Fig. 4: Development of a more accurate NOT gate for CRISPRa by employing the anti-CRISPR AcrIIA4. | Nature Communications

Fig. 4: Development of a more accurate NOT gate for CRISPRa by employing the anti-CRISPR AcrIIA4.

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

Fig. 4: Development of a more accurate NOT gate for CRISPRa by employing the anti-CRISPR AcrIIA4.The alternative text for this image may have been generated using AI.

a 293T cells were co-transfected with the AcrIIA4 in bacterial codons [“A”] or its human codon-optimized version [ACRmax, “Amax”] (both under CMV promoter), and the constitutively expressed SAM components. Thirty-six hours after transfection, cell lysates were harvested and subjected to IB for levels of EGFP and Flag-dCas9. b The p53-tet H1299 cells were transfected with a single promoter-driven CRISPRa expression unit targeting an EGFP reporter, and ACRmax led by PM2 (illustration on the left, PSuv: survivin promoter). The effects of DOX (10 ng/ml) treatment were analyzed by IB (middle). Quantitation of EGFP band intensities (normalized to those of GAPDH) is shown on the right (mean ± SEM, n = 3 measurements from independent experiments). c, d In c, the illustration shows the circuit featuring a strong CRISPRa actuator (CMV-dCas9 and U6-sgTGTa for EGFP), and an inhibitory module of PM2-ACRmax. In d, the circuit was introduced into A549 and H1299 cells. A non-targeting sgRNA (sgCon-a) was the control. A representative histogram shows the fluorescence pattern for EGFP+ cells (“Ta”: sgTGTa). The dotted lines mark high levels of EGFP positivity definitively attributed to CRISPRa. The quantitation is shown next to the histogram (EGFP+%×MFI, mean ± SEM, n = 3 biological replicates). e Isogenic clones (six each) of WT or p53-deficient A549 cells were prepared after CRISPR/Cas9-mediated genome editing. The circuit in c was introduced into the cells and their EGFP signals were determined by flow cytometry (left). The dotted lines highlight p53/PM2-ACR-driven inhibition of CRISPRa activities. The quantitation for fluorescence (EGFP+%×MFI) is presented on the right (mean ± SEM, n = 6 independent WT and p53−/− cell lines transfected in parallel). f WT or p53−/− MEFs were prepared. The inset shows a representative genotype analysis. The circuit in c was introduced to the cells (sgCon-a as a control). The cell lysates were subjected to IB. One-sided Student’s t-tests were used for statistical analyses in this figure (P values provided). Blotting results in (a–c) are representative of 2, 3, and 2 independent experiments, respectively. Source data are provided in the Source Data file.

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