All fluorescence measurements were taken from aliquots of non-selective NM minimal media subcultures grown with IPTG (100 µM) for 20 h at 37 °C. After mixing with a chilled PBS (0.5% FBS) solution, aliquots were kept on ice and analyzed with a SONY SH800 cell cytometer. Mean GFP values were calculated from 30,000 events per culture using compensation settings pre-calibrated with US0 derivatives constitutively expressing either GFP or mCherry alone. Each NM subculture was generated by dilution (1:2000) from independent post-exponential rich media cultures grown for 8–10 h at 37 °C in 2xYT supplemented with glucose (0.5%). Rich media cultures were inoculated from single colonies of US0 co-transformants harboring reporter and ω-dCas9 plasmids. In pilot experiments, 5 mL of each broth was used for culturing and subculturing in round-bottom tubes; otherwise, experiments were carried out with 1 mL of each broth and continuous shaking in 2 mL 96-well V-bottom assay blocks (Corning) that had been autoclaved after pre-seeding wells with 4 mM glass beads (Fisher). For activation or dual-activation/repression experiments, plots were generated by normalizing to the replicate-averaged mean value for Wt ω-dCas9 with a single-target NGG reporter obtained on the same day of the experiment, where applicable. For repression experiments, the single-target NCC reporter with Wt ω-dCas9 was used for normalization. Otherwise, values were plotted without normalization in arbitrary units.

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