Fig. 2: Deletion of Mettl3 by CRISPR-Cas9 alters mast cell responses.

a Optimization of gene editing in primary mast cells. Cells were transfected with Cas9 RNPs containing gRNAs against the Kit gene (schematic representation on top). One representative FACS plot showing the efficiency of deletion of the surface receptor c-Kit. Representative of at least N = 8 experiments. b Top, schematic representation of the Mettl3 locus, with indicated the location of the gRNAs and the PCRs used in T7 endonuclease I assays. Bottom: one representative FACS plot showing the deletion of the Mettl3 protein, by intracellular staining. Representative of at least N = 11 experiments. c T7 endonuclease I assay performed on genomic DNA from cells as in (b). The expected patterns of digestion, based on the locations of the sgRNAs and the PCR primers, are shown schematically on a side (see Supplementary Fig. 3b for further details). The effect of one gRNA or two gRNAs is shown. UT: untreated (no T7 enzyme). Representative of N = 4 experiments; M=marker. d Mast cells were transfected as in (c), followed by stimulation with IgE and antigen complexes for 6 h. Release of IL-13 in the culture supernatants was measured by ELISA. N = 3 independent experiments. Mean ± SD. Two-ways ANOVA. e Intracellular cytokine staining was performed on cells as in (c). N = 4-5 independent experiments. Mean ± SEM. Paired t test, two-tailed. f BrdU incorporation assay to measure proliferation 4-6 days post-transfection. N = 6 independent experiments (ratio compared to control samples). Mean ± SEM. Paired t test, two-tailed. One representative FACS plot is shown on the right. g Viability of mast cells transfected with Mettl3 RNPs or controls was measured by LIVE/DEAD staining. N = 5 independent experiments. Mean ± SEM. Paired t test, two-tailed. h Mast cells were injected into the ear pinna of Kit^W-sh/W-sh mice. Two weeks after, anti-DNP IgE antibodies were injected intradermally followed, 24 h later, by intravenous injection of HSA-DNP antigen together with Evan’s blue dye. The dye was then extracted and measured spectrophotometrically. Each dot represents one mouse (N = 12), N = 2 independent experiments. One representative experiment is shown on the left. Mean ± SD. Paired t test, two-tailed. Source data are provided as a Source data File.