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Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells

Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeed...

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Detalles Bibliográficos
Autores principales: Liu, Zhongliang, Hui, Yi, Shi, Lei, Chen, Zhenyu, Xu, Xiangjie, Chi, Liankai, Fan, Beibei, Fang, Yujiang, Liu, Yang, Ma, Lin, Wang, Yiran, Xiao, Lei, Zhang, Quanbin, Jin, Guohua, Liu, Ling, Zhang, Xiaoqing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032288/
https://www.ncbi.nlm.nih.gov/pubmed/27594587
http://dx.doi.org/10.1016/j.stemcr.2016.07.021
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author Liu, Zhongliang
Hui, Yi
Shi, Lei
Chen, Zhenyu
Xu, Xiangjie
Chi, Liankai
Fan, Beibei
Fang, Yujiang
Liu, Yang
Ma, Lin
Wang, Yiran
Xiao, Lei
Zhang, Quanbin
Jin, Guohua
Liu, Ling
Zhang, Xiaoqing
author_facet Liu, Zhongliang
Hui, Yi
Shi, Lei
Chen, Zhenyu
Xu, Xiangjie
Chi, Liankai
Fan, Beibei
Fang, Yujiang
Liu, Yang
Ma, Lin
Wang, Yiran
Xiao, Lei
Zhang, Quanbin
Jin, Guohua
Liu, Ling
Zhang, Xiaoqing
author_sort Liu, Zhongliang
collection PubMed
description Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs.
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spelling pubmed-50322882016-09-29 Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells Liu, Zhongliang Hui, Yi Shi, Lei Chen, Zhenyu Xu, Xiangjie Chi, Liankai Fan, Beibei Fang, Yujiang Liu, Yang Ma, Lin Wang, Yiran Xiao, Lei Zhang, Quanbin Jin, Guohua Liu, Ling Zhang, Xiaoqing Stem Cell Reports Article Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs. Elsevier 2016-09-01 /pmc/articles/PMC5032288/ /pubmed/27594587 http://dx.doi.org/10.1016/j.stemcr.2016.07.021 Text en © 2016 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Liu, Zhongliang
Hui, Yi
Shi, Lei
Chen, Zhenyu
Xu, Xiangjie
Chi, Liankai
Fan, Beibei
Fang, Yujiang
Liu, Yang
Ma, Lin
Wang, Yiran
Xiao, Lei
Zhang, Quanbin
Jin, Guohua
Liu, Ling
Zhang, Xiaoqing
Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
title Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
title_full Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
title_fullStr Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
title_full_unstemmed Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
title_short Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells
title_sort efficient crispr/cas9-mediated versatile, predictable, and donor-free gene knockout in human pluripotent stem cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032288/
https://www.ncbi.nlm.nih.gov/pubmed/27594587
http://dx.doi.org/10.1016/j.stemcr.2016.07.021
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