CRISPR/Cas9-mediated Generation ofCOL7A1-deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa

Abstract

AbstractObjectiveRecessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in theCOL7A1gene which is responsible for coding type VII collagen. Investigating the pathological mechanisms and novel candidate therapies for RDEB could be fostered by new cellular models. Here, we developed multiple immortalizedCOL7A1-deficient keratinocyte cell lines using CRISPR/Cas9 technology as RDEB cellular model.Materials and MethodsIn this experimental study, we used transient transfection to expressCOL7A1-targeting gRNA and Cas9 in HEK001 immortalized keratinocyte cell line followed by enrichment with fluorescent-activated cell 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 functional effect ofCOL7A1knockout.ResultsWe achieved 46.1% (p < 0.001) efficiency of indel induction in the enriched transfected cell population. Except for 4% of single nucleotide insertions, the remaining indels were deletions of different sizes. Out of nine single clones expanded, two homozygous and two heterozygousCOL7A1-deficient cell lines were obtained with defined mutation sequences. No off-target effect was detected in the knockout cell lines. Immunostaining and western blot analysis showed the lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed thatCOL7A1-deficient cells had higher motility compared with their wild-type counterparts.ConclusionWe reported the first isogenic immortalizedCOL7A1-deficient keratinocyte lines that provide a useful cell culture model to investigate aspects of RDEB biology and potential therapeutic options.