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Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing
- Source :
- Repisalud, Instituto de Salud Carlos III (ISCIII), Molecular Therapy, e-Archivo. Repositorio Institucional de la Universidad Carlos III de Madrid, instname
- Publication Year :
- 2019
- Publisher :
- Elsevier, 2019.
-
Abstract
- Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.<br />Highly efficient and safe approaches for precise correction of pathogenic mutations are required to realize the full potential of gene-editing protocols for clinical practice. Here we describe a single-step NHEJ-based CRISPR/Cas9 strategy of COL7A1 mutation-bearing exon deletion with unprecedented efficacy for ex vivo correction of recessive dystrophic epidermolysis bullosa.
- Subjects :
- Keratinocytes
Collagen Type VII
Medicina
Population
Biology
Gene mutation
Frameshift mutation
Mice
03 medical and health sciences
Exon
0302 clinical medicine
Genome editing
epidermal stem cells
Drug Discovery
Genetics
medicine
Animals
Humans
CRISPR
epidermolysis bullosa
Frameshift Mutation
education
Molecular Biology
Gene
CRISPR/Cas9
030304 developmental biology
Gene Editing
Pharmacology
0303 health sciences
education.field_of_study
High-Throughput Nucleotide Sequencing
Exons
medicine.disease
gene therapy
Epidermolysis Bullosa Dystrophica
Disease Models, Animal
030220 oncology & carcinogenesis
Molecular Medicine
Original Article
Epidermolysis bullosa
CRISPR-Cas Systems
RNA, Guide, Kinetoplastida
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Repisalud, Instituto de Salud Carlos III (ISCIII), Molecular Therapy, e-Archivo. Repositorio Institucional de la Universidad Carlos III de Madrid, instname
- Accession number :
- edsair.doi.dedup.....2f903e859eaa2881c8d991d6f99881dc