Mpk10025

DND41 PE^+/− cells were generated following a previously described protocol (59 (link)). Briefly, custom primers (Sigma-Aldrich) (Supplementary Table S3) were used to generate sgRNA DNA templates. Evaluation for potential off-targets was performed through CRISPRscan (https://www.crisprscan.org). In vitro transcription of sgRNAs from templates was performed following the protocol guidelines. 500ng of each purified sgRNA was independently incubated with 500ng of Cas9 (1074181, IDT) for 20 minutes at RT. Subsequently, one million DND41 cells were electroporated with 1μg of each complex per transfection using the Neon Transfection System (MPK5000, Thermo Fisher Scientific) and 100μL electroporating tips (MPK10025, Thermo Fisher Scientific). Electroporation conditions: pulse voltage 1350v, pulse width 10ms, pulse number 3, cell density 107/ml. Single cell clones were sorted using the Influx High Speed Sorter (BD Biosciences) and were subsequently grown in tissue culture and screened for PE loss by PCR using REDTaq ReadyMix (R2523–100RXN, Sigma Aldrich) following standard conditions and using custom primers (Supplementary Table S3).

To stimulate CASP11 noncanonical inflammasome activation, LPS was electroporated into the indicated cells using the Neon Transfection System (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, BMDMs were electroporated with LPS (1 μg) in buffer R (MPK10025, Thermo Fisher Scientific) under pulse voltage 1400 V, pulse width 10 ms, and pulse number 2.