ER-Hoxb8–immortalized WT and Gsdmd−/− C57BL/6N mouse–derived iMACs were previously reported (6, 36, 37). For CRISPR gRNA KO stable lines, parental Cas9+ iMAC lines were generated by retroviral transduction with human codon–optimized Streptococcus pyogenes Cas9 [cloned in pMX–green fluorescent protein (GFP) (Cell BioLabs)], which was followed by sorting and subcloning of GFP-positive cells. Cas9+ cells were transduced with gRNAs by lentiviral delivery with the pLKO.1 vector (Sigma), which was followed by the selection of gRNA-expressing cells with puromycin (10 μg/ml) (Gibco) and testing after 14 days. The gRNA sequences are as follows: Luciferase: 5′-GCATGCGAGAATCTCACGC, Gsdmd: 5′-AGAGGCGATCTCATTCCGG, Irf2 gRNA1: 5′-TAAATTCCAATACGATACC, and Irf2 gRNA2: 5′-GGATGCATGCGGCTCGGCA. For reconstitution of Gsdmd, the cDNA encoding mouse GSDMD was subcloned into the piggyBac vector (BH1.11, Genentech). IRF2 gRNA2 KO iMAC cells were co-electroporated with GSDMD/BH1.11 and the transposase vector (pBO, Transposagen Biopharmaceuticals) using the Amaxa shuttle with Solution V (Lonza). Cells were selected with blasticidin (6.25 μg/ml) (Gibco). For CRISPR-mediated, homology-directed repair to generate the IRF2-binding motif knock-in mutants, a protocol from IDT (Integrated DNA Technologies) was followed, in which a Cas9+ iMAC parental line was electroporated with synthetic [annealed crispr RNA (crRNA) + trans-activating crRNA (tracrRNA) from IDT] gRNA (5′-ACGTCGGGCTGAAGCTTTA) and single-stranded DNA (ssDNA) donor template (Ultramer Oligo DNA from IDT) with ~40 bp of homology arms on both sides containing the site mutation introducing a Sal I restriction site in place of the IRF2 consensus sequence and mutation to disrupt the protospacer adjacent motif (PAM) site preventing further CRISPR cuts (5′-CTCCTTGAAAGCACCGCGCCCGCTACGTCGGGCTGAAGCTTTACaGTgTCgacTTTGTGTCCTGCCGCCTGAGTTCCGCTCTTGGTCGTGGCTCCC; bold italics indicate the gRNA recognition sequence; lowercase letters represent mutations; the Sal I site is underlined). After 5 days, cells were plated to identify single-cell clone lines with integrated mutations. In brief, cells were collected in 96-well plates and genomic DNA was harvested with the Quick-DNA 96 Kit (Zymo Research). Genomic DNA was subjected to PCR with the Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) with either WT or mutant primers (WT forward: 5′-GCTGAAGCTTTACGGTTTCACTTTTG: mutant forward: 5′-GGCTGAAGCTTTACAGTGTCGAC, which were paired with the reverse primer: 5′-CGGGGAGGGGTCTATTGTCT) to identify a ~100-bp product corresponding to the WT or mutant sequences. For potential clones, a larger, 250-bp region around the target IRF2 motif was amplified by PCR with the following primers: forward: 5′-GAAAGCGAAGCTCCCGGAT; reverse: 5′-CGGGGAGGGGTCTATTGTCT. The amplified fragment was subjected to Sal I (New England Biolabs) digestion for prescreening and then subcloned into Zero Blunt TOPO (Thermo Fisher Scientific) for Sanger sequencing. From the same gRNA/ssDNA donor–transfected parental pool, three control cell lines and three IRF2 motif mutant lines were established (fig. S6A). For EA.hy926 CRISPR gRNA KO lines, parental Cas9+ EA.hy926 cells were electroporated with synthetic gRNA (annealed crRNA:tracrRNA from IDT) using the Neon Transfection System. The crRNA sequences are as follows: Luciferase: 5′-GCATGCGAGAATCTCACGC, GSDMD: 5′-GGTAGTCCGGAGAGTGGTCC, IRF1_a: 5′-TAATCTGCATCTCTAGCCA, IRF1_b: 5′-CATGCTGCCAAGCATGGCT, IRF2_a: 5′-GTTATCTGCTCCTCCAGCCA, and IRF2_b: 5′-GTCTAGCCGCATGCATCCAG.

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