> Details

Petković, Igor; Bischof, Johannes; Kocher, Thomas; March, Oliver Patrick; Liemberger, Bernadette; Hainzl, Stefan; Strunk, Dirk; Raninger, Anna Maria; Binder, Heide-Marie; Reichelt, Julia; Guttmann-Gruber, Christina; Wally, Verena; Piñón Hofbauer, Josefina; Bauer, Johann Wolfgang; Koller, Ulrich

COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa

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  • Media type: E-Article
  • Title: COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa
  • Contributor: Petković, Igor; Bischof, Johannes; Kocher, Thomas; March, Oliver Patrick; Liemberger, Bernadette; Hainzl, Stefan; Strunk, Dirk; Raninger, Anna Maria; Binder, Heide-Marie; Reichelt, Julia; Guttmann-Gruber, Christina; Wally, Verena; Piñón Hofbauer, Josefina; Bauer, Johann Wolfgang; Koller, Ulrich
  • imprint: Frontiers Media SA, 2022
  • Published in: Frontiers in Medicine
  • Language: Not determined
  • DOI: 10.3389/fmed.2022.976604
  • ISSN: 2296-858X
  • Keywords: General Medicine
  • Origination:
  • Footnote:
  • Description: <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 ∼38% 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>
  • Access State: Open Access