期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 23, 期 8, 页码 -出版社
MDPI
DOI: 10.3390/ijms23084410
关键词
congenital muscular dystrophies; collagen VI-related disorders; dominant negative mutations; allele-specific silencing; CRISPR; Cas9; gene editing
资金
- Fundacion Noelia
- Fundacion SOMOS UNO
- Instituto de Salud Carlos III
- FEDER una manera de hacer Europa [PI19/0122, IMP/00009]
- Spanish Center for Biomedical Network Research on Rare Diseases [CIBERER] [ER19P5AC728/2021, ACCI2018-02]
- Regional Government of Madrid [B2017/BMD3721]
Collagen VI-related disorders are a common type of congenital muscular dystrophy without available treatments. This study demonstrates the potential of CRISPR/Cas9-based genome editing to silence or correct pathogenic variants in the COL6A1 gene, leading to a recovery in collagen VI expression.
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 alpha 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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