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 (DMD) gene coding for the dystrophin protein, whi...

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Bibliographic Details
Published in:International journal of molecular sciences 2022-05, Vol.23 (11), p.6160
Main Authors: Happi Mbakam, Cédric, Rousseau, Joël, Tremblay, Guillaume, Yameogo, Pouiré, Tremblay, Jacques P
Format: Article
Language:English
Subjects:
Binding sites
CRISPR
CRISPR-Cas Systems - genetics
CRISPR/Cas9
DMD gene
Duchenne Muscular Dystrophy
Duchenne's muscular dystrophy
Dystrophin
Dystrophin - genetics
Dystrophin - metabolism
Editing
Electroporation
Exons
Gene Editing - methods
Genes
HEK293 Cells
Humans
Muscular dystrophy
Muscular Dystrophy, Duchenne - genetics
Muscular Dystrophy, Duchenne - metabolism
Muscular Dystrophy, Duchenne - therapy
Mutation
mutations
Myoblasts
Myotubes
Nucleotides
Patients
Plasmids
Point mutation
prime editing
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Summary: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 (DMD) gene coding for the dystrophin protein, which is absent in DMD patients. Up to 11% and 21% desired mutations of the DMD 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 DMD 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 DMD gene.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms23116160