COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa.
Front Med (Lausanne)
; 9: 976604, 2022.
Article
em En
| MEDLINE
| ID: mdl-36091706
ABSTRACT
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 â¼38% 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.
Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Front Med (Lausanne)
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
Áustria