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Mol Plant. 2018 Feb 5;11(2):245-256. doi: 10.1016/j.molp.2017.11.010. Epub 2017 Nov 29.

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

Author information

1
Department of Biology, East Carolina University, Greenville, NC 27858, USA.
2
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China.
3
Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA.
4
Department of Plant and Microbial Biology and Plants for Human Health Institute, North Carolina State University, North Carolina Research Campus, Kannapolis, NC 28081, USA.
5
Department of Genetics, Cell Biology and Development, Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
6
Department of Biotechnology, School of Life Science and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu 610054, China. Electronic address: zhangyong916@uestc.edu.cn.
7
Department of Biology, East Carolina University, Greenville, NC 27858, USA; Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA; Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA. Electronic address: yiping@umd.edu.

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.

KEYWORDS:

CRISPR; MS2-VP64; TALE-VP64; gRNA2.0; multiplex; transcriptional activation

PMID:
29197638
DOI:
10.1016/j.molp.2017.11.010
[Indexed for MEDLINE]
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