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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...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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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. |
format | Online Article Text |
id | pubmed-9025481 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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