A. for the use of our platform in physiological and pathological contexts. Graphical Abstract Open in a separate window Introduction The molecular machinery underlying the processes of protein transport FTY720 (Fingolimod) and secretion has been conserved from yeast to mammalian cells, being essential to maintain specificity and communication between organelles, exocytosis, and endocytosis. Fully one third of proteins navigate the secretory pathway, entering this system via the ER. They are then transported via cellular compartments, including the Golgi apparatus and transport carriers, to be targeted to their final destination (Lee et al., 2004). Recent demonstrations indicate that cell compartments establish cross-regulatory mechanisms through numerous membrane contact sites (Wu et al., 2018), are endowed with tightly regulated dynamics (Valm et al., 2017), and stand at the crossroad of signaling pathways where inputs and outputs are integrated and coordinated (Luini and Parashuraman, 2016). Thus, protein transport and secretion processes are clearly more complex than previously thought. Yeast genetic studies and in vitro biochemical approaches were initially used to discover the basal protein transport machinery (Novick et al., 1980; Braell et al., 1984). More recently, arrayed RNA interference screens at the genome scale or targeting kinases/phosphatases expanded the list of functional and regulatory components of secretory pathways in metazoan cells (Bard et al., 2006; Asensio et al., 2010; Wendler et al., 2010; Chia et FTY720 (Fingolimod) al., 2012; Simpson et al., 2012). In these studies, artificial secreted reporters preceded by a signal sequence such as HRP (ss-HRP) or firefly luciferase were used for detection (Bard et al., 2006; Wendler et al., 2010). Alternatively, secretory pathway organization or transport to the cell surface of fluorescently labeled exogenous transmembrane proteins such as vesicular stomatitis virus G (VSV-G) were analyzed using high throughput imaging or flow cytometry systems DNMT (Asensio et al., 2010; Chia et al., 2012; Simpson et al., 2012). Although these approaches were useful, their implementations in different cell types, with various cargo proteins and environmental conditions, remain difficult. Indeed, a current challenge is to understand how secretory pathways are adapted and regulated in response to intrinsic demands or environmental cues, and eventually altered in diseases. Toward this objective, versatile platforms are needed to uncover factors involved in protein transport in various physiological and pathophysiological contexts. Recent developments of the bacterial CRISPR-associated nuclease Cas9 technology combined with libraries of single-guide RNAs (sgRNAs) have been successfully used to perform FTY720 (Fingolimod) pooled genome-wide screening (Shalem et al., 2015; Kampmann, 2018), where targeted genes can be disrupted (Shalem et al., 2014) and gene manifestation can be inhibited or triggered (Gilbert et al., 2014). Here, we developed an efficient strategy using a pooled genome-wide CRISPR interference (CRISPRi) screen to identify genes involved in protein trafficking and secretion in mammalian cells, and we focus on the contribution of newly recognized factors in these processes. Results Dual fluorescent reporter for protein transport We first founded a HeLa cell collection stably expressing the GFP-tagged TAC protein (interleukin-2 receptor) and the catalytically inactive Cas9 (dCas9) fused to the KRAB repressor protein (HeLa TAC-GFP dCas9-KRAB cells). The TAC protein, which contains a single transmembrane website (TMD) FTY720 (Fingolimod) and localizes in the cell surface, represents a straightforward reporter to investigate protein transport along the ER-Golgi secretory pathway (Stanley and Lacy, 2010). A GFP transmission allows monitoring of the total manifestation of the TAC protein, whereas its cell surface manifestation can be assessed by immunofluorescence microscopy and circulation cytometry analysis using a phycoerythrin (PE)Cconjugated antibody, which recognizes the extracellular website of the TAC protein (Fig. 1, ACC; and Fig. S1). On the other hand, the dCas9-KRAB fusion protein in association to sgRNA focusing on the transcription start site (TSS) of specific genes (CRISPRi system) is a powerful tool to inhibit gene manifestation (Gilbert et al., 2013). We 1st tested our reporter system by incubating cells with brefeldin A.