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Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange. | LitMetric

Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange.

Anaerobe

Department of Biology, Texas A&M University, College Station, TX, 77843, USA. Electronic address:

Published: August 2024

AI Article Synopsis

  • Clostridioides difficile, a non-model enteropathogenic bacterium, presents challenges for targeted mutagenesis, which is essential for studying its disease-causing mechanisms.
  • Traditional mutagenesis methods, including uracil auxotrophy and CRISPR/Cas, have their own limitations, making mutation generation difficult.
  • The study introduces a new allelic exchange strategy using a theophylline-dependent riboswitch to improve mutation screening and selection, allowing for efficient mutant creation without the drawbacks of other methods.

Article Abstract

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.

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Source
http://dx.doi.org/10.1016/j.anaerobe.2024.102858DOI Listing

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