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.
Stem Cell Res
CRISPR/Cas9, Dopaminergic neurons, Human induced pluripotent stem cells, Knock-in, Digital droplet PCR, Fluorescent reporter, FACS, CRISPR-Cas Systems, Cell Line, Dopaminergic Neurons, Gene Editing, Gene Knock-In Techniques, Green Fluorescent Proteins, Humans, Induced Pluripotent Stem Cells, Microscopy, Fluorescence, Polymerase Chain Reaction, Transgenes