Cited by 232CRISPR and Genetic EngineeringPlant Molecular Biology ResearchTransgenic Plants and Applications

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Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems

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Lowder, Levi G., Zhou, Jianping, Zhang, Yingxiao, Malzahn, Aimee, Zhong, Zhaohui, Hsieh, Tzung-Fu, Voytas, Daniel F., Zhang, Yong, Qi, Yiping·2018·Molecular Plant

DOI 10.1016/j.molp.2017.11.010

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Abstract

User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR-associated protein 9 (dCas9) fused with four tandem repeats of the transcriptional activator VP16 (VP64) could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCas9-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator-like effector activation (mTALE-Act) system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.

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Authors: Lowder, L. G., Zhou, J., Zhang, Y., Malzahn, A., Zhong, Z., Hsieh, T.-F., Voytas, D. F., Zhang, Y., & Qi, Y.
Affiliations: East Carolina University (US)
University of Electronic Science and Technology of China (CN)
University of Maryland, College Park (US)
North Carolina State University (US)
University of Minnesota (US)
Corresponding author: Qi, Yiping
ORCID: Zhou, Jianping
Zhang, Yingxiao
Malzahn, Aimee
Hsieh, Tzung-Fu
Voytas, Daniel F.
Qi, Yiping
Venue: Molecular Plant
Publisher: Elsevier BV
Published: 2018-02
Volume: 11
Issue: 2
Pages: 245-256
ISSN: 1674-2052 (ISSN-L)
DOI: 10.1016/j.molp.2017.11.010
OpenAlex: W2768119869
Cited by: 232
References: 81
Citations by year: 2026: 20 · 2025: 22 · 2024: 13 · 2023: 28 · 2022: 45 · 2021: 29 · 2020: 32 · 2019: 36 · 2018: 7
FWCI: 8.37
Type: journal article
Language: en
Open access: bronze
doi.org
Licence: www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/open-access/userlicense/1.0/ (vor, 369 d)
Locations: Molecular Plant (publishedVersion)
PubMed (publishedVersion)
Funders: East Carolina University
University of Maryland-College Park
North Carolina Biotechnology Center
Syngenta Biotechnology (2016-CFG-8003)
Sichuan Youth Science and Technology Foundation (2017JQ0005)
National Science Foundation of China (31771486)
Fundamental Research Funds for the Central Universities (ZYGX2016J119)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Transgenic Plants and Applications
Keywords: transcriptional activation, transcription activator-like effectors, positive feedback loop, gene regulatory networks, Golden Gate cloning, Gateway cloning, plant genome editing
MeSH terms: Transcription Factors, Transcriptional Activation, Gene Expression Regulation, Plant, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems

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