A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile

Abstract

Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (P xyl ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile . Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.