title: Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.
abstract: Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP(+) myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.
authors.affiliation: Center for Stem Cell and Regenerative Medicine (CSCRM), Brown Foundation Institute of Molecular Medicine (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Wu
authors.affiliation: Center for Stem Cell and Regenerative Medicine (CSCRM), Brown Foundation Institute of Molecular Medicine (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Hunt
authors.affiliation: Center for Stem Cell and Regenerative Medicine (CSCRM), Brown Foundation Institute of Molecular Medicine (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA.|Department of Neurosurgery, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.affiliation: Center for Stem Cell and Regenerative Medicine (CSCRM), Brown Foundation Institute of Molecular Medicine (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA.|Department of Neurosurgery, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.|The Senator Lloyd &B.A. Bentsen Center for Stroke Research, The Brown Foundation Institute of Molecular Medicine (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Center for Stem Cell and Regenerative Medicine (CSCRM), Brown Foundation Institute of Molecular Medicine (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Radbod
authors.initials: R
authors.lastname: Darabi
mesh_terms: D015415:Biomarkers; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D002460:Cell Line; D054338:Cell Transdifferentiation; D065150:Cellular Reprogramming / Q000235:genetics; D000067470:Cellular Reprogramming Techniques; D058732:Embryoid Bodies; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D023061:Gene Order; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D059765:Homologous Recombination; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D015415:Biomarkers; D051787:Myogenic Regulatory Factor 5