Neuromuscular disorders (NMDs) are a group of >840 diseases affecting the peripheral nervous system and skeletal muscle. Despite increased identification of NMD disease genes, only half of patients receive a genetic diagnosis following testing. This is partially due to gaps in our knowledge on the coding and non-coding genes involved in muscle biology. Prioritizing potential muscle disease gene candidates first requires a comprehensive understanding of the genes that play a role in the healthy human “muscle-ome”.
Genetic variants tend to cause a phenotype in the tissues where the gene is highly expressed. We shall use CRISPR/Cas9 technology to delete genes that exhibit enriched expression in human skeletal muscle, but are not associated with disease. This will enable us to assess associated phenotypic and transcriptional changes, identify biological pathways that are affected, and infer the function of the target. We have selected candidate genes with enriched skeletal muscle expression within FANTOM5 data.
Initially targeting two long non-coding RNAs (lncRNAs) for deletion, we compared the resilience, transfection efficiency, and biological relevance of two myogenic cell lines: a clonal colony of immortalized Hu5/E18 myoblasts stably expressing Cas9-GFP, and primary human myoblasts transiently expressing Cas9-GFP. Results showed that the Hu5/E18s had significantly higher rates of survival, transfection and editing success than the primary myoblasts. However, the Hu5/E18s failed to differentiate into myotubes, making them less biologically relevant than the primary myoblasts. We showed the primary myoblast lines have a normal karyotype, whereas both the clonal Hu5/E18 populations and the originally supplied Hu5/E18 cells have abnormal ploidy.
Future directions include optimizing the methods of transfection, exploring the utility of single cell transcriptomic profiling and editing of iPSCs, which can then be differentiated down the muscle lineage.