Allelic exchange technologies for iClostridioides difficile/i research and their application in toxin studies

The ability to precisely manipulate the genome of iClostridioides difficile/i allows for the generation of mutants that can be characterised to unveil critical aspects relating to pathogenesis and virulence. Such findings can ultimately direct the development of antivirulence agents to combat iC. difficile/i infection, by small molecule inhibition. In this review, the mechanisms underpinning the most widely adopted allelic exchange technologies for generating precise genomic mutations in iC. difficile/i, are discussed. The advantages and disadvantages of each system are considered. In addition, a comprehensive review of the application of these tools for the study of iC. difficile/i toxins and genes residing within the toxin loci, is presented.