Generation of fluorescent reporter lines for hiPSCs derived midbrain dopaminergic neurons: using a CRISPR/Cas9 based knock-in approach to mark TH and GIRK2 expression in vitro

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a focal loss of class A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). The establishment of patient derived pluripotent stem cells (hiPSCs) and their efficient conversion into dopaminergic neurons fueled the possibilities for disease modelling and its implication in development and screening of novel drugs up to implementation of cell transplantation therapies. hiPSCs can be differentiated into dopaminergic neurons and support the study of neurological diseases in vitro. However, current neuronal differentiation protocols all lack the ability to produce a homogeneous population of midbrain DA neurons. This states an important limitation for the system as a predictive and translational application for in vitro disease model for PD. To overcome this limitation, I created knock-in hiPSCs that carry fluorescent reporter genes at two distinct loci, whose gene expression is characteristic of dedication to DA cell fate, which are TH gene for tyrosine hydroxylase (TH) and KCNJ6 gene for G-protein activated inward rectifier potassium channel 2 (GIRK2). The combined presence of these two proteins is marking the A9 subtype of DA neurons, most susceptible in PD. For CRISPR/Cas9 editing, specific sgRNAs were designed for targeting the two different loci. For both loci, a donor template was designed in order to be delivered at the target site and enable homologous directed repair (HDR) at the stop codon of two loci. A knock-in strategy was designed which implied replacing the stop codon of both loci with an approximately 1500bp fragment containing the sequence for a T2A peptide, a fluorescent protein and an antibiotic resistance cassette with a promoter flanked by loxP sites. The T2A peptide allows “cleavage” of the fluorescent protein from the endogenous gene, to ensure evading fusion protein formation. A specific digital droplet PCR (ddPCR) screening approach allowed the screening of HDR positive clones and subsequent manual selection. This engineering strategy allows the identification of DA neurons in vitro through an endogenously driven reporting system. The outcome of this work is the starting point for manifold applications, not last, because of the ability to recognize DA neurons with flow cytometry and enrich the population.