Skip to main content

Research Repository

Advanced Search

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

Ingle, Patrick; Groothuis, Daphne; Rowe, Peter; Huang, He; Cockayne, Alan; Kuehne, Sarah A.; Jiang, Weihong; Gu, Yang; Humphreys, Christopher M.; Minton, Nigel P.


Daphne Groothuis

Peter Rowe

He Huang

Alan Cockayne

Sarah A. Kuehne

Weihong Jiang

Yang Gu


© 2019, The Author(s). 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.


Ingle, P., Groothuis, D., Rowe, P., Huang, H., Cockayne, A., Kuehne, S. A., …Minton, N. P. (2019). Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system. Scientific Reports, 9, Article 8123.

Journal Article Type Article
Acceptance Date May 14, 2019
Online Publication Date May 31, 2019
Publication Date May 31, 2019
Deposit Date May 20, 2019
Publicly Available Date Jun 3, 2019
Journal Scientific Reports
Print ISSN 2045-2322
Electronic ISSN 2045-2322
Publisher Nature Publishing Group
Peer Reviewed Peer Reviewed
Volume 9
Article Number 8123
Public URL
Publisher URL
Additional Information Received: 28 January 2019; Accepted: 14 May 2019; First Online: 31 May 2019; : The authors declare no competing interests.


You might also like

Downloadable Citations