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.

人类诱导多能干细胞（hiPSC）已成为疾病建模不可或缺的一部分。它们是获取含有致病突变的患者细胞的重要资源。从 PD 患者的 hiPSC 中衍生中脑多巴胺能 (DAergic) 神经元是模拟人类患者无法获得的细胞类型生理过程的唯一选择。然而，分化不会产生同质的 DA 神经元群体，污染细胞类型可能会干扰体外系统的读数。在这里，我们使用 CRISPR/Cas9 为 DA 神经元生成新型敲入报告基因系，并采用内源性荧光酪氨酸羟化酶 - 增强型绿色荧光蛋白 (TH-eGFP) 报告基因进行改造。我们提出了一种可重复的敲入策略，结合使用数字液滴 PCR (ddPCR) 的高度特异性同源定向修复 (HDR) 筛选方法。我们创建的敲入细胞系显示了 DA 神经元的功能荧光报告系统，可通过流式细胞术识别。