Efficient genome editing of differentiated renal epithelial cells

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• 作者：Alexis Hofherr ; Tilman Busch ; Nora Huber…
• 关键词：CRISPR ; TALEN ; MDCK ; mIMCD3 ; PKD1 ; PKD2
• 刊名：Pflügers Archiv - European Journal of Physiology
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：469
• 期：2
• 页码：303-311
• 全文大小：4639KB
• 刊物主题：Human Physiology; Molecular Medicine; Neurosciences; Cell Biology; Receptors;
• 出版者：Springer Berlin Heidelberg
• ISSN：1432-2013
• 卷排序：469

文摘

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.