Abstract
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.