CRISPR/Cas9/AAV9-mediated in vivo editing identifies MYC regulation of 3D genome in skeletal muscle stem cell.

Skeletal muscle satellite cells (SCs) are stem cells responsible for muscle development and regeneration. Although CRISPR/Cas9 has been widely used, its application in endogenous SCs remains elusive. Here, we generate mice expressing Cas9 in SCs and achieve robust editing in juvenile SCs at the postnatal stage through AAV9-mediated short guide RNA (sgRNA) delivery. Additionally, we reveal that quiescent SCs are resistant to CRISPR/Cas9-mediated editing. As a proof of concept, we demonstrate efficient editing of master transcription factor (TF) Myod1 locus using the CRISPR/Cas9/AAV9-sgRNA system in juvenile SCs. Application on two key TFs, MYC and BCL6, unveils distinct functions in SC activation and muscle regeneration. Particularly, we reveal that MYC orchestrates SC activation through regulating 3D genome architecture. Its depletion results in strengthening of the topologically associating domain boundaries thus may affect gene expression. Altogether, our study establishes a platform for editing endogenous SCs that can be harnessed to elucidate the functionality of key regulators governing SC activities.

Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration.

Necroptosis, a form of programmed cell death, is characterized by the loss of membrane integrity and release of intracellular contents, the execution of which depends on the membrane-disrupting activity of the Mixed Lineage Kinase Domain-Like protein (MLKL) upon its phosphorylation. Here we found myofibers committed MLKL-dependent necroptosis after muscle injury. Either pharmacological inhibition of the necroptosis upstream kinase Receptor Interacting Protein Kinases 1 (RIPK1) or genetic ablation of MLKL expression in myofibers led to significant muscle regeneration defects. By releasing factors into the muscle stem cell (MuSC) microenvironment, necroptotic myofibers facilitated muscle regeneration. Tenascin-C (TNC), released by necroptotic myofibers, was found to be critical for MuSC proliferation. The temporary expression of TNC in myofibers is tightly controlled by necroptosis; the extracellular release of TNC depends on necroptotic membrane rupture. TNC directly activated EGF receptor (EGFR) signaling pathway in MuSCs through its N-terminus assembly domain together with the EGF-like domain. These findings indicate that necroptosis plays a key role in promoting MuSC proliferation to facilitate muscle regeneration.