Dataset Information

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.

SUBMITTER: Wu J

PROVIDER: S-EPMC4700424 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

Wu Jianbo J Hunt Samuel D SD Xue Haipeng H Liu Ying Y Darabi Radbod R

Scientific reports 20160105

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 ...[more]

PMID: 26729410

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Project description:Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Dataset Information

Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

Publications

Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

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