Synthetic gene activators consisting of nuclease‐dead Cas9 (dCas9) for single‐guide RNA (sgRNA)‐directed promoter binding and a transcriptional activation domain (TAD) represent new tools for gene activation from endogenous genomic locus in basic and applied plant research. However, multiplex gene coactivation by dCas9‐TADs has not been demonstrated in whole plants. There is also room to optimize the performance of these tools. Here, we report that our previously developed gene activator, dCas9‐TV, could simultaneously upregulate OsGW7 and OsER1 in rice by up to 3,738 fold, with one sgRNA targeting to each promoter. The gene coactivation could persist to at least the fourth generation. Astonishingly, the polycistronic tRNA‐sgRNA expression under the maize ubiquitin promoter, a Pol II promoter, could cause enormous activation of these genes by up to >40,000‐fold in rice. Moreover, the yeast GCN4 coiled coil‐mediated dCas9‐TV dimerization appeared to be promising for enhancing gene activation. Finally, we successfully introduced a self‐amplification loop for dCas9‐TV expression in Arabidopsis to promote the transcriptional upregulation of AtFLS2, a previously characterized dCas9‐TV‐refractory gene with considerable basal expression. Collectively, this work illustrates the robustness of dCas9‐TV in multigene coactivation and provides broadly useful strategies for boosting transcriptional activation efficacy of dCas9‐TADs in plants.

中文翻译：

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dCas9-TV介导的植物基因激活的多重优化

由用于单向导 RNA (sgRNA) 引导的启动子结合的核酸酶死亡 Cas9 (dCas9) 和转录激活域 (TAD) 组成的合成基因激活剂代表了基础和应用植物研究中从内源基因组位点进行基因激活的新工具。然而，dCas9-TADs 的多重基因共激活尚未在整个植物中得到证实。这些工具的性能也有优化空间。在这里，我们报告了我们之前开发的基因激活剂 dCas9-TV，可以同时将水稻中的OsGW7和OsER1上调高达 3,738 倍，其中一个 sgRNA 靶向每个启动子。基因共激活至少可以持续到第四代。令人惊讶的是，玉米泛素下的多顺反子 tRNA-sgRNA 表达启动子，Pol II 启动子，可以在水稻中引起这些基因的巨大激活，最高可达 40,000 倍以上。此外，酵母 GCN4 卷曲螺旋介导的 dCas9-TV 二聚化似乎有望增强基因激活。最后，我们成功地在拟南芥中引入了dCas9-TV表达的自我扩增环，以促进AtFLS2的转录上调，这是一种先前表征的 dCas9-TV 难治性基因，具有相当大的基础表达。总的来说，这项工作说明了 dCas9-TV 在多基因共激活中的稳健性，并为提高 dCas9-TAD 在植物中的转录激活功效提供了广泛有用的策略。