Abstract
Reporter cell lines based on human pluripotent stem cells (hPSCs) are highly desirable for studying differentiation, lineage tracing, and target cell selection. However, several technical bottlenecks, such as DNA transduction, low homology recombination rate (HDR), and single-cell cloning, have made this effort an arduous process in hPSCs. Here, we provide a step-by-step protocol and practical guide for generating reporter lines in hPSCs via CRISPR/Cas9-mediated HDR. We also elaborate on the process of generating a TBXT-GFP reporter line as an example.