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

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

Previously, we have shown transcriptional activation of endogenous genes in plants using dCas9-VP64. Here, we develop a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for CRISPR-Cas9 based transcriptional activation. Simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than our first generation dCas9-VP64 activators. In addition, we have built 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. Moreover, 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 may 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. The systems will enable transcriptional activation applications in both dicots and monocots, and the vectors in this new toolbox are publicly available to the research community through Addgene.