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.

Abstract

© 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.

• 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
• Electronic ISSN: 2045-2322
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 9
• Article Number: 8123
• DOI: https://doi.org/10.1038/s41598-019-44458-y
• Public URL: https://nottingham-repository.worktribe.com/output/2067863
• Publisher URL: https://www.nature.com/articles/s41598-019-44458-y
• Additional Information: Received: 28 January 2019; Accepted: 14 May 2019; First Online: 31 May 2019; : The authors declare no competing interests.