Fig. 5: An improved P_Suv/P_M2 AND-NOT logic circuit (v2) rewires p53-deficient tumor cells to produce IFNγ.

a–c In a, the p53-tet H1299 cells (±10 ng/ml DOX) were introduced with the circuit shown on the left (“v2”). The P_Suv-driven CRISPRa is programmed to activate transcription of endogenous IFNG, whereas P_M2-ACRmax forms an inhibitory module (compared to a non-expression construct, P_M2-Con). The non-targeting sgNC was used as a negative control for circuit actuation. The cell lysates were harvested 48 h after transfection and were subjected to IB. The data shown are representative of two independent experiments. b The conditioned media from the cells were also collected and were added to freshly prepared PBMC for 24 h. The cells were subjected to flow cytometry of Class II HLA levels (CD45⁺CD11b⁺-gated). The relative MFI values are marked on the histograms. The dotted lines denote the control levels. In c, p53-tet cells were transfected with the circuit at low confluency (~20%). For target activation, a construct containing a tandem of U6-dependent IFNG-targeting sgRNAs (sgIFN3 + 1) was used. Ninety-six hours after transfection, the cells were subjected to MTT assay. A quantitative summary from three independent experiments is presented (mean ± SEM, two-sided t-test, P values provided). d Co-cultures containing LLC cells (p53-deficient, labeled with mCherry) and their p53⁺ knock-in derivatives in equal proportion were established. The mixed cells were transfected with the P_Suv-CRISPRa/P_M2-ACRmax circuit (v2) targeting EGFP (sgTa: sgTGTa) for 24 h. Cells were subjected to flow cytometry analyses for mCherry and EGFP fluorescence (pseudo-color). The EGFP⁺ subpopulation in either the mCherry⁺ and mCherry^- gates are further resolved in an EGFP fluorescence histogram, respectively in red and blue. The insets in the histograms show the averaged EGFP levels (EGFP⁺%×MFI) in these different gates from two independent experiments. e The parental and p53⁺ LLC cells were respectively transfected with the P_Suv/P_M2 circuit (v2) for conditional mouse Ifng activation by CRISPRa. The mRNA levels for IFNγ and its target gene Irf1 are shown. Cdkn1a levels report p53 status. These qPCR results are presented as mean ± SEM (n = 3 biological replicates). Source data are provided in the Source Data file.