Department of Biochemistry, Molecular Biology, and Biophysics and Biotechnology Institute, University of Minnesota, Saint Paul, MN 55108, USA.
Center for Precision Plant Genomics, University of Minnesota, Saint Paul, MN 55108, USA.
Nucleic Acids Res. 2023 Jul 21;51(13):7083-7093. doi: 10.1093/nar/gkad458.
CRISPR/Cas-based transcriptional activators have been developed to induce gene expression in eukaryotic and prokaryotic organisms. The main advantages of CRISPR/Cas-based systems is that they can achieve high levels of transcriptional activation and are very easy to program via pairing between the guide RNA and the DNA target strand. SunTag is a second-generation system that activates transcription by recruiting multiple copies of an activation domain (AD) to its target promoters. SunTag is a strong activator; however, in some species it is difficult to stably express. To overcome this problem, we designed MoonTag, a new activator that works on the same basic principle as SunTag, but whose components are better tolerated when stably expressed in transgenic plants. We demonstrate that MoonTag is capable of inducing high levels of transcription in all plants tested. In Setaria, MoonTag is capable of inducing high levels of transcription of reporter genes as well as of endogenous genes. More important, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects. MoonTag is also able to efficiently activate genes in eudicotyledonous species such as Arabidopsis and tomato. Finally, we show that MoonTag activation is functional across a range of temperatures, which is promising for potential field applications.
基于 CRISPR/Cas 的转录激活子已被开发用于诱导真核和原核生物中的基因表达。基于 CRISPR/Cas 的系统的主要优势在于，它们可以实现高水平的转录激活，并且通过向导 RNA 与 DNA 靶链之间的配对非常容易编程。SunTag 是一种第二代系统，通过将多个激活结构域 (AD) 募集到其靶启动子上来激活转录。SunTag 是一种强激活剂；然而，在某些物种中，它很难稳定表达。为了克服这个问题，我们设计了 MoonTag，这是一种新的激活剂，其工作原理与 SunTag 相同，但在稳定表达于转基因植物中时，其组件的耐受性更好。我们证明 MoonTag 能够在所有测试的植物中诱导高水平的转录。在柳枝稷中，MoonTag 能够诱导报告基因和内源性基因的高水平转录。更重要的是，MoonTag 组件在转基因植物中以高水平表达，而没有任何有害影响。MoonTag 还能够有效地在拟南芥和番茄等双子叶植物物种中激活基因。最后，我们表明 MoonTag 激活在一系列温度下是有效的，这对于潜在的田间应用是有希望的。
