Fig. 7.

dCas9-mediated chromosome rewiring of an endogenous gene. a Schematic illustration of the gutRQ-norRVW loci of the engineered E. coli. Recombineering was used to replace the gutQ-norR genes with a module containing NtrC enhancer-binding sites and a kanamycin selection cassette (yellow). Gray boxes represent endogenous genes. b The principle of the rewiring experiment. Bivalent dCas9-mediated loop assistance is expected to bring the NtrC sites closer to the σ⁵⁴-controlled norVW promoter, leading to improved activation. c qRT-PCR showing the relative expression levels of the norV, norW, and rho genes in the wild-type (unshaded) or engineered (shaded) E. coli strains, either in the presence or absence of specific sgRNAs. Improved activation of the norV and norW genes (but not the rho control gene) is seen only when both specific guides are present. The E. coli gyrA gene was used as a reference gene for data normalization. Data are mean ± standard error (n = 3) and are expressed as a ratio relative to the engineered strain in the absence of sgRNAs. Asterisk indicates p < 0.05 evaluated by a paired one-tailed Student’s t test on the log₁₀ transformed data