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CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in inde...

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Autores principales: Ge, Xianglian, Xi, Haitao, Yang, Fayu, Zhi, Xiao, Fu, Yanghua, Chen, Ding, Xu, Ren-He, Lin, Ge, Qu, Jia, Zhao, Junzhao, Gu, Feng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155318/
https://www.ncbi.nlm.nih.gov/pubmed/27898094
http://dx.doi.org/10.1038/mtna.2016.100
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author Ge, Xianglian
Xi, Haitao
Yang, Fayu
Zhi, Xiao
Fu, Yanghua
Chen, Ding
Xu, Ren-He
Lin, Ge
Qu, Jia
Zhao, Junzhao
Gu, Feng
author_facet Ge, Xianglian
Xi, Haitao
Yang, Fayu
Zhi, Xiao
Fu, Yanghua
Chen, Ding
Xu, Ren-He
Lin, Ge
Qu, Jia
Zhao, Junzhao
Gu, Feng
author_sort Ge, Xianglian
collection PubMed
description Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.
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spelling pubmed-51553182016-12-20 CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells Ge, Xianglian Xi, Haitao Yang, Fayu Zhi, Xiao Fu, Yanghua Chen, Ding Xu, Ren-He Lin, Ge Qu, Jia Zhao, Junzhao Gu, Feng Mol Ther Nucleic Acids Original Article Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome. Nature Publishing Group 2016-11 2016-11-29 /pmc/articles/PMC5155318/ /pubmed/27898094 http://dx.doi.org/10.1038/mtna.2016.100 Text en Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-sa/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/
spellingShingle Original Article
Ge, Xianglian
Xi, Haitao
Yang, Fayu
Zhi, Xiao
Fu, Yanghua
Chen, Ding
Xu, Ren-He
Lin, Ge
Qu, Jia
Zhao, Junzhao
Gu, Feng
CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
title CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
title_full CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
title_fullStr CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
title_full_unstemmed CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
title_short CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells
title_sort crispr/cas9-aav mediated knock-in at nrl locus in human embryonic stem cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155318/
https://www.ncbi.nlm.nih.gov/pubmed/27898094
http://dx.doi.org/10.1038/mtna.2016.100
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