A CRISPR-screen in intestinal epithelial cells identifies novel factors for polarity and apical transport

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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.

The following data sets were generated

1. Vogel GF
(2022) NGS raw sgRNA-counts
Dryad Digital Repository, doi:10.5061/dryad.m0cfxpp62.

https://dx.doi.org/10.5061/dryad.m0cfxpp62

Article and author information

Author details

Katharina MC Klee

Institute of Cell Biology, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.
Michael W Hess

Institute of Histology and Embryology, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.
Michael Lohmüller

Institute of Developmental Immunology, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-7712-3143
Sebastian Herzog

Institute of Developmental Immunology, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.
Kristian Pfaller

Institute of Histology and Embryology, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.
Thomas Müller

Department of Paediatrics I, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.
Georg F Vogel

Institute of Cell Biology, Medical University of Innsbruck, Innsbruck, Austria

For correspondence
georg.vogel@i-med.ac.at

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-2515-4490
Lukas A Huber

Institute of Cell Biology, Medical University of Innsbruck, Innsbruck, Austria

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-1116-2120

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.

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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.