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 （d...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 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） 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 dCasg-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 transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector 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.展开更多
基金This work was supported by startup funds from East Carolina University and University of Maryland-College Park and a Collaborative Funding grant from North Carolina Biotechnology Center and Syngenta Biotechnology （2016-CFG-8003） to Y.Q. This work was also supported by grants, including the Sichuan Youth Science and Technology Foundation （2017JQ0005）, the National Science Foundation of China （31771486）, and the Fundamental Research Funds for the Central Universities （ZYGX2016J119） to Y.Z.
文摘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 （dCasg） fused with four tandem repeats of the transcriptional activator VP16 0/1=64） 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 dCasg-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 transcrip- tional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator- likeeffector 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.
