CRISPR/Cas9-Mediated Generation of COL7A1-Deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa

Objective: Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blisteringdisease caused by mutations in the COL7A1 gene which is responsible for coding type VII collagen. Investigating thepathological mechanisms and novel candidate therapies for RDEB could be fostered by new cellular models. Theaim of this study was to employ CRISPR/Cas9 technology in the development of immortalized COL7A1-deficientkeratinocyte cell lines intended for application as a cellular model for RDEB in ex vivo studies.Materials and Methods: In this experimental study, we used transient transfection to express COL7A1-targeting guideRNA (gRNA) and Cas9 in HEK001 immortalized keratinocyte cell line followed by enrichment with fluorescent-activatedcell sorting (FACS) via GFP expressing cells (GFP+ HEK001). Homogenous single-cell clones were then isolated,genotyped, and evaluated for type VII collagen expression. We performed a scratch assay to confirm the functionaleffect of COL7A1 knockout.Results: We achieved 46.1% (P lt;0.001) efficiency of in/del induction in the enriched transfected cell population.Except for 4% of single nucleotide insertions, the remaining in/dels were deletions of different sizes. Out of nine singleexpanded clones, two homozygous and two heterozygous COL7A1-deficient cell lines were obtained with definedmutation sequences. No off-target effect was detected in the knockout cell lines. Immunostaining and western blotanalysis showed lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed thatCOL7A1-deficient cells had higher motility compared to their wild-type counterparts.Conclusion: We reported the first isogenic immortalized COL7A1-deficient keratinocyte lines that provide a useful cellculture model to investigate aspects of RDEB biology and potential therapeutic options.