Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis Academic Article uri icon


  • 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.

altmetric score

  • 7.35

author list (cited authors)

  • McAllister, K. N., Bouillaut, L., Kahn, J. N., Self, W. T., & Sorg, J. A.

citation count

  • 46

complete list of authors

  • McAllister, Kathleen N||Bouillaut, Laurent||Kahn, Jennifer N||Self, William T||Sorg, Joseph A

publication date

  • December 2017