Fig. 3: Construction of a customized AND-NOT logic circuit (v1) targeting a malignant state. | Nature Communications

Fig. 3: Construction of a customized AND-NOT logic circuit (v1) targeting a malignant state.

From: Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors

Fig. 3: Construction of a customized AND-NOT logic circuit (v1) targeting a malignant state.The alternative text for this image may have been generated using AI.

a The left box contains a two-input table based on activities of an onco-TF(I) and a tumor-suppressive TF(II). The tumorous state can be gated by the status of this TFs using an AND–NOT logic (“ON” in green). The right box illustrates the strategy for an AND–NOT logic, tumor-targeting circuit based on the activities of HIF1α and p53. An output is engaged only in cells featuring high HIF1α activity in conjunction with p53 deficiency. b H1299 cells were transfected with a 3× HRE-luciferase reporter and treated with 150 μM CoCl2 for 24 h. Relative levels of luciferase activities were presented (mean ± SD, n = 3 biological replicates). c H1299 cells were introduced with tetracycline-inducible p53 via a lentiviral vector (“p53-tet”). These cells were transfected with a p53-responsive PM2-luciferase construct and treated with 50 ng/ml of DOX for 48 h. The cells were harvested for luciferase assay (mean ± SD, n = 3 biological replicates). d The illustration on the left shows the design for an AND–NOT circuit (“v1.1”). Each CRISPRa component (to activate EGFP transcription) is controlled individually by a 3× HRE promoter, whereas the sgOFF is driven by a p53-responsive promoter (PM2). In the results shown on the right, the effects of PM2-sg#4-i were compared to sg-Con-i in p53-tet H1299 cells. The circuit-introduced cells were treated with ±150 μM CoCl2 and ±DOX (with indicated doses) for 24 h. Cell lysates were examined by IB. The dCas9 levels were represented by their Flag tag (Flag-dC). e All CRISPRa components were assembled into one single 3× HRE-controlled unit, as shown in the illustration on the top. Other designs and experiments were similar to those in (d). According to its stage of development, the circuit is named “v1.2”. In d, e, the blotting results are representative of two independent experiments. In addition, quantitation for the basal, CoCl2, and co-addition (DOX at 50 ng/ml) groups pooled from four independent experiments are marked below the corresponding EGFP panel (E/G, EGFP normalized to GAPDH, mean ± SEM). Source data are provided in the Source Data file.

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