A xylose-inducible expression system and a CRISPRi-plasmid for targeted knock-down of gene expression in Clostridioides difficile

Abstract

Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile. The xylose-inducible expression vector allows for ~100-fold induction of an mCherryOpt 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 Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively-expressed single guide RNA (sgRNA). Expression of dCas9 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 C. difficile, we knocked-down expression of three genes involved in biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected phenotypes associated with 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.\n\nImportanceClostridioides 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 (Pxyl) 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.