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CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts

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...

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Autores principales: López-Márquez, Arístides, Morín, Matías, Fernández-Peñalver, Sergio, Badosa, Carmen, Hernández-Delgado, Alejandro, Natera-de Benito, Daniel, Ortez, Carlos, Nascimento, Andrés, Grinberg, Daniel, Balcells, Susanna, Roldán, Mónica, Moreno-Pelayo, Miguel Ángel, Jiménez-Mallebrera, Cecilia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9025481/
https://www.ncbi.nlm.nih.gov/pubmed/35457228
http://dx.doi.org/10.3390/ijms23084410
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author López-Márquez, Arístides
Morín, Matías
Fernández-Peñalver, Sergio
Badosa, Carmen
Hernández-Delgado, Alejandro
Natera-de Benito, Daniel
Ortez, Carlos
Nascimento, Andrés
Grinberg, Daniel
Balcells, Susanna
Roldán, Mónica
Moreno-Pelayo, Miguel Ángel
Jiménez-Mallebrera, Cecilia
author_facet López-Márquez, Arístides
Morín, Matías
Fernández-Peñalver, Sergio
Badosa, Carmen
Hernández-Delgado, Alejandro
Natera-de Benito, Daniel
Ortez, Carlos
Nascimento, Andrés
Grinberg, Daniel
Balcells, Susanna
Roldán, Mónica
Moreno-Pelayo, Miguel Ángel
Jiménez-Mallebrera, Cecilia
author_sort López-Márquez, Arístides
collection PubMed
description 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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spelling pubmed-90254812022-04-23 CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts López-Márquez, Arístides Morín, Matías Fernández-Peñalver, Sergio Badosa, Carmen Hernández-Delgado, Alejandro Natera-de Benito, Daniel Ortez, Carlos Nascimento, Andrés Grinberg, Daniel Balcells, Susanna Roldán, Mónica Moreno-Pelayo, Miguel Ángel Jiménez-Mallebrera, Cecilia Int J Mol Sci Article 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. MDPI 2022-04-16 /pmc/articles/PMC9025481/ /pubmed/35457228 http://dx.doi.org/10.3390/ijms23084410 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
López-Márquez, Arístides
Morín, Matías
Fernández-Peñalver, Sergio
Badosa, Carmen
Hernández-Delgado, Alejandro
Natera-de Benito, Daniel
Ortez, Carlos
Nascimento, Andrés
Grinberg, Daniel
Balcells, Susanna
Roldán, Mónica
Moreno-Pelayo, Miguel Ángel
Jiménez-Mallebrera, Cecilia
CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts
title CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts
title_full CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts
title_fullStr CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts
title_full_unstemmed CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts
title_short CRISPR/Cas9-Mediated Allele-Specific Disruption of a Dominant COL6A1 Pathogenic Variant Improves Collagen VI Network in Patient Fibroblasts
title_sort crispr/cas9-mediated allele-specific disruption of a dominant col6a1 pathogenic variant improves collagen vi network in patient fibroblasts
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9025481/
https://www.ncbi.nlm.nih.gov/pubmed/35457228
http://dx.doi.org/10.3390/ijms23084410
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