Application of CRISPR Base Editing in Dysferlinopathies
Recent advances in CRISPR technology has allowed for the precise and efficient edit of a single base in the genome. To address some of the challenges of gene replacement therapy and develop an alternative approach, we aim to perform CRISPR base editing on five dysferlinopathy mutations in patient fibroblast cells. 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 design and validate sgRNA that will target the patient mutation site in the respective cells. This milestone will indirectly produce knockout cells with insertion/deletion mutations. After validation we will use the respective sgRNA with CRISPR-Cas9 technology fused to a nucleic acid base editor to correct five single-base dysferlinopathy mutations in patient fibroblasts. Lastly, the corrected fibroblast will be converted to myoblast using MyoD conversion and levels of dysferlin assessed with western blot.