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Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform

Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral...

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Detalles Bibliográficos
Autores principales: Morita, Sumiyo, Horii, Takuro, Kimura, Mika, Hatada, Izuho
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084704/
https://www.ncbi.nlm.nih.gov/pubmed/32106616
http://dx.doi.org/10.3390/ijms21051574
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author Morita, Sumiyo
Horii, Takuro
Kimura, Mika
Hatada, Izuho
author_facet Morita, Sumiyo
Horii, Takuro
Kimura, Mika
Hatada, Izuho
author_sort Morita, Sumiyo
collection PubMed
description Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9–VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9–SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9–SunTag format synergistically activates gene expression using a single sgRNA.
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spelling pubmed-70847042020-03-24 Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform Morita, Sumiyo Horii, Takuro Kimura, Mika Hatada, Izuho Int J Mol Sci Communication Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9–VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9–SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9–SunTag format synergistically activates gene expression using a single sgRNA. MDPI 2020-02-25 /pmc/articles/PMC7084704/ /pubmed/32106616 http://dx.doi.org/10.3390/ijms21051574 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Communication
Morita, Sumiyo
Horii, Takuro
Kimura, Mika
Hatada, Izuho
Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform
title Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform
title_full Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform
title_fullStr Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform
title_full_unstemmed Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform
title_short Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9–SunTag Platform
title_sort synergistic upregulation of target genes by tet1 and vp64 in the dcas9–suntag platform
topic Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084704/
https://www.ncbi.nlm.nih.gov/pubmed/32106616
http://dx.doi.org/10.3390/ijms21051574
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