COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa
Authors
Igor Petković, Johannes Bischof, Thomas Kocher, Oliver Patrick March, Bernadette Liemberger, Stefan Hainzl, Dirk Strunk, Anna Maria Raninger, Heide-Marie Binder, Julia Reichelt, Christina Guttmann-Gruber, Verena Wally, Josefina Piñón Hofbauer, Johann Wolfgang Bauer, Ulrich Koller
Institution
EB House Austria, University Hospital of the Paracelsus Medical University Salzburg
Country
Austria
Year
2022
Journal
Frontiers in Medicine
Abstract
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 COL17A1 in the treatment of junctional epidermolysis bullosa (JEB). 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. 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.