Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotides

Yunpeng Dong, Tao Peng, Weijing Wu, Donghui Tan, Xue Z Liu, Dinghua Xie

Research output: Contribution to journalArticle

1 Scopus citations


Background: Heterozygous purinergic receptor p2x gene (P2RX2) c.178G>T (p.V60L) mutations can lead to progressive hearing loss (HL) and increased susceptibility to noise. However, the underlying mechanisms remain unclear. A combination of human induced pluripotent stem cell (hiPSC) technology with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9-mediated gene editing may provide a promising tool to study gene function and treat hereditary deafness in humans. Methods: hiPSC technology and CRISPR/Cas9-mediated gene editing were used to generate heterozygous and homozygous P2RX2 c.178G>T (p.V60L) cell models. Results: We generated non-integrative hiPSCs from urine samples derived from three members of a large Chinese family carrying heterozygous P2RX2 c.178G>T mutations (designated P2RX2 +/– ) as a model to study P2RX2-mediated hereditary HL. Furthermore, we used CRISPR/Cas9 and single-stranded donor oligonucleotides to genetically establish homozygous P2RX2 c.178G>T hiPSCs (designated P2RX2 –/– ) from heterozygous patient-specific hiPSCs as a control to further study the pathological gene function. Conclusions: Heterozygous and homozygous P2RX2-mutated hiPSC lines are good models to investigate the pathological mechanisms of P2RX2 mutations in HL pathogenesis. Our findings confirmed our hypothesis that it is feasible and convenient to introduce precise point mutations into genomic loci of interest to generate gene-mutated hiPSC models.

Original languageEnglish (US)
Pages (from-to)1717-1730
Number of pages14
JournalJournal of International Medical Research
Issue number4
StatePublished - Apr 1 2019
Externally publishedYes



  • CRISPR/Cas9
  • gene editing
  • hearing loss
  • iPS cells
  • P2RX2
  • point mutations

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biochemistry, medical

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