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

Here we introduce plasmids for xylose-regulated expression and repression of genes in The xylose-inducible expression vector allows for ∼100-fold induction of an 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 Cas9 protein () that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of 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 , we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene , the S-layer protein gene , and the peptidoglycan synthase gene 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. 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 infections. Here we introduce new plasmid vectors that allow for titratable induction (P ) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in 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.