Efficient Fluorescent Labeling of Human Trophoblast Stem Cells via a CRISPR/Cas9-Mediated Knock-In Approach in a Safe Harbor Locus.

Labeling cells with reporter genes allows researchers to visually identify specific cells and observe how they interact with each other in dynamic biological systems. Even though various labeling methods are now available, a specific description of gene knock-in labeling methods for human trophoblast stem cells (hTSCs) has not been reported. Here, we present a streamlined protocol for labeling hTSCs with the green fluorescent protein (GFP) reporter gene via CRISPR/Cas9-mediated knock-in of the gene into the adeno-associated virus site 1 (AAVS1) safe harbor locus. A commonly used hTSC cell line, CT29, was transfected with a dual plasmid system encoding the Cas9 endonuclease and an AAVS1-targeted guide RNA in one plasmid and a donor plasmid encoding a puromycin resistance gene and GFP reporter gene flanked by AAVS1 homology arms. Puromycin-resistant clonal cells were isolated, and AAVS1 integration was confirmed via PCR and sequencing of the PCR products. The labeled cells are proliferative and can give rise to extravillous cytotrophoblast cells (EVT) and the syncytiotrophoblast (ST). To our knowledge, this is the first report using the CRISPR/Cas9 system for AAVS1 integration of a reporter gene in human trophoblast stem cells. It provides an efficient tool to facilitate the study of human trophoblast development and function in co-culture systems and will be highly useful in developing clinical gene therapy-related plasmid constructs. Key features • First report to constitutively express a fluorescent label in hTSCs by applying a CRISPR/Cas9 knock-in approach and an AAVS1 safe harbor locus. • Provides an efficient tool to facilitate the study of human trophoblast development and function, particularly in heterologous co-culture systems. • Offers an approach for developing clinical gene therapy-related plasmid constructs that allow insertion of therapeutic genes without associated disruption of essential genes. • Widely applicable approach to label other human cell lines.