ArticleScientific reports2019
Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system.
Article in Scientific reports, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed.
- Development and Evaluation of Exosporium-Anchored Bioluminescent and Fluorescent Reporters for TrackingACS synthetic biology · 2026Article
- Botulinum neurotoxins exploit host digestive proteases to boost their oral toxicity via activating OrfXs/P47.Nature structural & molecular biology · 2025Article
- Inducible promoters of bacterial microcompartments improve the CRISPR/Cas9 tools for efficient metabolic engineering ofApplied and environmental microbiology · 2025Article
- Expression of Recombinant Clostridial Neurotoxin byMicroorganisms · 2024Article
- RNA-Based Vaccines and Therapeutics Against Intracellular Pathogens.Methods in molecular biology (Clifton, N.J.) · 2024Review
- Removal of mobile genetic elements from the genome ofFrontiers in microbiology · 2024Article
- A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile.PLoS pathogens · 2023Article
- Article
- Required Gene Set for Autotrophic Growth ofApplied and environmental microbiology · 2022Article
- Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.Scientific reports · 2022Article
- Genetic and metabolic engineering challenges of C1-gas fermenting acetogenic chassis organisms.FEMS microbiology reviews · 2021Review
- Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection.Nature communications · 2021Article
- CRISPR-Cas9-Based Toolkit forFrontiers in microbiology · 2021Article
- Efficient Secretion of Murine IL-2 From an Attenuated Strain ofFrontiers in microbiology · 2021Article
- Towards continuous industrial bioprocessing with solventogenic and acetogenic clostridia: challenges, progress and perspectives.Journal of industrial microbiology & biotechnology · 2020Review
- Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacteriumMetabolic engineering communications · 2020Article
- A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences.Genes · 2020Article
- Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile.Applied and environmental microbiology · 2019Article
- Article
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Authors and funding
10 authors.
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Abstract
Understanding the molecular pathogenesis of Clostridioides difficile has relied on the use of ermB-based mutagens in erythromycin-sensitive strains. However, the repeated subcultures required to isolate sensitive variants can lead to the acquisition of ancillary mutations that affect phenotype, including virulence. CRISPR-Cas9 allows the direct selection of mutants, reducing the number of subcultures and thereby minimising the likelihood of acquiring additional mutations. Accordingly, CRISPR-Cas9 was used to sequentially remove from the C. difficile 630 reference strain (NCTC 13307) two ermB genes and pyrE. The genomes of the strains generated (630Δerm* and 630Δerm*ΔpyrE, respectively) contained no ancillary mutations compared to the NCTC 13307 parental strain, making these strains the preferred option where erythromycin-sensitive 630 strains are required. Intriguingly, the cas9 gene of the plasmid used contained a proximal frameshift mutation. Despite this, the frequency of mutant isolation was high (96% and 89% for ermB and pyrE, respectively) indicating that a functional Cas9 is still being produced. Re-initiation of translation from an internal AUG start codon would produce a foreshortened protein lacking a RuvCI nucleolytic domain, effectively a 'nickase'. The mutation allowed cas9 to be cloned downstream of the strong P
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