COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa

• Published in: Frontiers in medicine Vol. 9; p. 976604
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 25-08-2022
• Subjects: COL17A1; CRISPR/Cas9; gene editing; gene therapy; homology-directed repair (HDR); junctional epidermolysis bullosa (JEB); Medicine
• ISSN: 2296-858X; 2296-858X
• DOI: 10.3389/fmed.2022.976604

Background
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).
Methods
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
COL17A1
gene.
Results
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.
Conclusion
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.