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

Abstract

17Here we introduce plasmids for xylose-regulated expression and repression of genes in 18 Clostridioides difficile. The xylose-inducible expression vector allows for ~100-fold 19 induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell-20 to-cell. The gene repression plasmid is a CRISPR-interference (CRISPRi) system 21 based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes 22 Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively-expressed 23 single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing 24 investigators to control the timing and extent of gene-silencing, as demonstrated here 25 by dose-dependent repression of a chromosomal gene for a red fluorescent protein 26 (maximum repression ~100-fold). To validate the utility of CRISPRi for deciphering gene 27 function in C. difficile, we knocked-down expression of three genes involved in 28 biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA 29 and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected 30 phenotypes associated with loss of FtsZ and SlpA, and revealed that the previously 31 uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell 32 division and protection against lysis. 33 34 Importance 35 Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in 36 developed countries. A better understanding of the basic biology of this devastating 37 pathogen might lead to novel approaches for preventing or treating C. difficile infections.38 3 Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or 39 knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy 40 depletion of target proteins in C. difficile. Besides bypassing the lengthy process of 41 mutant construction, CRISPRi can be used to study the function of essential genes, 42 which are particularly important targets for antibiotic development.43 44 48 diarrhea (1). The symptoms are caused by toxins that damage the intestinal epithelium 49 and can range in severity from mild diarrhea to life threatening conditions such as 50 pseudomembranous colitis and toxic megacolon (2-4). A recent study estimated that C.