Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-Based Transcriptional Activators

Stem Cell Reports. 2020 May 12;14(5):755-769. doi: 10.1016/j.stemcr.2020.03.026. Epub 2020 Apr 23.

Abstract

Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.

Keywords: CRISPR; differentiation; epigenome editing; iPSCs; myoblasts; myogenesis; pluripotent; satellite cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Cell Differentiation
  • Cell Lineage*
  • Cells, Cultured
  • Cellular Reprogramming Techniques / methods*
  • Dystrophin / genetics
  • Dystrophin / metabolism
  • Epigenesis, Genetic
  • Female
  • Gene Editing / methods
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism*
  • Infant, Newborn
  • Male
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • Muscle Development
  • Myoblasts / cytology
  • Myoblasts / metabolism*
  • PAX7 Transcription Factor / genetics
  • PAX7 Transcription Factor / metabolism*

Substances

  • Dystrophin
  • PAX7 Transcription Factor
  • PAX7 protein, human