Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system

Research output: Contribution to journalArticlepeer-review


  • Patrick Ingle
  • Daphne Groothuis
  • Peter Rowe
  • He Huang
  • Alan Cockayne
  • Weihong Jiang
  • Yang Gu
  • Christopher M. Humphreys
  • Nigel P. Minton

Colleges, School and Institutes

External organisations

  • University of Nottingham


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 Pthl promoter. It may find application elsewhere where the use of strong, constitutive promoters is preferred.


Original languageEnglish
Article number8123
Number of pages11
JournalScientific Reports
Publication statusPublished - 31 May 2019