Designing a CRISPR-Cas9 pipeline to investigate the effects of putative genetic modifiers on Duchenne Muscular Dystrophy (DMD)

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about 1 in 3,500 live male births (Bushby et al. 2010). DMD is caused by a mutation of the Dystrophin (or DMD) gene that causes loss of dystrophin, a structural protein. Loss of dystrophin increases muscle's susceptibility to damage leading to muscle atrophy and loss of muscle function (Bass et al. 2016). Disease progression is variable in patients with DMD. Genome-wide association studies (GWAS) have identified putative genetic modifiers of DMD that may influence disease severity and variability (Bello et al. 2016; Flangian et al. 2013; Flanigan et al. 2021; Wess et al. 2018). I am using zebrafish, an established model organism to study DMD, to validate and further investigate these putative modifiers. CRISPR-Cas9 mutagenesis is highly efficient in zebrafish and can be used for rapid genetic screens by injecting Cas9 protein and a gene-specific sgRNA, targeting vital protein domains, to knock-out gene function and generate crispant fish. I designed sgRNAs to target zebrafish orthologs of DMD, LTBP4, THBS1, ETAA1, and PARD6G. Microinjection of zebrafish embryos with Cas9 and sgRNA targeting dmd causes loss of dystrophin protein, disorganization of muscle when assayed by birefringence, impaired motility, and decreased life-span of injected fish. To assay the effect of these genetic modifiers on dmd, multiplexed injections of dmd sgRNA + sgRNA targeting putative modifiers were carried out. To validate mutagenesis of putative modifiers hRMA was conducted after injection. Fish that show a dmd phenotype and mutagenesis of the putative modifier can then be used for subsequent analysis of muscle birefringence, motility, and lifespan. My data lays the foundational work to rapidly screen and identify modifiers of DMD for the development of new DMD therapeutics.Undergraduate Research Apprenticeship ProgramNationwide Children's HospitalOSU President's Research Excellence Accelerator AwardAcademic Major: Biolog