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

Abstract

Here we introduce plasmids for xylose-regulated expression and repression of genes in <i>Clostridioides difficile</i> The xylose-inducible expression vector allows for ∼100-fold induction of an <i>mCherryOpt</i> reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the <i>Streptococcus pyogenes</i> Cas9 protein (<i>dCas9</i>) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of <i>dCas9</i> is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in <i>C. difficile</i>, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene <i>ftsZ</i>, the S-layer protein gene <i>slpA</i>, and the peptidoglycan synthase gene <i>pbp-0712</i> CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis.<b>IMPORTANCE</b><i>Clostridioides difficile</i> 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 <i>C. difficile</i> infections. Here we introduce new plasmid vectors that allow for titratable induction (P <i>_xyl</i> ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in <i>C. difficile</i> 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.