Targeted gene editing, especially with CRISPR, has revolutionized science and has many applications in various fields, mainly in cell line development, cell and gene therapy.

Gene editing to obtain a cell line, like the p53 knockout described in this application note, involves several steps. The steps involve stable transfection, edited cell pool generation, single cell printing, and screening for a stable protein-expressing monoclonal cell line.

The CloneSelect® Imager FL (CSI-FL) offers great assistance in upstream process development of cell line development workflows. The stringent regulations of cell line development monoclonality can be easily met using the fluorescence detection capabilities of CSI-FL to confirm a single cell and to provide a day zero assurance of monoclonality.

In this application note, we present a fluorescent method for identifying monoclonal HEK-293 cells after gene editing with CRISPR-based p53 knockout plasmids that have a red fluorescent protein (RFP) marker for puromycin selection. The edited cells were seeded into 96-well plates with a single-cell printer. These were then monitored using CSI-FL to assess monoclonality and monitor growth through RFP and white light channels.

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