Xeroderma pigmentosum group C (XPC) is a multifunctional DNA damage recognition protein involved in the global genome nucleotide excision repair (GG-NER) pathway. Mammalian cells employ this pathway to eliminate bulky DNA lesions that induce double helix distortion caused by mutagens such as UV radiation or cisplatin. Loss-of-function mutations in the XPC gene lead to various malignancies, including skin cancers. Photosensitivity and the buildup of DNA damage can both define the phenotype of XPC patients’ cells. To date, there is no relevant and reproducible cellular model available that can mimic the course of the human XPC disease. We carried out, for the first time, XPC gene editing in several immortalized skin cells (keratinocytes, fibroblasts, and melanocytes) using RNP (ribonucleoprotein) CRISPR-Cas9 technology where we had a tremendous editing efficacy. Characterization of the XPC gene mutation was validated at the mRNA and protein expression levels via qRT-PCR, western blot, and immunofluorescence and at the DNA level via automated Sanger sequencing. The viability of XP-C cells post UV radiation was further quantified by Presto Blue staining and the repair activity of DNA damage was assessed by Immunocytochemistry (ICC) via single-cell analysis. We showed that skin cells edited for the XPC gene recapitulate the major phenotypes of XPC mutations: photosensitivity and impairment of UV-damage repair. These cells will be used to investigate the etiology of skin cancers linked to XPC disease, unravel new protein biomarkers required for skin cancer genesis, and eventually design an innovative treatment or preventative measures for this disease. Finally, we will develop, for the first time, full XP-C skin organoids using the 3 cell types (keratinocytes, fibroblasts, and melanocytes) harboring XPC gene mutation.

ESDR267 - Generation and Characterization of a CRISPR-Cas9-Mediated XPC gene Knockout in Human Skin Cells

The scientific meeting of the European Society for Dermatological Research (ESDR) is held yearly, usually in September. With an international attendance of more than 1000 skin scientists from around the globe, the four-day event includes short oral presentations from peer-reviewed abstracts, keynote lectures from international experts, sponsored symposia, satellite programs, educational events as well as an important focus on posters, networking and building collaborations amongst scientists. For more information about this year’s meeting visit our website.