1 A)

1 A). Open in a separate window Figure 1. CRISPR-mediated genome-wide screen using an autophagic flux reporter. protein 1 (VMP1), which is induced in acute pancreatitis, is an ER-localized transmembrane protein that is required for autophagosome formation (Ropolo et al., 2007). lacks some of these factors including ATG101 (Hosokawa et al., 2009a; Mercer et al., 2009), VMP1 (Ropolo et al., 2007), EPG5 (Wang et al., 2016), and EI24 (Tian et al., 2010). Therefore, it is possible that more autophagy factors not revealed in yeast screens remain to be discovered in metazoans. Until recently, techniques for genetically perturbing mammalian cells were limited, and genome-wide screens targeting autophagy could only be performed using siRNA-mediated gene silencing (Chan et al., 2007; Orvedahl et al., 2011; Hale et al., 2016; Jung et al., 2017). However, the recent emergence of the Mouse Monoclonal to VSV-G tag CRISPR-Cas9 system and the development of high-speed sequencing techniques have enabled knockout (KO)-based genome-wide screening in mammalian cells (Shalem et al., 2014; Wang, 2014). In the autophagy field, a CRISPR-based genome-wide screen identified the UFMylation pathway as a regulator of expression of SQSTM1/p62, a substrate of autophagy (DeJesus et al., 2016). Another group discovered an autophagy-independent lysosomal targeting pathway by a genome-wide screen using NCOA4, an adaptor involved in ferritin autophagy, as an indicator (Goodwin et al., 2017). In this study, we performed an independent genome-wide screen using our novel autophagic flux reporter (Kaizuka et al., 2016) and identified as a novel autophagy-related gene. TMEM41B localizes on the ER and functions together with VMP1, a structurally related autophagy protein. Results and discussion A genome-scale CRISPR screen using an autophagic flux reporter To perform CRISPR-based genome-wide screening, we used a novel autophagic flux reporter recently established by our group (Kaizuka et al., 2016). In this system, GFP-LC3-RFP is cleaved by endogenous ATG4 proteins to yield equimolar amounts of GFP-LC3 and RFP (Fig. 1 A). GFP-LC3 is then conjugated with phosphatidylethanolamine (PE) on isolation membranes and is quenched/degraded when delivered to the lysosome by BAPTA tetrapotassium autophagy. Meanwhile, RFP stays in the cytosol, serving as an internal control. Thus, the reduction in the GFP:RFP fluorescence ratio represents autophagic flux (Fig. 1 A). Open in a separate window Figure 1. CRISPR-mediated genome-wide screen using an autophagic flux reporter. (A) Schematic representation of the autophagic flux reporter GFP-LC3-RFP. GFP-LC3-RFP is cleaved by endogenous ATG4 family proteins to yield equimolar amounts of GFP-LC3 (autophagy substrate) and RFP (internal control). Reduction in the GFP:RFP ratio BAPTA tetrapotassium indicates autophagic activity. (B) HEK293T BAPTA tetrapotassium cells expressing Cas9 and GFP-LC3-RFP were transduced with or without sgRNAs targeting ATG9A and FIP200 and selected with puromycin. The GFP and RFP intensities were determined by flow cytometry under nutrient-rich and starvation conditions. The autophagy-deficient population is indicated by the region of interest (ROI). (C) Schematic representation of the CRISPR-mediated genome-wide screen. An sgRNA library (GeCKO) was introduced to HEK293T cells expressing Cas9 and GFP-LC3-RFP. The cell population that did not respond to starvation (indicated by the ROI) was collected by FACS and expanded. After repeating this enrichment process three times, genomic DNA was extracted and subjected to next-generation sequencing. The proportion (%) of the autophagy-deficient population is indicated by the ROI. (D) Scatterplot of the results of two replicates. Data represent log2 (fold change) of read counts of individual sgRNAs before versus after enrichment. Enriched sgRNAs are shown in the separate panel. Canonical genes and known autophagy-related genes (green), genes encoding HOPS and ESCRT components (blue), negative regulators of mTORC1 (yellow), and high-scoring genes not previously linked to autophagy (magenta) are indicated. We established a HEK293T cell line stably expressing GFP-LC3-RFP and Cas9. When these cells were starved, the GFP signal decreased, while the RFP signal remained constant (Fig. 1 B). This specific reduction of the GFP signal was inhibited following introduction of single-guide RNAs (sgRNAs) targeting and (also known as (scored highly in both replicates, confirming that the screens were effective and almost saturated (Fig. 1 D and Table S1). In addition to canonical and and ((Hs) TMEM41A, HsTMEM41B, HsVMP1,.