Generation of Duchenne muscular dystrophy cell lines harboring mutation in exon 52 and 53 by CRISPR/Cas9-mediated genome editing

Duchenne muscular dystrophy (DMD) is the most serious childhood form of muscular dystrophy. DMD is a lethal X-linked disorder made by mutations in the dystrophin gene. There is currently no cure to this sickness but multiple treatment procedures are under investigation and have shown positive promise for the treatment of DMD. Dystrophin gene-replacement approaches, genetic modification procedures to restore dystrophin expression, and modulation of the dystrophin homologue (utrophin) as a surrogate to restore muscle activity. The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-planned for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an economical, simplistic, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Duchenne muscular dystrophy cell lines with specific mutations can enable the study of the effectiveness of these therapeutic strategies. In order to Generate of Duchenne muscular dystrophy cell lines, we use CRISPR/Cas9-mediated genome editing to create two double strand breaks (DSBs) in exon 52 and 53 in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Existing deletion has been identified by using the GAP-PCR method in the clones and finally the exon 52 and 53 deletions will be identified by using the sequencing. Dystrophin protein expression in the cell line Will be evaluated by reverse transcriptase PCR and western blot techniques.

Keywords:

Duchenne muscular dystrophy , cell lines , genome editing , CRISPR/Cas9