Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System

Shenglan Li, Haipeng Xue, Jianbo Wu, Mahendra S. Rao, Dong H. Kim, Wenbin Deng, Ying Liu

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2925-2942
Number of pages18
JournalStem Cells and Development
Volume24
Issue number24
DOIs
StatePublished - Dec 15 2015

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CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Induced Pluripotent Stem Cells
Gene Targeting
Transcription Factors
Stem Cells
Technology
Preclinical Drug Evaluations
Regenerative Medicine
Microbial Drug Resistance
Transcriptional Activation
Genetic Recombination
Flow Cytometry
Proteins
Clone Cells
Binding Sites
Cell Line

ASJC Scopus subject areas

  • Hematology
  • Developmental Biology
  • Cell Biology

Cite this

Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System. / Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S.; Kim, Dong H.; Deng, Wenbin; Liu, Ying.

In: Stem Cells and Development, Vol. 24, No. 24, 15.12.2015, p. 2925-2942.

Research output: Contribution to journalArticle

Li, Shenglan ; Xue, Haipeng ; Wu, Jianbo ; Rao, Mahendra S. ; Kim, Dong H. ; Deng, Wenbin ; Liu, Ying. / Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System. In: Stem Cells and Development. 2015 ; Vol. 24, No. 24. pp. 2925-2942.
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