Development of a CRISPR-Cas9-Based Cellular Model for SGCB Gene Mutation: A Platform for Investigating Gene Therapy Strategies in LGMD2E

Background Limb-Girdle Muscular Dystrophy Type 2E (LGMD2E) is one of the most prevalent phenotypes within the limb-girdle muscular dystrophies (LGMDs). This myopathy is caused by pathogenic mutations in the SGCB gene, which encodes β-type sarcoglycan. LGMD2E is recognized as the most prevalent sarcoglycanopathy among the Iranian population specially within the Baloch ethnic group. To develop innovative gene therapy strategies based on accessible gene delivery and gene editing techniques, it is essential to have a cell line harboring genomic mutations in the SGCB gene. These cell models are crucial for the preliminary evaluation of the efficacy of gene-editing-based methods. In this study, we utilized the clustered regularly interspaced short palindromic repeats (CRISPR) system and the approach of inducing indel mutations to generate an HEK-293T cell model harboring a frameshift mutation in SGCB gene. Methods Two distinct SGCB exon 2 targeting single guide RNAs (sgRNAs) were cloned into PX458 plasmids containing spCas9, and recombinant plasmids were transduced into HEK293T cells. The mutagenesis efficiency was evaluated using the TIDE program on Sanger sequencing data of transduced cells. Mutated cell clones were obtained through serial dilution techniques. Results Our results demonstrated the substantial efficacy of the CRISPR-Cas9 system in inducing mutations within the SGCB gene. This capability presents significant potential for precise editing of the SGCB gene in muscular cells, thereby establishing a robust cellular model for LGMD2E gene-editing strategies. Conclusions The application of CRISPR-Cas9 technology in this study highlights its potential for targeted gene editing in LGMD2E research. These findings pave the way for further investigations into the development of precision therapy for LGMD2E, ultimately contributing to the development of effective therapeutic strategies.