Fig. 2: GBS CRISPR interference (CRISPRi) downregulates expression of a firefly luciferase (ffluc) reporter gene consistent with the previously established collision model of transcriptional blockade.

Multiple sites were targeted with complementary sgRNA protospacer sequences cloned into p3015b and transformed into CNCTC 10/84 and A909 dcas9 strains, followed by standardized luminescence assays performed on the transformed strains. Targets were selected along the sense and antisense strands of DNA and were located in the promoter and along the coding sequence. Luminescence measurements revealed significant repression of Ffluc expression when the sgRNA-complementary target was on the antisense strand, with more repression caused by interference nearer the transcription start site than downstream (A, ****p < 0.001, one-way ANOVA with Bonferroni correction; comparison of each bar is to triplicate luminescence readings in the same strain transformed with the pFfluc plasmid and p3015b bearing a sham protospacer). In a set of point-mutated ffluc target genes in which all four possible PAM sequences were tested with the same sgRNA protospacer targeting nucleotide position 315 (see inset), expression knockdown was observed in all conditions, although significantly less downregulation was observed in both 10/84 and A909 when the target sequence was adjacent to the AGG PAM (B, ***p < 0.005 ****p < 0.001, one-way ANOVA with Bonferroni correction, ns = not significant). All data shown from n = 3 biological replicates with technical triplicates in each case; each data point is the mean of technical triplicates for that experiment.