Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC)
Objective(s): Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and c...
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| Format: | Article |
| Language: | English |
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Mashhad University of Medical Sciences
2021-08-01
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| Series: | Iranian Journal of Basic Medical Sciences |
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| Online Access: | https://ijbms.mums.ac.ir/article_18498_d988ada858f045a656efe749ff211929.pdf |
| _version_ | 1818381287833993216 |
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| author | Mahintaj Dara vahid razban Mohsen Mazloomrezaei Maryam Ranjbar Marjan Nourigorji Mehdi Dianatpour |
| author_facet | Mahintaj Dara vahid razban Mohsen Mazloomrezaei Maryam Ranjbar Marjan Nourigorji Mehdi Dianatpour |
| author_sort | Mahintaj Dara |
| collection | DOAJ |
| description | Objective(s): Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and cardiac muscles, premature death is inevitable. There is no curative treatment available for DMD. In recent years, advances in genetic engineering tools have made it possible to manipulate gene sequences and accurately modify disease-causing mutations. CRISPR/Cas9 technology is a promising tool for gene editing because of its ability to induce double-strand breaks in the DNA. Materials and Methods: In this study for the exon-skipping approach, we designed a new pair of guide RNAs (gRNA) to induce large deletion of exons 48 to 53 in the DMD gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene.Results: Data showed successful editing of DMD gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited DMD gene, the data of real-time PCR, immune florescent staining demonstrated successful expression of truncated dystrophin in edited cells.Conclusion: This study demonstrated that the removal of exons 48-53 by the CRISPR / Cas9 system did not alter the expression of the DMD gene due to the preservation of the reading frame of the gene. |
| first_indexed | 2024-12-14T02:32:11Z |
| format | Article |
| id | doaj.art-6f9a6cd08d234e868458083a9b2a0ba3 |
| institution | Directory Open Access Journal |
| issn | 2008-3866 2008-3874 |
| language | English |
| last_indexed | 2024-12-14T02:32:11Z |
| publishDate | 2021-08-01 |
| publisher | Mashhad University of Medical Sciences |
| record_format | Article |
| series | Iranian Journal of Basic Medical Sciences |
| spelling | doaj.art-6f9a6cd08d234e868458083a9b2a0ba32022-12-21T23:20:14ZengMashhad University of Medical SciencesIranian Journal of Basic Medical Sciences2008-38662008-38742021-08-012481153115810.22038/ijbms.2021.54711.1226918498Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC)Mahintaj Dara0vahid razban1Mohsen Mazloomrezaei2Maryam Ranjbar3Marjan Nourigorji4Mehdi Dianatpour5Department of Molecular Medicine, School of Advanced Medical Science and Technology, Shiraz University of Medical Science, Shiraz, IranDepartment of Molecular Medicine, School of Advanced Medical Science and Technology, Shiraz University of Medical Science, Shiraz, IranStudent Research Committee, Shiraz University of Medical Science, Shiraz, IranDepartment of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IranDepartment of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IranDepartment of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IranObjective(s): Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the DMD gene, resulting in the absence of dystrophin expression leading to membrane fragility and myofibril necrosis in the muscle cells. Because of progressive weakness in the skeletal and cardiac muscles, premature death is inevitable. There is no curative treatment available for DMD. In recent years, advances in genetic engineering tools have made it possible to manipulate gene sequences and accurately modify disease-causing mutations. CRISPR/Cas9 technology is a promising tool for gene editing because of its ability to induce double-strand breaks in the DNA. Materials and Methods: In this study for the exon-skipping approach, we designed a new pair of guide RNAs (gRNA) to induce large deletion of exons 48 to 53 in the DMD gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene.Results: Data showed successful editing of DMD gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited DMD gene, the data of real-time PCR, immune florescent staining demonstrated successful expression of truncated dystrophin in edited cells.Conclusion: This study demonstrated that the removal of exons 48-53 by the CRISPR / Cas9 system did not alter the expression of the DMD gene due to the preservation of the reading frame of the gene.https://ijbms.mums.ac.ir/article_18498_d988ada858f045a656efe749ff211929.pdfcrispr/cas9dmddystrophingene editinghskmc |
| spellingShingle | Mahintaj Dara vahid razban Mohsen Mazloomrezaei Maryam Ranjbar Marjan Nourigorji Mehdi Dianatpour Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC) Iranian Journal of Basic Medical Sciences crispr/cas9 dmd dystrophin gene editing hskmc |
| title | Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC) |
| title_full | Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC) |
| title_fullStr | Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC) |
| title_full_unstemmed | Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC) |
| title_short | Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC) |
| title_sort | dystrophin gene editing by crispr cas9 system in human skeletal muscle cell line hskmc |
| topic | crispr/cas9 dmd dystrophin gene editing hskmc |
| url | https://ijbms.mums.ac.ir/article_18498_d988ada858f045a656efe749ff211929.pdf |
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