Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression

Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vectorRetroviral vectors production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR)Nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFRNerve growth factor receptor (NGFR) protein. To eradicate the high background NGFRNerve growth factor receptor (NGFR) expression in future retroviral vectorRetroviral vectors packaging cellsPackaging cells, we here employed the CRISPR/Cas9CRISPR/Cas9 system to generate human suspension 293-F293-f NGFRNerve growth factor receptor (NGFR) knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFRNerve growth factor receptor (NGFR) targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F293-f cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.