Skipping of exons 11 and 12 of the DMD gene for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, recessive X-linked disease caused by pathogenic variants in the DMD gene. Nonsense and frameshift variants typically result in a more severe phenotype, DMD, whereas non-frameshift mutations produce a truncated but partially functional dystrophin, leading to a milder phenotype known as Becker muscular dystrophy. The primary therapeutic strategy for DMD involves frameshift correction to achieve a Becker-like phenotype. This study aimed to disrupt splice sites in exons 11 and 12 of the DMD gene to restore the reading frame using CRISPR-Cas gene editing. Six guide RNAs (gRNAs) for SpCas9 and SaCas9 nucleases were selected. In the initial screening phase conducted in HEK293T cells, three gRNAs targeting exon 11 and one targeting exon 12 were identified as the most effective. Experiments in myoblasts derived from a DMD patient with deletions of exons 12–18 revealed that the efficiency of exon 11 skipping was low, likely due to the introduction of extensive deletions near the canonical splicing site. Conversely, the skipping efficiency of exon 12 in immortalized myoblasts from a healthy donor averaged 9.2% of alleles. Further research is needed to optimize exon 12 skipping in patient-specific cells to confirm successful restoration of dystrophin protein production.

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