Fig. 4: eCRISPR inhibition of a native QS signal and multiplexed control of QS communication.

a Schematic of luxS eCRISPRi to inhibit AI-2 QS signaling. luxS specific gRNA LuxS1 is expressed under oxyS promoter in pSC-LuxS1. Both pSC-LuxS1 and pdCas9ω were transformed into NB101 allowing expression of gRNA and dCas9ω. b Measured AI-2 activity of the filtered media samples collected at various times post-induction. In-film eCRISPRi cells were electroinduced for 0 (purple) or 30 min (green) 3 h after deposition. Comparisons used two-sided mixed effect analysis. c Schematic of multiplexed eCRISPR for engineering a ‘bilingual’ strain. gRNAs sg108 and LuxS1 were flanked with the DR sequence and placed downstream of the oxyS promoter in pSC-sg108+LuxS1. tracrRNA was individually expressed under a synthetic constitutive promoter. Plasmids pSC-sg108+LuxS1, pdCas9ω, and pMC-lasI-LAA were all transformed into NB101 allowing multiplexed gRNA expression. d Measured AI-1 levels and AI-2 activity (by luminescent reporter cells) of the filtered media samples collected 7 h post-deposition. In-film ‘bilingual’ cells were electro-induced for 0 or 30 min at 0 or 0 and 3 h post-deposition. Comparisons used ordinary one-way ANOVA. Exact P values are provided in Source data. In (b) and (d), data are presented as mean ± s.d. (b: n = 3, d: n = 4). Individual replicates are indicated as open circles.