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

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.

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

“…CRISPR‐Cas9 can be converted to nickases for promoting targeting specificity (Fauser et al ., ; Ran et al ., ), stimulating HDR (Cermak et al ., ; Miki et al ., ; Sun et al ., ; Wang et al ., 2017a), and repurposing for base editing (Gaudelli et al ., ; Komor et al ., ; Shimatani et al ., ). Furthermore, deactivated Cas9 (dCas9) has been used for engineering synthetic transcriptional regulators (Lowder et al ., , ; Piatek et al ., ; Tang et al ., ) and DNA labelling in plants (Dreissig et al ., ). It is conceivable that our STU‐Cas9 2.0 systems can be applied in all these applications.…”

“…In some cases, homology directed repair (HDR) was used for targeted gene replacement (Endo et al ., 2016b; Gil‐Humanes et al ., ; Li et al ., ; Miki et al ., ; Schiml et al ., ; Svitashev et al ., ). Cas9 nickase (Fauser et al ., ; Ran et al ., ) and base editors (Gaudelli et al ., ; Komor et al ., ; Li et al ., ; Shimatani et al ., ) have further expanded the applications of Cas9‐based plant genome editing (Hua et al ., ; Lowder et al ., ; Lu and Zhu, ; Ren et al ., , ; Yan et al ., ; Zong et al ., ).…”

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…Through recruitment of multiple transcriptional activators, the dCas‐based transcriptional activator system can be improved further (Konermann et al ; Zalatan et al ), as shown using the SunTag system (Tanenbaum et al ). Furthermore, indirect fusions of transcriptional activators, like VP64 to modified gRNAs, have allowed robust transcriptional multiplex gene activation, in planta (Lowder et al ).…”

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

“…More recent approaches using the dCas9 system were based on the recruitment of effectors via the sgRNA. Recently published data show that in plants a combination of the direct and indirect fusion system currently represents the most robust system for transcriptional activation [59]. The fusion of regulators to the RBPs enable an indirect recruitment to the site of interest [56].…”