Deciphering the function of candidate genes in skeletal muscle aging using AAV-CRISPR/Cas9

Sarcopenia is the progressive loss of muscle mass and function, associated with biological aging. However, the precise molecular mechanisms that functionally impair the neuromuscular system are not yet fully understood. The advent of –omics approaches has allowed molecular profiling of sarcopenic muscle and shed light on the molecular remodeling of muscle at high age. Traditionally, forward and reverse genetic studies are applied to relate molecular events to phenotypes. In many tissues, CRISPR became the method of choice to generate loss-of-function mouse models. However, when it comes to skeletal muscle, CRISPR is not applicable so far due to particular challenges of skeletal muscle, such as its size and molecular organization. Hence, transgenic mice generated via the Cre-loxP system remain the method of choice for functional gene interrogation studies in skeletal muscle, which makes the profiling of multiple genes cumbersome and highly time consuming. Hence, an efficient, multiplexable and muscle fiber-specific gene editing approach is sorely needed to characterize the underlying molecular drivers of sarcopenia. To facilitate efficient characterization of candidate genes in sarcopenia, we established a versatile tool for local and systemic gene knockout, specifically in skeletal muscle fibers by coupling the advantages of CRISPR/Cas9 with novel myotropic AAV vectors. We initially engineered Cas9mKI mice, which express Cas9 specifically in skeletal muscle fibers and are morphologically and functionally indistinguishable from their littermate controls. AAVMYO-mediated delivery of multiple sgRNA yielded potent transduction and editing efficiencies, resulting in local and systemic loss of function. As a proof-of-principle, we demonstrated that this system is capable of reproducing traditional Cre-loxP-mediated gene knockout phenotypes, including potent signaling pathway alteration, NMJ deterioration or muscle hypertrophy stimulation. With having such a powerful and versatile tool for gene function interrogation in the back, we further characterized the molecular mechanism of genes, potentially contributing to sarcopenia. By comparing molecular profiles of multiple denervation-associated conditions, including sarcopenia, surgical denervation and sustained mTORC1 hyperactivation, we identified a conserved gene signature. Among those, we selected a small Ras-related GTPase Rrad for functional characterization. Rrad expression in healthy skeletal muscle induced a prominent loss of muscle mass and function. Interestingly, Rrad expression induced a strong gene expression signature, reminiscent of denervation and sarcopenia, without causing muscle denervation. Whereas Rrad induced muscle wasting, AAV-CRISPR/Cas9-mediated depletion of Rrad in muscle fibers exacerbated mitochondrial and muscle dysfunction upon surgical denervation.
Taken together, this thesis established an AAV-CRISPR/Cas9 system for fast, highly efficient and multiplexable interrogation of gene function in skeletal muscle and characterized the functional role of Rrad in the context of skeletal muscle denervation.