Dystrophin gene editing by CRISPR/Cas9 system in human skeletal muscle cell line (HSkMC).

OBJECTIVES

Duchene muscular dystrophy (DMD) is a progressive neuromuscular disease caused by mutations in the 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 gene in the human skeletal muscle cell line (HSkMC), in order to correct the frame of the gene.

RESULTS

Data showed successful editing of gene by deletion of exons 48 to 53 and correction of the reading frame in edited cells. Despite a large deletion in the edited 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 gene due to the preservation of the reading frame of the gene.