Discovering regulators of ER autophagy using CRISPR screening

Autophagy, the process by which cellular contents are degraded via the lysosome, is critical for cellular homeostasis and regulation. ER autophagy (ER-phagy) is the process by which pieces of the endoplasmic reticulum (ER) are degraded by the lysosome. This process was only recently discovered to occur in mammalian cells, and it has been suggested that misregulation manifests in neuropathy conditions. In order to understand this process, we developed genome-editing tools to aid in uncovering regulators of ER autophagy (ER-phagy).We developed and improved multiple genome-editing tools. In chapter 2, I describe how to use CasRNPs (ribonucleotide proteins) to genome edit cell lines. This protocol is ideal for individual desired edits or to genome-edit in arrayed screening fashion. This chapter details how to make single guide RNAs (sgRNAs), how to purify Cas proteins, and how to use those reagents to edit a cell line.In chapter 3, we used various genome-editing tools (including the protocols described in chapter 2) to uncover regulators of ER autophagy. We conducted a genome-wide screen to identify factors that inhibit or enhance ER-phagy when knocked down. Our screen yielded 200 high-confidence hits. We mechanistically followed up on two pathways: mitochondrial oxidative phosphorylation (OXPHOS) and ER-localized DDRGK1 and UFMylation. This work advances our understanding of the regulatory mechanisms of ER-phagy, and my hope is that the CRISPR tools and ER-phagy tools we generated will allow the ER-phagy field to progress quickly