Fig. 2: DAMAGE responds to RSV infection and induces pyroptosis.
From: A synthetic system for RNA-responsive pyroptosis based on type III-E CRISPR nuclease-protease
a Schematic of DAMAGE-RSV. b The pyroptotic activity of DAMAGE-RSV. In HEK293T cells, RSV-N as tgRNA. OFF: dCas7-11, Csx29, GSDMs-Csx30, RSV-N CR-Mix and Vector; ON: replace Vector with RSV-N; N+: positive control, replace GSDMs-Csx30 with GSDMs-Csx30-N in the ON groups. The p values between the ON group and the OFF group of each GSDMs-Csx30 are labeled in the figure. c LDH release assay of DAMAGE-RSV in HeLa cells. d: dCas7-11 or dCsx29. d Immunoblotting and immunoprecipitation (IP) of supernatants to validate the pyroptosis-targeting activity of DAMAGE-RSV. Left bands: cell-lysate; right bands: supernatant-IP. e Immunofluorescence analysis of DAMAGE-RSV in HeLa cells. OFF: uninfected; ON: RSV-infected. RSV-N antibody indicates RSV infection; Flag-tag antibody indicates B-SL. Scale bar, 10 μm. f, g Gradient analysis of tgRNA in DAMAGE-RSV. Left: FCM assay, “-” represents PI-negative cells; “+” represents PI-positive cells; right: LDH release assay. RSV-N plasmid gradient (f). RSV multiplicity of infection (MOI) gradient (g). h LDH release assay of DAMAGE-RSV in A549 cells. Left: RSV-N plasmid transfection; right: RSV infection. i Cell imaging of DAMAGE-RSV in A549 cells. White arrows indicate pyroptotic cells. Scale bar, 50 μm. B-SL as effector, RSV-N as tgRNA (c–i). All experiments were repeated at least three times (b–i). Data are means ± s.e.m. (b: n = 3) or means ± s.d. (c, f–h: n = 3). p values were calculated by two-way ANOVA with Tukey’s multiple comparisons test (b), two-way ANOVA with Dunnett’s multiple comparisons test (c) and two-tailed unpaired t-test (h). Source data are provided as a Source Data file.
