Fig. 2: Construction of a preliminary dual-input, AND–NOT logic circuit.

a An EGFP reporter whose expression as a fusion protein would be initiated by Cas9/sgRNA-mediated upstream cleavage (frameshift) is illustrated on top. A ribozyme-based, Pol II sgRNA expression system is illustrated right below. 293T cells were transfected with Cas9 and the reporter, as well as the CMV-driven sgRNA for cutting the reporter construct (sgCUT), its Pol III counterpart, or the corresponding control non-targeting sgRNAs (sgCon). Cells were harvested (48 h) and subjected to flow cytometry. Percentages of EGFP⁺ cells(mRuby⁺ gate) were marked on the histograms. b The illustration on the top shows a Pol II promoter-controlled CRISPRa SAM complex. The dCas9 and targeting sgRNA for activation (sgTGTa) are both led by 5× ISRE, while the co-activator MPH is constitutively expressed. In the reporter, 3× target sites proceed a minimal promoter-led EGFP. After plasmid transfections, 293T cells were treated with human IFNα (1000 IU/ml) for 24 h. EGFP levels were determined by fluorescence microscopy (inset, scale: 100 μm), and by immunoblotting (IB, lower left). c, d In c, the illustration shows a circuit with one Pol II promoter (5× ISREs) controlling all three CRISPRa components (activating an EGFP reporter) and another (CMV) driving an off-switching sgRNA (sgOFF, targeting all three CRISPRa components). A non-targeting sgRNA (sgCon-i) serves as a control for the sgOFF. Transfected cells were treated (1000 IU/ml IFN, 48 h) and harvested for IB. In d, some cells were subjected to flow cytometry and followed by quantitation (EGFP⁺%×MFI, mean ± range, n = 2 from independent experiments). e, f The circuit was similar to (c), except that the sgOFF was led by 5× NκRE. The cells were treated ±IFN (1000 IU/ml) and ±TNF (50 ng/ml) for 48 h and harvested either for IB analyses (e), or for flow cytometry (f). In f, the histogram shows the fluorescence pattern for EGFP⁺ cells. The dotted lines mark high levels of EGFP positivity definitively attributed to CRISPRa activity. Relative levels of EGFP⁺%×MFI are marked on the histogram. The IB results in b, c, e are representative of two independent experiments. Source data are provided in the Source Data file.