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

Stem Cell Reports. 2021 Oct 12;16(10):2442-2458. doi: 10.1016/j.stemcr.2021.08.011. Epub 2021 Sep 16.


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.

Keywords: 3D chromatin; CRISPR/Cas9; MYC; muscle stem cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Chromatin / metabolism*
  • Gene Editing / methods
  • Gene Expression Regulation
  • Genes, myc*
  • Genome*
  • Mice
  • MyoD Protein / genetics
  • MyoD Protein / metabolism*
  • Nucleic Acid Conformation
  • Proto-Oncogene Proteins c-bcl-6 / genetics
  • Proto-Oncogene Proteins c-bcl-6 / metabolism*
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism*
  • Satellite Cells, Skeletal Muscle / physiology*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism


  • Bcl6 protein, mouse
  • Chromatin
  • MyoD Protein
  • MyoD1 myogenic differentiation protein
  • Proto-Oncogene Proteins c-bcl-6
  • RNA, Guide, CRISPR-Cas Systems
  • Transcription Factors