Plex 307 vector

cDNA encoding the full-length mouse Nlrp1b gene was cloned from RAW 264.7 macrophages. The full-length gene was shuttled into a pLEX_307 vector (Addgene) with a C-terminal V5 tag using Gateway technology (Thermo Fisher Scientific). Two C-terminal constructs of Nlrp1b starting at S984 and M986 were generated by PCR amplification using specific primers, which contained a 5’ sequence overlap with ubiquitin. The ubiquitin (Ub) sequence was PCR amplified (Addgene 12647) with primers containing a 3’ overlap with corresponding Nlrp1b C-termini. The partially overlapping Ub and Nlrp1b C-termini products were mixed and assembled by PCR to yield the ubiquitin fused constructs. These products were then shuttled into a pLEX_307 vector (Addgene) with a C-terminal V5 tag using Gateway technology (Thermo Fisher Scientific). cDNA encoding SMAC was cloned from The Broad Institute’s ORFeome (Yang et al., 2011 (link)), and similarly amplified to generate the ubiquitin fused constructs. These products were then shuttled into a modified pLEX_307 vector (Addgene) with a C-terminal FLAG tag and a pET-DEST42 vector (Addgene) using Gateway technology (Thermo Fisher Scientific).

sgRNAs were designed using the Broad Institute’s web portal ^{33 (link)} (http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design) and cloned into the lentiGuide-Puro vector (Addgene #52963) as described previously^{34 (link)}. The sgRNA sequences are listed in Supplementary Table 4. cDNA coding for full length human CASP1 (Origene) was cloned with a C-terminal HA tag into pInducer20 vector (Addgene) and with a stop codon into a modified version of the pLEX_307 vector (Addgene) with a hygromycin resistance marker using Gateway technology (Thermo Fisher Scientific). cDNA coding for full length human GSDMD (Dharmacon) was cloned into the pLEX_307 vector (Addgene) with a C-terminal V5 tag. cDNAs coding for full length NLRP1 and CARD8 were purchased from Origene and RFP from Addgene and cloned with stop codons into the pLEX_307 vector. The C-terminal and N-terminal region of CARD8 were cloned with stop codons into pLEX_307 and pDEST27, respectively. The S297A single amino acid point mutation was generated with the QuickChange site-directed mutagenesis kit (Agilent).