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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| Veröffentlicht in: | International journal of molecular sciences 2020-02, Vol.21 (5), p.1574 |
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| 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. |
| doi_str_mv | 10.3390/ijms21051574 |
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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. 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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.</description><subject>A549 Cells</subject><subject>Amino acids</subject><subject>Basic-Leucine Zipper Transcription Factors - genetics</subject><subject>Basic-Leucine Zipper Transcription Factors - metabolism</subject><subject>Cloning vectors</subject><subject>Communication</subject><subject>CRISPR-Associated Protein 9 - genetics</subject><subject>CRISPR-Associated Protein 9 - metabolism</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Expression vectors</subject><subject>Format</subject><subject>Gene expression</subject><subject>Gene therapy</subject><subject>Genes</subject><subject>Genetic Engineering - methods</subject><subject>Genomes</subject><subject>Humans</subject><subject>Mixed Function Oxygenases - genetics</subject><subject>Mixed Function Oxygenases - metabolism</subject><subject>Molecular weight</subject><subject>Peptides</subject><subject>Promoter Regions, Genetic</subject><subject>Proteins</subject><subject>Proto-Oncogene Proteins - genetics</subject><subject>Proto-Oncogene Proteins - metabolism</subject><subject>RNA, Guide, CRISPR-Cas Systems - genetics</subject><subject>RNA, Guide, CRISPR-Cas Systems - metabolism</subject><subject>Single-Chain Antibodies - genetics</subject><subject>Single-Chain Antibodies - metabolism</subject><subject>Tethering</subject><subject>Therapeutic applications</subject><subject>Tiling</subject><subject>Transcription activation</subject><subject>Transcription factors</subject><subject>Up-Regulation</subject><issn>1422-0067</issn><issn>1661-6596</issn><issn>1422-0067</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>8G5</sourceid><sourceid>BENPR</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNpdkc9LHTEQx0OpVKveei6BXjx0Nb82u7kI5WGtICi4eg3Z3cmax27yTHYL7783ViuvhYEZmM98Z4YvQl8oOeVckTO3nhKjpKRlJT6gAyoYKwiR1cedeh99TmlNCOOsVJ_QPs8DUlJ5gB7uth7i4NLsOny_iTAso5ld8DhY3Jg4wIwvwUPC7RY3Fw3Fxvf44VYK7DyeHwH3K5NUcbf4xgz4Ng_bEKcjtGfNmOD4LR-i-58XzepXcX1zebX6cV10oqrnggtWWqk4QFsrVpu2s5VRtaikyWFk37V9LZWCqreqFZRSIKwqW0Wtba3s-CE6f9XdLO0EfQd-jmbUm-gmE7c6GKf_7Xj3qIfwW1ckbyEiC5y8CcTwtECa9eRSB-NoPIQlacalEoyXJcvot__QdViiz-_9oYjkStWZ-v5KdTGkFMG-H0OJfjFM7xqW8a-7D7zDfx3izw6tkMo</recordid><startdate>20200225</startdate><enddate>20200225</enddate><creator>Morita, Sumiyo</creator><creator>Horii, Takuro</creator><creator>Kimura, Mika</creator><creator>Hatada, Izuho</creator><general>MDPI AG</general><general>MDPI</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20200225</creationdate><title>Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform</title><author>Morita, Sumiyo ; 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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.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>32106616</pmid><doi>10.3390/ijms21051574</doi><oa>free_for_read</oa></addata></record> |
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| subjects | A549 Cells Amino acids Basic-Leucine Zipper Transcription Factors - genetics Basic-Leucine Zipper Transcription Factors - metabolism Cloning vectors Communication CRISPR-Associated Protein 9 - genetics CRISPR-Associated Protein 9 - metabolism CRISPR-Cas Systems Deoxyribonucleic acid DNA Expression vectors Format Gene expression Gene therapy Genes Genetic Engineering - methods Genomes Humans Mixed Function Oxygenases - genetics Mixed Function Oxygenases - metabolism Molecular weight Peptides Promoter Regions, Genetic Proteins Proto-Oncogene Proteins - genetics Proto-Oncogene Proteins - metabolism RNA, Guide, CRISPR-Cas Systems - genetics RNA, Guide, CRISPR-Cas Systems - metabolism Single-Chain Antibodies - genetics Single-Chain Antibodies - metabolism Tethering Therapeutic applications Tiling Transcription activation Transcription factors Up-Regulation |
| title | Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform |
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