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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 wit...

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Autores principales: Dong, Yunpeng, Peng, Tao, Wu, Weijing, Tan, Donghui, Liu, Xuezhong, Xie, Dinghua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: SAGE Publications 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460615/
https://www.ncbi.nlm.nih.gov/pubmed/30819013
http://dx.doi.org/10.1177/0300060519829990
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author Dong, Yunpeng
Peng, Tao
Wu, Weijing
Tan, Donghui
Liu, Xuezhong
Xie, Dinghua
author_facet Dong, Yunpeng
Peng, Tao
Wu, Weijing
Tan, Donghui
Liu, Xuezhong
Xie, Dinghua
author_sort Dong, Yunpeng
collection PubMed
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.
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spelling pubmed-64606152019-04-19 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 Dong, Yunpeng Peng, Tao Wu, Weijing Tan, Donghui Liu, Xuezhong Xie, Dinghua J Int Med Res Pre-Clinical Research Reports 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. SAGE Publications 2019-02-28 2019-04 /pmc/articles/PMC6460615/ /pubmed/30819013 http://dx.doi.org/10.1177/0300060519829990 Text en © The Author(s) 2019 http://creativecommons.org/licenses/by-nc/4.0/ Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).
spellingShingle Pre-Clinical Research Reports
Dong, Yunpeng
Peng, Tao
Wu, Weijing
Tan, Donghui
Liu, Xuezhong
Xie, Dinghua
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 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
topic Pre-Clinical Research Reports
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460615/
https://www.ncbi.nlm.nih.gov/pubmed/30819013
http://dx.doi.org/10.1177/0300060519829990
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