used and essential tools in molecular biology. However, plasmids often impose a metabolic burden and are only temporarily useful for genetic engineering, bio-sensing and characterization purposes. While numerous techniques for genetic manipulation exist, a universal tool enabling rapid removal of plasmids from bacterial cells is lacking.
resultsBased on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have constructed a universal plasmid-curing system (pFREE) and developed a one-step protocol and PCR procedure that allow for identification of plasmid-free clones within 24 h. While the context of the targeted replicons affects efficiency, we obtained curing efficiencies between 40 and 100% for the plasmids most widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both Escherichia coli and the promising cell factory chassis Pseudomonas putida.
conclusionAs a fast and freely available plasmid-curing system, targeting virtually all vectors used for cloning and expression purposes, we believe that pFREE has the potential to eliminate the need for individualized vector suicide solutions in molecular biology. We envision the application of pFREE to be especially useful in methodologies involving multiple plasmids, used sequentially or simultaneously, which are becoming increasingly popular for genome editing or combinatorial pathway engineering.
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