Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system
| Autor: | Yang Gu, Daphne Groothuis, Christopher M. Humphreys, Nigel P. Minton, Peter Rowe, Patrick Ingle, He Huang, Weihong Jiang, Alan Cockayne, Sarah A. Kuehne |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Genetic Vectors Mutant Codon Initiator lcsh:Medicine Mutagenesis (molecular biology technique) Biology medicine.disease_cause Polymerase Chain Reaction Article Frameshift mutation 03 medical and health sciences 0302 clinical medicine Plasmid Start codon Escherichia coli medicine CRISPR Frameshift Mutation Promoter Regions Genetic lcsh:Science Gene Gene Editing Genetics Mutation Multidisciplinary Clostridioides difficile lcsh:R Methyltransferases Culture Media Erythromycin Phenotype 030104 developmental biology Mutagenesis lcsh:Q Pathogens CRISPR-Cas Systems Microbial genetics Gene Deletion Genome Bacterial 030217 neurology & neurosurgery Plasmids |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-11 (2019) |
| ISSN: | 2045-2322 |
| DOI: | 10.1038/s41598-019-44458-y |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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