Identification of organelle-specific autophagy regulators from tandem CRISPR screens

Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation autophagy is largely non-selective, promoting indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles. We hypothesized that selective autophagy may be regulated by distinct upstream signaling from starvation induced autophagy to promote organelle turn-over. To address this question, we conducted kinome-wide CRISPR screens using the DsRed-IRES-GFP-p62 reporter line to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. The Brunello kinome library was designed to enhance on-target activity while minimizing off-target effects, ensuring the effectiveness and efficiency of our screens. These parallel screens identified established and novel autophagy shared regulators under these conditions, as well as kinases specifically required for ER-phagy or pexophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as activator or inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling specificity, separating regulation of selective autophagy and bulk autophagy. Following exposure to stress conditions (starvation, ER stress, or peroxisomal stress), DsRed-IRES-GFP-p62 HEK293A reporter line was fixed, sorted into high GFP or low GFP populations by Fluorescence-activated cell sorting (FACS), and analized by NextSeq 500