Application of CRISPR Prime Editing in Dysferlinopathies
Objective: Recent advances in CRISPR technology has allowed for the precise and efficient edit of single nucleotide variants and small insertions and deletions. To address some of the challenges of gene replacement therapy and develop an alternative approach, we aim to perform CRISPR prime editing on five dysferlinopathy mutations in patient fibroblast cells harboring three unique homozygous loss of function variants. This alternative approach will produce regulated DYSF expression of the wild-type form. This will be achieved across three progressive milestones/aims. Firstly, we will perform an analysis to identify known DYSF pathogenic variants that can be targeted by CRISPR prime editing. Second, we will design and validate sgRNA that will target the patient mutation site in the respective cells. The corrected fibroblast will be converted to myoblast using MyoD conversion and levels of dysferlin assessed with western blot. Lastly, we will knock in mutations in an appropriate mouse cell line as a preliminary proof of concept that the approach can then be further validated in vivo with the corresponding DYSF mouse model.