Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa.
J Invest Dermatol
; 140(10): 1985-1993.e5, 2020 10.
Article
em En
| MEDLINE
| ID: mdl-32142798
ABSTRACT
End-joiningâbased gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joiningâmediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Epidermólise Bolhosa Distrófica
/
Colágeno Tipo VII
/
Sistemas CRISPR-Cas
/
Edição de Genes
Tipo de estudo:
Prognostic_studies
/
Risk_factors_studies
Limite:
Female
/
Humans
Idioma:
En
Revista:
J Invest Dermatol
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Áustria