CRISPR-mediated generation and comprehensive phenotyping of Duchenne Muscular Dystrophy mouse models

Duchenne Muscular Dystrophy (DMD) is a severe, progressive muscle-wasting disorder predominantly affecting boys, with an incidence of 1 in 5,000 births. It is caused by loss-of-function mutations in the X-linked DMD gene, which encodes the dystrophin protein essential for muscle integrity. CRISPR-Cas9 gene-editing technology has enabled precise and permanent modifications to DNA, facilitating the generation of diverse animal models for studying genetic disorders such as DMD. This study aims to establish a protocol for generating mouse models of DMD using CRISPR-Cas9, alongside comprehensive guidelines for their phenotypic and molecular characterisation, to support researchers investigating potential treatments for DMD.

We employed CRISPR-Cas9 microinjection of mouse embryos to produce murine models with exon-51 and exon-52 deletions, mimicking common DMD mutations seen in patients. The DMD models were validated through DNA, RNA, and protein analyses. The protocol also includes functional assessments such as forelimb grip strength testing, histological examination, and serum creatine kinase (CK) testing to evaluate muscle damage and dystrophic pathology.

Generation of murine models with DMD-associated mutations was confirmed through molecular analysis, including Western blot and immunostaining, which revealed the absence of dystrophin. Functional assays demonstrated hallmark DMD characteristics, such as reduced muscle strength and elevated CK levels. Histological analysis of muscle tissue consistently showed dystrophic features, including centrally nucleated myofibers, fibrotic deposition, and variability in myofiber size, all consistent with DMD pathology. These findings confirm the utility of our DMD mouse models in replicating disease traits.

This comprehensive protocol provides a standardised and accessible framework for generating and characterising DMD mouse models, addressing the need for cohesive methodologies. Our approach enables in-depth investigation of DMD pathophysiology and offers a reliable platform for testing therapeutic strategies. By establishing these protocols, we aim to accelerate DMD research and therapeutic development, with applications extending to the study of other genetic disorders.