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Gene activation by a CRISPR-assisted trans enhancer

The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high effi...

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Autores principales: Xu, Xinhui, Gao, Jinliang, Dai, Wei, Wang, Danyang, Wu, Jian, Wang, Jinke
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478495/
https://www.ncbi.nlm.nih.gov/pubmed/30973327
http://dx.doi.org/10.7554/eLife.45973
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author Xu, Xinhui
Gao, Jinliang
Dai, Wei
Wang, Danyang
Wu, Jian
Wang, Jinke
author_facet Xu, Xinhui
Gao, Jinliang
Dai, Wei
Wang, Danyang
Wu, Jian
Wang, Jinke
author_sort Xu, Xinhui
collection PubMed
description The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators.
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spelling pubmed-64784952019-04-25 Gene activation by a CRISPR-assisted trans enhancer Xu, Xinhui Gao, Jinliang Dai, Wei Wang, Danyang Wu, Jian Wang, Jinke eLife Biochemistry and Chemical Biology The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators. eLife Sciences Publications, Ltd 2019-04-11 /pmc/articles/PMC6478495/ /pubmed/30973327 http://dx.doi.org/10.7554/eLife.45973 Text en © 2019, Xu et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Xu, Xinhui
Gao, Jinliang
Dai, Wei
Wang, Danyang
Wu, Jian
Wang, Jinke
Gene activation by a CRISPR-assisted trans enhancer
title Gene activation by a CRISPR-assisted trans enhancer
title_full Gene activation by a CRISPR-assisted trans enhancer
title_fullStr Gene activation by a CRISPR-assisted trans enhancer
title_full_unstemmed Gene activation by a CRISPR-assisted trans enhancer
title_short Gene activation by a CRISPR-assisted trans enhancer
title_sort gene activation by a crispr-assisted trans enhancer
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478495/
https://www.ncbi.nlm.nih.gov/pubmed/30973327
http://dx.doi.org/10.7554/eLife.45973
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