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
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 wi...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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SAGE Publishing
2019-04-01
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| Series: | Journal of International Medical Research |
| Online Access: | https://doi.org/10.1177/0300060519829990 |
| _version_ | 1819143480734121984 |
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| author | Yunpeng Dong Tao Peng Weijing Wu Donghui Tan Xuezhong Liu Dinghua Xie |
| author_facet | Yunpeng Dong Tao Peng Weijing Wu Donghui Tan Xuezhong Liu Dinghua Xie |
| author_sort | Yunpeng Dong |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-22T12:26:55Z |
| format | Article |
| id | doaj.art-b1bcca70289640aab096eceecff44c7c |
| institution | Directory Open Access Journal |
| issn | 0300-0605 1473-2300 |
| language | English |
| last_indexed | 2024-12-22T12:26:55Z |
| publishDate | 2019-04-01 |
| publisher | SAGE Publishing |
| record_format | Article |
| series | Journal of International Medical Research |
| spelling | doaj.art-b1bcca70289640aab096eceecff44c7c2022-12-21T18:25:48ZengSAGE PublishingJournal of International Medical Research0300-06051473-23002019-04-014710.1177/0300060519829990Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotidesYunpeng DongTao PengWeijing WuDonghui TanXuezhong LiuDinghua XieBackground 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.https://doi.org/10.1177/0300060519829990 |
| spellingShingle | Yunpeng Dong Tao Peng Weijing Wu Donghui Tan Xuezhong Liu Dinghua Xie 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 Journal of International Medical Research |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| url | https://doi.org/10.1177/0300060519829990 |
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