biostudies-literatureIngle PSwiss National Science FoundationMedical Research CouncilNational Institute for Health Research (NIHR)Biotechnology and Biological Sciences Research Council8123https://www.ebi.ac.uk/biostudies/studies/S-EPMC6544763biostudies-literatureUnknown9(1)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 preScientific reportsGeneration of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system.PMC6544763BB/L01081X/1147603BB/L502030/1BB/M012336/1NIHR-INF-0155BB/K00283X/1BB/L013940/1G0601176Ingle PCockayne AKuehne SAGu YJiang WHuang HGroothuis DRowe PHumphreys CMMinton NPfalseGeneration of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system.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 pre2019-01-01T00:00:00Z2019 May2026-05-07T02:08:50.495Z2019-07-24T07:07:55ZS-EPMC65447633114854810.1038/s41598-019-44458-y