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Title: CRISPR/Cas9-mediated allele-specific disruption of a dominant COL6A1 pathogenic variant improves collagen VI network in patient fibroblasts
Author: López-Márquez, Arístides
Morín, Matías
Fernández-Peñalver, Sergio
Badosa, Carmen
Hernández-Delgado, Alejandro
Natera-de Benito, Daniel
Ortez, Carlos
Nascimento, Andrés
Grinberg Vaisman, Daniel Raúl
Balcells Comas, Susana
Roldán Molina, Mònica
Moreno-Pelayo, Miguel Ángel
Jiménez-Mallebrera, Cecilia
Keywords: Distròfia muscular
Mutació (Biologia)
Muscular dystrophy
Mutation (Biology)
Issue Date: 16-Apr-2022
Publisher: MDPI
Abstract: Collagen VI-related disorders are the second most common congenital muscular dystrophies for which no treatments are presently available. They are mostly caused by dominant-negative pathogenic variants in the genes encoding α chains of collagen VI, a heteromeric network forming collagen; for example, the c.877G>A; p.Gly293Arg COL6A1 variant, which alters the proper association of the tetramers to form microfibrils. We tested the potential of CRISPR/Cas9-based genome editing to silence or correct (using a donor template) a mutant allele in the dermal fibroblasts of four individuals bearing the c.877G>A pathogenic variant. Evaluation of gene-edited cells by next-generation sequencing revealed that correction of the mutant allele by homologous-directed repair occurred at a frequency lower than 1%. However, the presence of frameshift variants and others that provoked the silencing of the mutant allele were found in >40% of reads, with no effects on the wild-type allele. This was confirmed by droplet digital PCR with allele-specific probes, which revealed a reduction in the expression of the mutant allele. Finally, immunofluorescence analyses revealed a recovery in the collagen VI extracellular matrix. In summary, we demonstrate that CRISPR/Cas9 gene-edition can specifically reverse the pathogenic effects of a dominant negative variant in COL6A1.
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It is part of: International Journal of Molecular Sciences, 2022, vol. 23, num. 8, p. 4410
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ISSN: 1661-6596
Appears in Collections:Articles publicats en revistes (Genètica, Microbiologia i Estadística)

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