Theophylline-based Control of Repa on a Clostridioides Difficile Plasmid for Use in Allelic Exchange

Historically, mutagenesis in the non-model enteropathogenic bacterium Clostridioides difficile has been challenging. Developing a versatile and reliable method of generating targeted mutations in C. difficile is important to further our understanding of its pathogenesis. Some of the most common targeted mutagenesis systems rely on allelic exchange mediated by either uracil auxotrophy combined with a toxic uracil precursor, a toxin / anti-toxin system, group II introns, or CRISPR/Cas mutagenesis. However, each of these methods suffers from its own issues. Here, we develop and test an allelic exchange strategy which better facilitates screening for integration and selecting for excision than previous systems. This is achieved by controlling plasmid replication with a theophylline-dependent riboswitch cloned upstream of repA, the gene whose product controls plasmid replication. This allows efficient mutant generation, can be performed in a wild-type strain of C. difficile, does not have the off-target effects inherent to group II introns, and alleviates the problem of testing multiple gRNA targets in CRISPR mutagenesis.