{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T21:37:19Z","timestamp":1790026639509,"version":"4.0.1"},"reference-count":25,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,8,25]],"date-time":"2022-08-25T00:00:00Z","timestamp":1661385600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002428","name":"Austrian Science Fund","doi-asserted-by":"publisher","award":["P 32769-B"],"award-info":[{"award-number":["P 32769-B"]}],"id":[{"id":"10.13039\/501100002428","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011852","name":"Paracelsus Medizinische Privatuniversit\u00e4t","doi-asserted-by":"publisher","award":["A-20\/02\/041-KOU"],"award-info":[{"award-number":["A-20\/02\/041-KOU"]}],"id":[{"id":"10.13039\/501100011852","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Med."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>Epidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR\/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in <jats:italic>COL17A1<\/jats:italic> in the treatment of junctional epidermolysis bullosa (JEB).<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the <jats:italic>COL17A1<\/jats:italic> gene.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>As analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of \u223c38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>Here we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR\/Cas9-based gene editing approach for JEB.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmed.2022.976604","type":"journal-article","created":{"date-parts":[[2022,8,25]],"date-time":"2022-08-25T13:49:02Z","timestamp":1661435342000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":16,"title":["COL17A1 editing via homology-directed repair in junctional epidermolysis 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