Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis. Issue 1 (December 2017)

• Record Type: Journal Article
• Authors: McAllister, Kathleen; Bouillaut, Laurent; Kahn, Jennifer; Self, William; Sorg, Joseph
• Is Part Of: Scientific reports. Volume 7:Issue 1(2017)
• Page Start: 1
• Page End: 12
• Publication Date: 2017-12
• Subjects: Natural history -- Research -- Periodicals; Biology -- Research -- Periodicals; Physical sciences -- Research -- Periodicals; Earth sciences -- Research -- Periodicals; Environmental sciences -- Research -- Periodicals; 502.85
• DOI: 10.1038/s41598-017-15236-5
• Languages: English
• ISSNs: 2045-2322

Abstract

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 inC .difficile Stickland metabolism and found that a TargeTron insertion intoselD, 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 forC .difficile . This system rapidly and efficiently introduces site-specific mutations into theC .difficile genome (20–50% mutation frequency). TheselD CRISPR deletion mutant had a growth defect in protein-rich medium and mimicked the phenotype of a generated TargeTronselD 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 theC .difficile genome.