Figure 1 –

Regulation of AAC(6′)-Ib copy number by CRISPRi. A. Schematic representation of the pJHCMW1 plasmid and a zoomed view on the gene of interest, the aminoglycoside acetyltransferase AAC(6′)-Ib tagged with mNeonGreen (mNG) under its native promoter. Boxes above the gene mark the regions targeted by the dCas9, which include the promoter (Pro.), coding sequence (CDS), and linker. B. Schematic representation of the dCas9-coding gene under the P_Llac promoter and the guide RNA (gRNA) with its constitutive promoter. C. Illustration of the workflow for AAC(6’)-Ib protein copy number quantification. The copy number per cell is estimated based on the optical density (OD₆₀₀), bacterial count (colony forming units (CFU)/OD₆₀₀/mL), and intensity measured with a spectrofluorometer from an overnight LB culture of the strain that carries the pJHCMW1 derivative plasmid and expresses the dCas9 protein. D. Average AAC(6′)-Ib copy number per cell obtained with the spectrofluorometer for strains harboring gRNAs with different complementarity to either the coding (cod.) or the non-coding strand (non-cod.), varying lengths and targeting the promoter sequence, the CDS (E.) or the linker (F.) of the gene. The native expression level represents AAC(6’)-Ib copy number per cell in the absence of dCas9 repression. Error bars represent the standard deviation.