Torin1

Single and combinatorial autophagy gRNA screens for single or synergistic autophagy inhibition were performed in biological triplicates in the monoclonal RPE1 cell line stably expressing SpCas9 and the autophagic flux probe (GFP-LC3-RFP) (10 (link)). For each replicate, 20 million cells (10 million for each, end time point and day 2 control) were transduced with lentiviral supernatant of the autophagy multiplex library with an MOI of 0.5 and a 1,000- or 20-fold library coverage for single or combinatorial autophagy library screening, respectively. The control time points were harvested 2 days post-transduction. The remaining cells were kept in growing conditions until day 7, at which point the cells were passaged, pooled and reseeded at a density maintaining library diversity. After 13, 14 and 15 days, the cells were treated with the mTOR inhibitor Torin1 (250 nM, InvivoGen) for 24 h to induce autophagy. Cells were collected and 150,000 to 300,000 cells for single, or 4.5–6.75 million cells for combinatorial screening were FACS-sorted to enrich cells with blocked autophagy. The sorted cells were reseeded and expanded for seven days before harvesting, pooled and stored at –20°C until their genomic DNA was extracted and processed for sequencing.

Cells were plated at 3 × 10⁶ cells/ml in a 24‐well plate (500 μl) for western blotting and at 1.33 × 10⁶ cells/ml in a 96‐well plate (150 μl) for ELISA and flow cytometry. Stimuli were used at the following concentrations, if not indicated otherwise: 20 μg/ml 2′‐3′‐cGAMP (synthesized in‐house, detailed synthesis instructions available upon request) for ELISA and WB, and 40 μg/ml 2′‐3′‐cGAMP for flow cytometry; 5:1 cells:beads αCD3/CD28 (Gibco, #11132D), 1 μM ABT737 (Selleck Chemicals, #S1002), 1 μM S63845 (Selleck Chemicals, #S8383), 6,000 IU/ml IFNα2a (Miltenyi Biotec, #130‐093‐874), 20 μM zVAD (R&D Systems, #FMK001), 2 μg/ml PHA (Sigma‐Aldrich, #61764), 50 ng/ml PMA (Enzo Life Sciences, #BML‐PE160‐0005), 1 IU/ml ionomycin (Alomone Labs, #I‐700), 10 ng/ml IFNγ (PeproTech, #300‐02), 10 ng/ml TNFα (PeproTech, #300‐01A), 50 IU/ml IL2 (R&D Systems, #202‐IL‐010), TPCA‐1 (R&D Systems, #2559/10), Dasatinib (Selleck Chemicals, #S1021), Tacrolimus (Astellas PHArma, #06896463), Torin1 (InvivoGen, #inh‐tor1), and 2 μΜ Thapsigargin (Sigma‐Aldrich, #T9033‐.5MG). For HT‐DNA delivery, 200 ng of HT‐DNA (Sigma‐Aldrich, #D6898‐1G) per condition was complexed with 0.5 μl of Lipofectamine 2000 (Thermo Fisher Scientific, #11668019) per condition in Opti‐MeM (Gibco, #10149832) for 5 min at RT.