FIG 3.

Suppression of rfp expression by CRISPRi. A set of CRISPRi plasmids was introduced into a C. difficile strain that expresses rfp constitutively. The CRISPRi plasmids express sgRNAs constitutively under P_gdh control and dCas9 alleles under P_xyl control. Cultures grown overnight were diluted into TY Thi₁₀ medium containing xylose, as indicated. When cultures reached an OD₆₀₀ of 0.5 (∼5 h), cells were fixed and processed to allow RFP development. (A) Cultures were inoculated to a starting OD₆₀₀ of 0.05, and the CRISPRi plasmids expressed dCas9 together with rfp^sgRNA or neg^sgRNA (negative control). (B) The dCas9 gene was replaced with a codon-optimized dCas9 gene. (C) The same CRISPRi plasmids as in panel B but with cultures inoculated to a starting OD₆₀₀ of 0.05 or 0.01 (lower inoculum). Data are graphed as the means and standard deviations of data from triplicate cultures and are representative of results from at least two independent experiments. The host strain was UM275. The plasmids were pAI25 (P_gdh::neg^sgRNA P_xyl::dCas9), pAI28 (P_gdh::rfp^sgRNA P_xyl::dCas9), pAI33 (P_gdh::rfp^sgRNA P_xyl::dCas9-opt), and pAI34 (P_gdh::neg^sgRNA P_xyl::dCas9-opt).