Fig. 3: DAMAGE selectively targets and eliminates HPV-infected cervical cancer cells.
From: A synthetic system for RNA-responsive pyroptosis based on type III-E CRISPR nuclease-protease
a Schematic of DAMAGE-HPV. b Screening of crRNAs targeting HPV18-E6/E7 mRNA from the HeLa genome. Mock: CR-NT. The Flag-tag antibody detects D-X; endogenous GSDMD antibody detects D-X-N. c, d Assessment of pyroptotic activity mediated by various GSDMs-Csx30 effectors in DAMAGE-HPV. OFF: dCas7-11, Csx29, GSDMs-Csx30 and CR-NT; ON: replace CR-NT with HPV18 CR-Mix. Quantitative assays of EGFP green fluorescence intensity (c) and ATP-based cell viability assays (d) to evaluate cell viability. The p values for each group under ON and OFF conditions are shown in the figure. e–g Time gradient analysis of DAMAGE-HPV. FCM analysis (e). LDH release assay. ctrl (-): negative control; ctrl (+): positive control, transfected with GSDMD-Csx30-N. p values indicate the statistical differences between each group and the control group (ctrl (-)) at the 48 h time point (f). Immunoblot analysis (g). h Cell imaging of DAMAGE-HPV in HeLa cells. Pyroptotic cells are indicated by white arrows. Scale bar, 100 μm, zoom inset, 25 μm. i Cell imaging of DAMAGE-HPV in HeLa and C33-A cells. Scale bar, 100 μm. All experiments were repeated at least three times, in HeLa cells or C33-A cells, endogenous HPV18-E6/E7 mRNA as tgRNA (b–i), B-FL as the effector (e–i). Data are means ± s.d. (c, d: n = 3) and means ± s.e.m. (f: n = 3). p values were calculated by two-way ANOVA with Sidak’s multiple comparisons test (c, d) and two-way ANOVA with Dunnett’s multiple comparisons test (f). Source data are provided as a Source Data file.
