Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells

• Published in: STAR protocols Vol. 1; no. 3; p. 100158
• Main Authors: Xi, Haibin, Young, Courtney S., Pyle, April D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.12.2020; Elsevier
• Subjects: 3' Untranslated Regions - genetics; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cell Count; Cell Differentiation; Conserved Sequence; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Drug Resistance, Microbial; Genes, Reporter; Genotype; Humans; Mammals; Mesoderm - embryology; MicroRNAs - genetics; MicroRNAs - metabolism; Muscle Development; PAX7 Transcription Factor - chemistry; PAX7 Transcription Factor - metabolism; Plasmids - genetics; Pluripotent Stem Cells - metabolism; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Protocol; Reproducibility of Results; RNA, Guide, CRISPR-Cas Systems - genetics; Somites - embryology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100158

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020). [Display omitted] •Establish reporter hPSCs for myogenic transcription factor PAX7 via CRISPR/Cas9•Fast reporter validation by dCas9-VPR artificial activation of PAX7 promoter•Physiological reporter validation through hPSC directed myogenic differentiation This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis.