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

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene, leading to progressive muscle degeneration and premature death.
• Current treatments for DMD are limited, highlighting the need for innovative therapeutic strategies like gene editing.
• CRISPR/Cas9 technology offers a precise method for DNA modification, showing promise for correcting disease-causing mutations.

Purpose of the Study:

• To investigate the efficacy of CRISPR/Cas9 gene editing for correcting mutations in the DMD gene associated with Duchenne muscular dystrophy.
• To assess the feasibility of an exon-skipping approach targeting exons 48-53 of the DMD gene.
• To evaluate the expression of dystrophin following gene editing in a human skeletal muscle cell line.

Main Methods:

• Designed a CRISPR/Cas9 system with specific guide RNAs to induce a large deletion of exons 48-53 in the DMD gene.
• Utilized a human skeletal muscle cell line (HSkMC) for the exon-skipping gene editing experiments.
• Employed real-time PCR and immune fluorescent staining to confirm gene editing and assess dystrophin expression.

Main Results:

• Successfully achieved deletion of exons 48-53 in the DMD gene, correcting the reading frame in the edited cells.
• Demonstrated successful expression of a truncated dystrophin protein in edited cells, despite the large deletion.
• Confirmed the precision of the CRISPR/Cas9 system in targeting and modifying the specific gene segments.

Conclusions:

• CRISPR/Cas9-mediated deletion of exons 48-53 in the DMD gene is a viable strategy for restoring the reading frame.
• The preservation of the reading frame allows for the expression of functional, albeit truncated, dystrophin.
• This exon-skipping approach holds potential as a therapeutic avenue for Duchenne muscular dystrophy.