Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy
The Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (<i>DMD</i>) gene coding for the dyst...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-05-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/11/6160 |
| _version_ | 1797493107660423168 |
|---|---|
| author | Cédric Happi Mbakam Joël Rousseau Guillaume Tremblay Pouiré Yameogo Jacques P. Tremblay |
| author_facet | Cédric Happi Mbakam Joël Rousseau Guillaume Tremblay Pouiré Yameogo Jacques P. Tremblay |
| author_sort | Cédric Happi Mbakam |
| collection | DOAJ |
| description | The Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (<i>DMD</i>) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the <i>DMD</i> gene in HEK293T cells were obtained with the PRIME Editor 2 (PE2) and PE3, respectively. Three repeated treatments increased the percentage of specific mutations with PE2 to 16%. An additional mutation in the protospacer adjacent motif (PAM) sequence improved the PE3 result to 38% after a single treatment. We also carried out the correction of c.428 G>A point mutation in exon 6 of the <i>DMD</i> gene in a patient myoblast. Myoblast electroporation showed up to 8% and 28% modifications, respectively, for one and three repeated treatments using the PE3 system. The myoblast correction led to dystrophin expression in myotubes detected by Western blot. Thus, prime editing can be used for the correction of point mutations in the <i>DMD</i> gene. |
| first_indexed | 2024-03-10T01:15:16Z |
| format | Article |
| id | doaj.art-7e42e300697648e7a4794ffa71301676 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T01:15:16Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-7e42e300697648e7a4794ffa713016762023-11-23T14:10:38ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-05-012311616010.3390/ijms23116160Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular DystrophyCédric Happi Mbakam0Joël Rousseau1Guillaume Tremblay2Pouiré Yameogo3Jacques P. Tremblay4Centre de Recherche du CHUQ-Université Laval, Québec, QC G1V4G2, CanadaCentre de Recherche du CHUQ-Université Laval, Québec, QC G1V4G2, CanadaCentre de Recherche du CHUQ-Université Laval, Québec, QC G1V4G2, CanadaCentre de Recherche du CHUQ-Université Laval, Québec, QC G1V4G2, CanadaCentre de Recherche du CHUQ-Université Laval, Québec, QC G1V4G2, CanadaThe Prime editing technique derived from the CRISPR/Cas9 discovery permits the modification of selected nucleotides in a specific gene. We used it to insert specific point mutations in exons 9, 20, 35, 43, 55 and 61 of the Duchenne Muscular Dystrophy (<i>DMD</i>) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the <i>DMD</i> gene in HEK293T cells were obtained with the PRIME Editor 2 (PE2) and PE3, respectively. Three repeated treatments increased the percentage of specific mutations with PE2 to 16%. An additional mutation in the protospacer adjacent motif (PAM) sequence improved the PE3 result to 38% after a single treatment. We also carried out the correction of c.428 G>A point mutation in exon 6 of the <i>DMD</i> gene in a patient myoblast. Myoblast electroporation showed up to 8% and 28% modifications, respectively, for one and three repeated treatments using the PE3 system. The myoblast correction led to dystrophin expression in myotubes detected by Western blot. Thus, prime editing can be used for the correction of point mutations in the <i>DMD</i> gene.https://www.mdpi.com/1422-0067/23/11/6160prime editingCRISPR/Cas9Duchenne Muscular Dystrophy<i>DMD</i> genemutations |
| spellingShingle | Cédric Happi Mbakam Joël Rousseau Guillaume Tremblay Pouiré Yameogo Jacques P. Tremblay Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy International Journal of Molecular Sciences prime editing CRISPR/Cas9 Duchenne Muscular Dystrophy <i>DMD</i> gene mutations |
| title | Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy |
| title_full | Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy |
| title_fullStr | Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy |
| title_full_unstemmed | Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy |
| title_short | Prime Editing Permits the Introduction of Specific Mutations in the Gene Responsible for Duchenne Muscular Dystrophy |
| title_sort | prime editing permits the introduction of specific mutations in the gene responsible for duchenne muscular dystrophy |
| topic | prime editing CRISPR/Cas9 Duchenne Muscular Dystrophy <i>DMD</i> gene mutations |
| url | https://www.mdpi.com/1422-0067/23/11/6160 |
| work_keys_str_mv | AT cedrichappimbakam primeeditingpermitstheintroductionofspecificmutationsinthegeneresponsibleforduchennemusculardystrophy AT joelrousseau primeeditingpermitstheintroductionofspecificmutationsinthegeneresponsibleforduchennemusculardystrophy AT guillaumetremblay primeeditingpermitstheintroductionofspecificmutationsinthegeneresponsibleforduchennemusculardystrophy AT pouireyameogo primeeditingpermitstheintroductionofspecificmutationsinthegeneresponsibleforduchennemusculardystrophy AT jacquesptremblay primeeditingpermitstheintroductionofspecificmutationsinthegeneresponsibleforduchennemusculardystrophy |