Fig. 3: CRISPRi allows targeted knockdown of nonessential GBS genes.

CRISPRi against nonessential genes was demonstrated with the cyl operon, which generates the cytolytic toxin β-hemoylsin/cytolysin. In A909, the cyl operon is downregulated at baseline by CovR binding at a binding site upstream of the transcription start locus. In 10/84, the covR gene is minimally transcribed due to a promoter polymorphism (see ref. ⁵³), which renders it hyperhemolytic due to relatively increased cyl expression (A). CRISPRi targeting of covR and cyl in 10/84 and A909 dcas9 strains led to expected repression or upregulation of genes by qRT-PCR (B, n = 2 biological replicates, indicated by data point shape; the second replicate was performed in technical triplicate, all data points shown). Confirmatory hemolysis assay results and plating on blood agar solid medium matched phenotype expectations from the regulatory model and qRT-PCR (C, ****p < 0.001, one-way ANOVA with Bonferroni correction, n = 3 biological replicates, each performed with technical triplicates; data points indicate technical triplicate means for each separate experiment).