Bicistronic 2A-peptide-based co-expression reporter knock-in strategy by CRISPR/Cas9 system: application to the labeling of specific cell lineages and gene expression monitoring.

Fluorescent cell labeling is used to identify the specific cell lineages in a tissue or a whole organism. Transgenic organisms with fluorescent reporter genes have been created to visualize specific cell lineages and to investigate cell specific morphologies, motilities, gene expressions, neural activities, intracellular signaling, etc. However, in human cells, transgenes are often silenced during cell differentiation, and so knock-in technology was adopted to label the specific human cell lineages, although the establishment of knock-in human pluripotent stem cells (hPSCs) required considerable efforts. Genome editing technology paved the way to more efficient and useful knock-in methods. Also, we applied a bicistronic 2A-peptide-based co-expression (B2AC) system to the knock-in strategy for the fluorescent cell labeling. By using these technologies, knock-in hPSC lines were established, and the expression of Crx, a specific photoreceptor marker, was revealed during three-dimensional retinal organoid culture. The Crx expression and fluorescent intensity in the cells were positively correlated, suggesting that the B2AC reporter system functioned during human retinal development. The immunohistochemistry of Crx and the maturation of fluorescent reporter cells after long-term differentiation culture indicated that knock-in of the reporter gene did not affect the function of the target Crx gene. B2AC reporter cells successfully represented Crx upregulation by DAPT, a Notch signal inhibitor, during retinal differentiation from hPSC. These results indicated that the B2AC reporter knock-in system could be used to investigate cell transplantation, developmental mechanisms, disease signaling, drug screening, and intracellular signaling.