Figure 2.
RNA-mediated transcriptional repression with the repurposed Type I-E CRISPR-Cas system in E. coli K-12. (A) Targeted silencing of plasmid-based GFP expression. The gfp gene is under the control of the lacZ promoter in the low-copy plasmid pUA66-lacZ. Each spacer sequence (blue line) and PAM (black circle) match the closest strand of the protospacer. RBS, ribosome-binding site. (B) Location-dependent and strand-dependent repression of GFP expression. BW25113 Δcas3::cat harboring the medium-copy pUA66-lacZ and the indicated single-spacer plasmid were subjected to flow cytometry analysis following induction with IPTG and L-arabinose. The non-targeting mviM spacer serves as a negative control. Repression is calculated as the ratio of the autofluorescence-subtracted fluorescence for the inducible no-spacer plasmid (pcrRNA.ind) and each single-spacer plasmid. See Supplementary Figure S3A for representative histograms from the flow cytometry analysis. (C) Reversibility of gene silencing. BW25113 Δcas3::cat cells harboring pUA66-lacZ and either the no-spacer plasmid (pcrRNA.ind, white circles) or the T2 single-spacer plasmid (T2, blue circles) either were pre-induced with only IPTG and switched to both IPTG and L-arabinose (left) or were pre-induced with both IPTG and L-arabinose and switched to only IPTG (right). Following the addition or removal of L-arabinose at t = 0, the autofluorescence-subtracted fluorescence for individual cells and the turbidity of the culture were followed over time. GFP fluorescence was ∼10-fold lower for cells with the targeting plasmid versus the spacer-free plasmid, which we attribute to leaky expression from the ParaB promoter under these growth conditions.