Abstract IA08: Pooled CRISPR screens for novel autophagy-related factors identify TMEM41, an ER-resident protein predicted to be a channel

Abstract

Abstract The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in metazoans has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new CRISPR/Cas9-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded and improved toolkit of autophagy reporters for CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known ATG factors as well as previously uncharacterized factors including VPS37A, TMEM251, ALS2, ACP1, and TMEM41B. To validate this dataset, we used quantitative microscopy and biochemical analyses to show that one novel hit, TMEM41B, is required for a late step in phagophore maturation. We found that TMEM41B is an integral ER membrane protein distantly related to the established autophagy factor VMP1, and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms. Citation Format: Vladimir Denic. Pooled CRISPR screens for novel autophagy-related factors identify TMEM41, an ER-resident protein predicted to be a channel [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA08.