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

OBJECTIVE

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in the gene 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. The aim of this study was to employ CRISPR/Cas9 technology in the development of immortalized COL7A1-deficient keratinocyte cell lines intended for application as a cellular model for RDEB in studies.

MATERIALS AND METHODS

In this experimental study, we used transient transfection to express -targeting guide RNA (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 of knockout.

RESULTS

We achieved 46.1% (P<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 single expanded clones, two homozygous and two heterozygous -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 lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed that -deficient cells had higher motility compared to their wild-type counterparts.

CONCLUSION

We reported the first isogenic immortalized -deficient keratinocyte lines that provide a useful cell culture model to investigate aspects of RDEB biology and potential therapeutic options.