A genetic assay for gene essentiality in Clostridium

Essential genes of pathogens are potential therapeutic targets, but are difficult to verify. Here, gene essentiality was determined by targeted knockout following engineered gene duplication. Null mutants of candidate essential genes of Clostridium difficile were viable only in the presence of a stable second copy of the gene.
Highlights • A rapid assay to demonstrate gene essentiality. • Based on ClosTron targeted knockout following engineered gene duplication. • Gene duplication rapidly achieved at the pyrE locus using Allele-Coupled Exchange. • trpS, metK, and CD0274 shown to be essential in Clostridium difficile 630Δerm. • CD0274 (dhaT) could represent a potential drug target.