Figure 3.

Activation of gene expression in E. coli using dCas9 fused to the ω subunit of RNAP. (A) dCas9 is directed to the promoter region and is fused to the ω subunit of RNAP, which recruits the polymerase by interacting with the β′ subunit. A host with a deletion of rpoZ, encoding ω, is used. (B) Either N- or C-terminal fusions of the ω subunit to dCas9 were directed to four regions of the top strand upstream of the −35 element of the lacZ gene. (C) lacZ gene expression levels in the different strains were measured as β-galactosidase activity (Miller units). Activation is reported as the relative Miller units normalized against the units obtained with cells expressing a C-terminal dCas9-ω fusion but no crRNA guide (Ø). The average of three independent experiments is indicated; error bars indicate one standard deviation. Asterisks indicate the P-values associated with each measurement, compared with the no crRNA guide control (Ø). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001.