title: Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.
abstract: Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.
authors.affiliation: 1 Department of Neurosurgery, Medical School, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.|2 Center for Stem Cell and Regenerative Medicine, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
authors.forename: Shenglan
authors.initials: S
authors.lastname: Li
authors.affiliation: 1 Department of Neurosurgery, Medical School, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.|2 Center for Stem Cell and Regenerative Medicine, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xue
authors.affiliation: 1 Department of Neurosurgery, Medical School, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.|2 Center for Stem Cell and Regenerative Medicine, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Wu
authors.affiliation: 3 The New York Stem Cell Foundation , New York, New York.
authors.forename: Mahendra S
authors.initials: MS
authors.lastname: Rao
authors.affiliation: 1 Department of Neurosurgery, Medical School, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.|2 Center for Stem Cell and Regenerative Medicine, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
authors.forename: Dong H
authors.initials: DH
authors.lastname: Kim
authors.affiliation: 4 Department of Biochemistry and Molecular Medicine, School of Medicine, University of California , Davis, California.|5 Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children , Sacramento, California.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Deng
authors.affiliation: 1 Department of Neurosurgery, Medical School, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.|2 Center for Stem Cell and Regenerative Medicine, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.|6 The Senator Lloyd and B.A. Bentsen Center for Stroke Research, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D055879:Gene Knock-In Techniques / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D009419:Nerve Tissue Proteins; C491183:NEUROG2 protein, human