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  1. Evaluation Summary:

    Here, the authors performed a CRISPR/Cas9 loss of function screen in polarized human epithelial cells to identify novel regulators of epithelial polarization and polarized membrane trafficking. This study provides a powerful resource for future investigations to unravel the complexity and diversity of mechanisms underlying epithelial polarization and polarized cargo transport. Furthermore, this dataset may represent an essential contribution to investigating novel congenital diseases associated with these processes, e.g., microvillus inclusion disease and necrotizing enterocolitis. A few experiments are suggested to bolster the authors' conclusions regarding the roles of key hits.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript, Klee and Hess et al. present the first unbiased, large-scale genetic screen for regulators of apical membrane protein trafficking in human polarized epithelial cells. Designing a sensitive and quantitative assay is critical for the successful completion of a high-throughput genetic screen of this type. To this end, the authors used fluorescence-activated cell sorting of CaCo2 cells stained with a DPP4 (a model apical membrane protein) antibody, selecting for mutant cells with a ~90% reduction in plasma membrane-localized DPP4. The authors' stringent screening criteria led to the identification of 89 genes that function in many cellular processes. For validation, the authors selected 7 target genes for more detailed phenotypic characterization.

    The main conclusion of the paper, that the screen identified novel genes required for apical membrane protein localization, is supported by the presented data. Additionally, the dozens of novel genes required for apical trafficking will be an important resource for epithelial biologists. However, phenotypic characterization of the selected target genes should be extended and better described. In particular, a quantitative analysis should be included to describe the qualitative phenotypes shown in the manuscript.

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  3. Reviewer #2 (Public Review):

    The authors developed a FACS-based assay for detecting endogenous plasma membrane cargo, which is easy to apply and can be adapted to various transmembrane proteins, given that specific antibodies are available. For our purposes, the authors used this assay and the highly efficient CRISPR screening system to study genes involved in plasma membrane targeting of the apical model cargo DPP4. The CRISPR screen in polarized enterocytes identified 89 genes critically involved in apical targeting of their model cargo, DPP4. This relatively moderate number of enriched genes resulted from the high stringency in the screening assay, namely sorting for cells with a drastic reduction of surface DPP4 (90%). Then, they selected seven factors for phenotypic and morphological characterization, focusing mainly on organelles associated with protein transport. Finally, they demonstrated that the KO of all selected candidates causes disturbed epithelial polarization. This effect was demonstrated by 3D cyst assays and EM analyses of filter-grown polarized monolayers. These results highlight yet uncharacterized regulators for epithelial polarization and propose potentially novel mechanisms for this process. It is fascinating the formation of striking, enlarged Lamp1, Lamp3, and Cathepsin-D-positive endolysosomal/lysosomal structures, upon KO of all cell lines that were additionally positive for DPP4 and stx3 in ANO8-, ARHGAP33- and TM9SF4-KOs.

    In this study, the effort to work with several cell lines simultaneously and the presentation of the results in an organized and understandable way are meritorious. Nonetheless, it is a pity that no further analysis of any identified proteins and pathways has been done. In this sense, the functional analyses carried out are superficial and very little informative. Characterizing the endolysosomal pathway as one of the most common phenotypes among the specific KOs of screening suggests a general mechanism associated with this endocytic degradative pathway that could be relevant in the processes of polarization and epithelial morphogenesis. Indeed, something to be explored in the future.

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  4. Reviewer #3 (Public Review):

    The manuscript by Klee and colleagues builds on work from this group and others mapping the molecular membrane trafficking machinery that ensures the delivery of apical-membrane proteins to the apical surface. This mapping has been essential as it has occurred in parallel to, and facilitated, the identification of how mutations in these machineries underpin enteropathies and congenital diarrhoea syndromes, such as Microvillus Inclusion Disease (MVID). This has revealed distinct profiles of dependency for differential apical membrane proteins on trafficking machineries: those that depend on Rab8/11-Myo5b-Stx3 (and others) and those that do not. An example cargo for this latter pathway is DPP4. The current work uses a genome-wide CRISPR screen in an intestinal epithelial cell line to identify novel machineries that regulate this 'Rab8/11-Myo5b-Stx3'-independent trafficking pathway, using DPP4 as a model cargo. The authors identify and characterise a number of new players in such an apical transport pathway.

    Using an approach based on lentiviral transduction of a genome-wide CRISPR screen into intestinal epithelial cells, followed by multi-day differentiation into a monolayer, the authors then dissociate the previously polarised cells into single cell suspensions and use antibody staining and cell sorting of individual cells to enrich for those with a defect in cell surface labelling of DPP4. The aim of this approach is to find machineries that, upon knockout, present a defect in apical delivery of DPP4. The central assumption in this approach is that cell surface levels of DPP4 measured by cell sorting in these post-monolayer single cells will be directly related to, and maintained from, their previous state of apical labelling of DPP4 in polarised monolayers. Evidence for this is provided in Figure 1B wherein sorting for surface DPP4 in single cells derived from polarised monolayers, versus unpolarised cells, is increased in the former compared to the latter.

    The authors identify a number of factors from the genome-wide screen for follow-up validation, focusing on 7 factors that have previously ascribed roles in membrane transport (though not apical membrane-specific transport pathways). The authors characterise that in addition to showing defects in DPP4 transport, a number of these factors do not disrupt the ability to form monolayers with some level of barrier function (as detected by transepithelial-resistance), but rather these present defects in collective cell polarisation related to the formation of an apical lumen. Such factors present defects in endo-lysosomal morphogenesis and defects in the formation and placement of microvillar structures.

    A strengthening of the work could result from examining the molecular mechanisms of how any of these factors regulate membrane transport, as well as independent validation of whether the identified factors are bona fide regulators of apical transport versus potential off-target effects, which could be achieved through rescue experiments or multiple independent knockout approaches to the same target. Despite these avenues for expansion, the work provides a resource for the identification of new potential regulators of membrane transport and provides a methodology for the identification of other transport pathways related to the surface levels of other proteins from polarised cells. It may open avenues for screening of the identified potential machineries for mutations in congenital diarrhoea syndromes or enteropathies, as was successfully applied by the authors and others to machineries of the Rab8/11-Myo5b-Stx3 pathway.

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