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An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells
Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner i...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Higher Education Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413595/ https://www.ncbi.nlm.nih.gov/pubmed/28116670 http://dx.doi.org/10.1007/s13238-016-0360-8 |
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author | Guo, Jianying Ma, Dacheng Huang, Rujin Ming, Jia Ye, Min Kee, Kehkooi Xie, Zhen Na, Jie |
author_facet | Guo, Jianying Ma, Dacheng Huang, Rujin Ming, Jia Ye, Min Kee, Kehkooi Xie, Zhen Na, Jie |
author_sort | Guo, Jianying |
collection | PubMed |
description | Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13238-016-0360-8) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5413595 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Higher Education Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-54135952017-05-18 An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells Guo, Jianying Ma, Dacheng Huang, Rujin Ming, Jia Ye, Min Kee, Kehkooi Xie, Zhen Na, Jie Protein Cell Research Article Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13238-016-0360-8) contains supplementary material, which is available to authorized users. Higher Education Press 2017-01-23 2017-05 /pmc/articles/PMC5413595/ /pubmed/28116670 http://dx.doi.org/10.1007/s13238-016-0360-8 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Research Article Guo, Jianying Ma, Dacheng Huang, Rujin Ming, Jia Ye, Min Kee, Kehkooi Xie, Zhen Na, Jie An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells |
title | An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells |
title_full | An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells |
title_fullStr | An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells |
title_full_unstemmed | An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells |
title_short | An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells |
title_sort | inducible crispr-on system for controllable gene activation in human pluripotent stem cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413595/ https://www.ncbi.nlm.nih.gov/pubmed/28116670 http://dx.doi.org/10.1007/s13238-016-0360-8 |
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