Generation 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 (630 Delta erm* and 630 Delta erm* Delta pyrE, 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-thl promoter. It may find application elsewhere where the use of strong, constitutive promoters is preferred.