Using Crispr-Cas9-Mediated Genome Editing To Generate C. Difficile Mutants Defective In Selenoproteins Synthesis

Abstract

Clostridium difficile is a significant concern as a nosocomial pathogen, and genetic tools are important when analyzing the physiology of such organisms so that the underlying physiology/pathogenesis of the organisms can be studied. Here, we used TargeTron to investigate the role of selenoproteins in C. difficile Stickland metabolism and found that a TargeTron insertion into selD, encoding the selenophosphate synthetase that is essential for the specific incorporation of selenium into selenoproteins, results in a significant growth defect and a global loss of selenium incorporation. However, because of potential polar effects of the TargeTron insertion, we developed a CRISPR-Cas9 mutagenesis system for C. difficile. This system rapidly and efficiently introduces site-specific mutations into the C. difficile genome (20-50% mutation frequency). The selD CRISPR deletion mutant had a growth defect in protein-rich medium and mimicked the phenotype of a generated TargeTron selD mutation. Our findings suggest that Stickland metabolism could be a target for future antibiotic therapies and that the CRISPR-Cas9 system can introduce rapid and efficient modifications into the C. difficile genome.

Publication Date

12-1-2017

Publication Title

Scientific Reports

Volume

7

Issue

1

Document Type

Article

Personal Identifier

scopus

DOI Link

https://doi.org/10.1038/s41598-017-15236-5

Socpus ID

85033500780 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/85033500780

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