title: Efficient genome editing of differentiated renal epithelial cells.
abstract: Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany. alexis.hofherr@uniklinik-freiburg.de.|Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany. alexis.hofherr@uniklinik-freiburg.de.|Faculty of Biology, University of Freiburg, Freiburg, Germany. alexis.hofherr@uniklinik-freiburg.de.
authors.forename: Alexis
authors.initials: A
authors.lastname: Hofherr
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.
authors.forename: Tilman
authors.initials: T
authors.lastname: Busch
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Nora
authors.initials: N
authors.lastname: Huber
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Nold
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Bohn
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.
authors.forename: Amandine
authors.initials: A
authors.lastname: Viau
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Bienaimé
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany.
authors.forename: E Wolfgang
authors.initials: EW
authors.lastname: Kuehn
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany.|BIOSS Centre of Biological Signalling Studies, Albert-Ludwigs-University, Freiburg, Germany.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Arnold
authors.affiliation: Renal Division, Department of Medicine, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany. michael.koettgen@uniklinik-freiburg.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Köttgen
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods; D016678:Genome / Q000235:genetics*; D005838:Genotype; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D007690:Polycystic Kidney Diseases / Q000235:genetics; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D000069896:Transcription Activator-Like Effector Nucleases
keywords: CRISPR; MDCK; PKD1; PKD2; TALEN; mIMCD3