Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons

oleh: Christa Überbacher, Julia Obergasteiger, Mattia Volta, Serena Venezia, Stefan Müller, Isabella Pesce, Sara Pizzi, Giulia Lamonaca, Anne Picard, Giada Cattelan, Giorgio Malpeli, Michele Zoli, Dayne Beccano-Kelly, Rowan Flynn, Richard Wade-Martins, Peter P. Pramstaller, Andrew A. Hicks, Sally A. Cowley, Corrado Corti

Format: Article
Diterbitkan: Elsevier 2019-12-01

Deskripsi

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase – enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry. Keywords: Human induced pluripotent stem cells, Dopaminergic neurons, CRISPR/Cas9, Knock-in, Digital droplet PCR, Fluorescent reporter, FACS