Epigenome Engineering for Precise Genetic Perturbation of Skeletal Muscle Gene Expression and Metabolism

Disruptions in muscle metabolism are a major driver of complex disease. Significant advances have been made using replacement genes or gene editing to treat monogenic disorders of metabolism in skeletal muscle. However, mechanisms of many complex diseases that impact skeletal muscle function, aging, and metabolism remain elusive. Recent advances in genome engineering tools have provided methods for precise gene activation and gene silencing. These tools have enabled functional genomic screening of the non-coding genome and high-throughput screening of gene regulatory elements at an unprecedented scale. Mice that allow inducible expression of these tools will allow higher throughput study of individual genes that contribute to muscle metabolism and overall health and enable multiplex genetic screening. In this proposal we aim to establish these mouse lines at the University of Arkansas and demonstrate the flexibility of genetic screening provided by these models. The power of these mouse models will be further enhanced by our development and usage of a recently described MyoAAV, which allows for efficient, safe, and highly specific delivery of guide RNAs to skeletal muscle.

Aim 1: Establish muscle-specific and inducible dCas9-p300 mice (CRISPR-ON) for targeted epigenetic activation. We will benchmark this approach with muscle-specific overexpression of c-Myc (Myc), a key regulator of ribosome biogenesis and, based on our preliminary data, potential metabolic shifts in muscle.

Aim 2: Establish dCas9-KRAB mice (CRISPR-OFF) for targeted epigenetic silencing and gene inhibition. We will expand this approach by simultaneously knocking down several paralogs of Myc in muscle. Currently, we only have the capability to overexpress c-Myc genetically, and inducible knockdown of multiple forms will be useful for loss-offunction experiments where other paralogs of Myc (l-Myc and n-Myc) may compensate.

Both aims will use the AIMRC Bioenergetics core to evaluate the veracity of our approaches with respect to changes in muscle metabolism using high-resolution respirometry. Establishing these mouse models will eliminate the need for generating muscle-specific floxed mice through numerous rounds of breeding, which is costly, labor-intensive, and time-consuming. The end result of this work will be a powerful new genetic screening tool with high collaborative potential and a robust proof-of-concept dataset for follow-on funding.