Fig. 1 | Nature Communications

Fig. 1

From: Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria

Fig. 1

Design and function of eukaryote-like CRISPR activation in E. coli. a The circuit design and structure of the CRISPRa device. The circuit includes a sgRNA generator driven by Plux2 (light blue block, inducible by AHL), a dCas9 generator driven by Ptet (blue block, inducible by aTc), an activator generator driven by PrhaB (red block, inducible by rhamnose), and a sfGFP reporter driven by PpspA with wild-type or heterologous UAS. b The activation mechanism of a eukaryote-like long distance regulation in our CRISPRa design, based on the IHF-dependent DNA loop structure. c, d Test of necessity of the three components in CRISPRa. c Combinations of components were achieved via presence (+) or absence (ø) of genetic part in the strain. The mismatch sgRNA had a random sequence as its spacer (sgRNA-LEA3). All strains were cultured with AHL (1.6 µM), rhamnose (0.4 mm) and aTc (2.5 ng mL−1). Statistical difference was determined by a two-tailed Welch’s t-test: (sgRNA cognate/mismatch) p = 0.0002, t = 74.98. d All genetic components for CRISPRa were present in the strain and combinations were achieved via presence (+) or absence (−) of inducers. Inducer concentrations: AHL (1.6 µM), rhamnose (0.4 mm) and aTc (2.5 ng mL−1). Statistical difference was determined by a two-tailed Welch’s t test: (+++/−−−), p = 0.0014, t = 26.82, (+++/−+−), p = 0.0015, t = 25.98. e A PAM inserted PpspA with wild-type UAS (referred as mut) or heterologous UAS (referred as G6) was tested against different spacers for activation in a pspF knocked out strain E. coli MC1061ΔpspF. Concentrations of inducers: aTc (2.5 ng mL−1), AHL (1.6 µM). Error bars, s.d. (n = 3); a.u., arbitrary units; p value summary: ****p value < 0.0001, 0.0001 < ***p value < 0.001, 0.001 < **p value < 0.01, 0.01 < *p value < 0.05, p value ≥ 0.05: n.s. Source data are provided as a Source Data file

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