Research ArticleHEALTH AND MEDICINE

A versatile genetic control system in mammalian cells and mice responsive to clinically licensed sodium ferulate

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Science Advances  07 Aug 2020:
Vol. 6, no. 32, eabb9484
DOI: 10.1126/sciadv.abb9484
  • Fig. 1 Design and characterization of the FAROFF switch.

    (A) Schematic design of the FAROFF switch. The binding domain PadR was fused to the transactivation domain VP64 to create an FA-dependent transactivator aPadR (VP64-PadR), which is driven by the simian virus 40 promoter. In the absence of FA (−FA), aPadR binds to a chimeric target promoter PaPadR [(OPadR)6-PhCMVmin] and activates the expression of reporter gene SEAP. In the presence of FA (+FA), aPadR is released from PaPadR, and SEAP expression is switched off. (B to D) Dose-dependent SEAP production profile of the FAROFF switch. SEAP expression of HEK-293 cells cotransfected with pLS125 and pLS74 was assessed after cultivation with different concentrations of FA (B), SF (C), or SF tablets (D). (E) FA-induced SEAP expression in different mammalian cell lines. (F) Time-dependent SEAP expression kinetics of the FAROFF switch. SEAP levels of stable cell line HEKFAR-OFF-SEAP, exposed to 500 μM SF for different time periods (0 to 72 hours), were profiled at specific time points (X axis, 6 to 72 hours). (G) Reversibility of the FAROFF switch. SEAP expression of HEKFAR-OFF-SEAP cells, alternating the SF concentrations (500 μM: OFF; 0 μM: ON) every 2 days, was profiled every 12 hours. Data are means ± SD; n = 3 independent experiments.

  • Fig. 2 Design and characterization of the FARON switch.

    (A) Schematic design of the FARON switch. PadR was fused to a trans-silencing domain KRAB to obtain the FA-dependent transrepressor iPadR (KRAB-PadR) driven by the constitutive simian virus 40 promoter. In the absence of FA (−FA), iPadR binds to a chimeric target promoter PiPadR [OPadR-PSV40-OPadR(RC)] and represses PSV40-driven SEAP expression. In the presence of FA (+FA), iPadR is dissociated from the target promoter PiPadR and derepresses SEAP expression. (B to D) Dose-dependent SEAP expression profile of the FARON switch. SEAP expression of HEK-293 cells cotransfected with pLS163 and pLS64 was assessed after cultivation with different concentrations of FA (B), SF (C) or SF tablets (D). (E) Dose-dependent SEAP expression kinetics of the stable cell line HEKFAR-ON-SEAP. (F) Time-dependent SEAP expression kinetics of the FARON switch. SEAP levels of HEKFAR-ON-SEAP cells, incubated with 500 μM SF for different time periods (0 to 72 hours), were profiled at different time points (X axis, 6 to 72 hours). (G) Reversibility of the FARON switch. SEAP expression of HEKFAR-ON-SEAP cells, alternating the SF concentrations (500 μM: ON; 0 μM: OFF) every 2 days, was profiled every 12 hours. Data are means ± SD; n = 3 independent experiments.

  • Fig. 3 FA-controlled CRISPR-Cas9 devices for genome and epigenome editing.

    (A) Schematic design of FA-controlled PadR-mediated activation (PadRa). In the presence of SF, the transactivator MS2-P65-HSF1 is produced and recruited by constitutively expressed dCas9 and sgRNAMS2 (sgRNA with MS2 loop) to form a transcriptional activation complex (sgRNAMS2-dCas9-MS2-P65-HSF1). (B) Dose-dependent SF-inducible SEAP expression for PadRa. (C) Dose-dependent PadRa-mediated activation of different endogenous genes, including ASCL1, IL1RN, and RHOXF. (D) Schematic design of SF-controlled PadR-mediated inhibition (PadRi). In the presence of SF, the expression of sgRNA is induced to allow assembly of a repressive complex (sgRNA-dCas9-KRAB) targeting sgRNA-specific DNA sites to inhibit gene expression. (E) Dose-dependent SF-repressible SEAP expression by PadRi. (F) PadRi-mediated repression of different endogenous genes, including CD71 and CXCR4. (G) Schematic design of FA-controlled PadR-mediated gene deletion (PadRdel). Cas9 expression is induced by SF and enables Cas9-mediated target gene deletion. (H) FA-induced gene editing of exogenous gene d2EYFP. The data [(B), (C), (E), (F), and (H)] represent the means ± SD; n = 3 independent experiments. (I) PadRdel-mediated genome CCR5 and CXCR4 editing. Red arrowheads indicate the expected cleavage bands. N.D.: not detectable; bp: base pair. See table S1 for detailed description of genetic components and table S6 for detailed transfection mixtures.

  • Fig. 4 SF- and BA-controlled programmable biocomputers in human cells.

    Processing performance of the (A) AND, (B) NAND, (C) NOR, (D) SF NIMPLY BA, and (E) BA NIMPLY SF logic gates in HEK-293 cells. The schematic design of the five logic gates is displayed on the left panels. The processing output performance of five logic gates is shown on the right panels. HEK-293 cells (8 × 104) were cotransfected with (A) pLS25, pMX34, and pDL55; (B) pLS56, pMX43, and pDL58; (C) pLS125, pDL41, and pDL68; (D) pMX43, pLS25, and pDL73; and (E) pLS125, pMX34, and pDL77 and cultivated with different combinations of the two input signals SF (1 mM) and BA (0.75 mM) in accordance with the truth tables. d2EYFP expression in the cells was quantified by fluorescence microscopy and flow cytometric analysis 48 hours after the addition of input signals. Data are means ± SD; n = 3 independent experiments. See table S1 for detailed description of genetic components and table S7 for detailed transfection mixtures for each logic gate.

  • Fig. 5 Performance of the FARON switch in mice.

    (A) Schematic showing the mouse experimental design and procedure for assessing SF-controlled transgene expression in vivo. HEK-293 cells engineered for FARON-inducible SEAP production were microencapsulated into alginate-poly-(l-lysine)-alginate beads allowing free diffusion of oxygen, nutrients, and secreted proteins across the membrane while simultaneously shielding encapsulated cells from the immune system. SEAP production in microencapsulated HEKFAR-ON-SEAP cells implanted in the peritoneum of mice could be controlled either via injection of SF or via oral administration of SF tablets. (B and C) Dose-dependent SF-inducible SEAP expression in mice. Eight-week-old male C57BL/6J mice were intraperitoneally implanted with 2 × 106 microencapsulated HEKFAR-ON-SEAP cells (200 cells per capsule) and received intraperitoneal administration of SF (B) or oral administration of clinically licensed SF tablets (C) three times per day (0 to 1000 mg/kg per day). SEAP levels in the bloodstream of mice were quantified on days 2, 4, 8, and 15 after implantation. Data are expressed as means ± SEM; n = 5 to 6 mice. n.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control.

Supplementary Materials

  • Supplementary Materials

    A versatile genetic control system in mammalian cells and mice responsive to clinically licensed sodium ferulate

    Yidan Wang, Shuyong Liao, Ningzi Guan, Yuanxiao Liu, Kaili Dong, Wilfried Weber, Haifeng Ye

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