title: Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.
abstract: Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system.
authors.affiliation: Center for Stem Cell and Regenerative Medicine (CSCRM), The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (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), The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (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), The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA; Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), 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), The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA; Department of Neurosurgery, The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), 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 for the Prevention of Human Diseases (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), The Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas Health Science Center at Houston, Houston, TX 77030, USA. Electronic address: radbod.darabi@uth.tmc.edu.
authors.forename: Radbod
authors.initials: R
authors.lastname: Darabi
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D051763:PAX7 Transcription Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: D051763:PAX7 Transcription Factor; C497321:PAX7 protein, human
keywords: CRISPR; Cas9; Cas9 nickase; GFP reporter; Homologous recombination; Human iPS cells; Knock-in; PAX7; dCas9