A CRISPR-screen in intestinal epithelial cells identifies novel factors for polarity and apical transport
- Medical University of Innsbruck, Austria
Abstract
Epithelial polarization and polarized cargo transport are highly coordinated and interdependent processes. In our search for novel regulators of epithelial polarization and protein secretion, we used a genome-wide CRISPR/Cas9 screen and combined it with an assay based on fluorescence-activated cell sorting (FACS) to measure the secretion of the apical brush border hydrolase dipeptidyl peptidase 4 (DPP4). In this way, we performed the first CRISPR screen to date in human polarized epithelial cells. Using high-resolution microscopy, we detected polarization defects and mislocalization of DPP4 to late endosomes/lysosomes after knock-out of TM9SF4, anoctamin 8, and ARHGAP33, confirming the identification of novel factors for epithelial polarization and apical cargo secretion. Thus, we provide a powerful tool suitable for studying polarization and cargo secretion in epithelial cells. In addition, we provide a dataset that serves as a resource for the study of novel mechanisms for epithelial polarization and polarized transport and facilitates the investigation of novel congenital diseases associated with these processes.
Data availability
Next generation sequencing data was made available in Dryadhttps://doi.org/10.5061/dryad.m0cfxpp62.Source Data files have been provided for Figure 3.
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NGS raw sgRNA-countsDryad Digital Repository, doi:10.5061/dryad.m0cfxpp62.
Article and author information
Author details
Funding
Austrian Science Fund (P35805-B)
- Georg F Vogel
Austrian Academy of Science (DOC-Scholarship)
- Katharina MC Klee
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Michel Bagnat, Duke University, United States
Publication history
- Received: May 10, 2022
- Preprint posted: May 17, 2022 (view preprint)
- Accepted: January 19, 2023
- Accepted Manuscript published: January 20, 2023 (version 1)
- Accepted Manuscript updated: January 23, 2023 (version 2)
- Version of Record published: January 31, 2023 (version 3)
Copyright
© 2023, Klee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A CRISPR-screen in intestinal epithelial cells identifies novel factors for polarity and apical transport
eLife 12:e80135.
https://doi.org/10.7554/eLife.80135
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