Fig. 4: DAMAGE identifies single nucleotide mutations and specifically triggers pyroptosis in KRAS-mutant cancer cells.
From: A synthetic system for RNA-responsive pyroptosis based on type III-E CRISPR nuclease-protease
a Schematic of the design of KRAS-G12C crRNAs. Red lowercase indicates the mutated base. b, c Characterization of all KRAS-G12C crRNAs in DAMAGE-KRAS. OFF: KRAS-WT; ON: KRAS-G12C. p values for OFF vs. ON comparisons of CR-16, CR-23, and CR-24 were all less than 0.0001. PI staining and FCM analysis (b). LDH release assay, data are means with s.d., n = 6 (c). d FCM analysis of KRAS-G12C crRNA-23. PI+ pyroptotic cells were shown by contour plots in each group. e Immunoblotting assay of all KRAS-G12C crRNAs. ON groups showed significant GSDMs-Csx30 cleavage. f Cell imaging of DAMAGE-KRAS. In HEK293T stable cell lines, crRNA-23 to target KRAS-G12C mRNA. White arrows indicate pyroptotic cells. Scale bar, 25 μm. g LDH release assay of DAMAGE-KRAS in HEK293T stable cell lines. Mock: only treated with transfection reagent. h LDH release assay of DAMAGE-KRAS in HeLa stable cell lines. Mock: only treated with transfection reagent. i PI-positive cell statistics by FCM analysis (left) and LDH release rate (right) of the optimal crRNA for all KRAS-G12 mutant isoforms. j, k Characterization of DAMAGE-KRAS in cancer cell lines harboring primary KRAS mutations. NCI-H23: KRAS-G12C mutant (j). A549: KRAS-G12S mutant (k). All experiments were repeated at least three times, B-FL as the effector (b–k). Data are means ± s.d. (b, g–k: n = 3). p values were calculated by one-way ANOVA with Dunnett’s multiple comparisons test (g, h) or two-way ANOVA with Sidak’s multiple comparisons test (b, c, i) and two-tailed unpaired t-test (j, k). Heatmap data are presented as the mean of three independent biological replicates (b: n = 3). Source data are provided as a Source Data file.
