The project spanning a two-year grant period aimed to explore the applicability of two novel CRISPR methods, base editing and prime editing using DYSF patient fibroblast cell lines harboring various mutation classes that can be potentially targeted for editing. We were able to create immortalized MyoD converted patient fibroblast. In addition, we were able to create a dysferlin knock out on a control human myoblast cell line (MB135). For these cell lines, we were able to validate the reduction of dysferlin protein expression in differentiated myotubes to varying levels depending on mutation. These cells are an available resource to use for the research community.
CRISPR base editing is limited to correcting base transitions and have a small window of efficient editing relative to the downstream PAM, thus the patient cell lines selected for this project were based on these restrictions and resulted in the selection of DYSF mutations that were compound heterozygous. We were able to design sgRNA and target the genomic location of each of the patient mutations using HEK293 cells. The CRISPR base editing achieved in patient cells were too low to be detected by Sanger sequencing in a population of edited cells, thus we employed next generation sequencing and observed low levels of editing in HEK293, control and patient fibroblast. The ability to observe low levels of editing aided in the design of optimal nucleofection conditions and the use of higher efficiency base editors and smaller plasmids.
Patient cell lines used in CRISPR prime editing, harbor homozygous small insertion/deletions that resulted in loss of function and are not amendable to CRISPR base editing approaches. Thus, we designed various CRISPR prime editing strategies by varying RTT and PBS lengths (i.e. template sequence used by reverse transcriptase) but unfortunately delivery by nucleofection yielded no detectable editing by next generation sequencing. Our computational analysis revealed the potential to edit more known dysferlin mutations compared to CRISPR base editing. However, difficulties in optimizing design and efficiency for CRISPR prime editing on each mutation site will need to be further researched in general for this to be a viable approach for dysferlin editing. Future studies should be focused on DYSF mutation sites that can be targeted by base editing as they have more translation potential due to higher efficiency in editing and the ability to be delivered by AAV.