Efficient gene activation in plants by the MoonTag programmable transcriptional activator.

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.

Efficient gene activation in plants by the MoonTag programmable transcriptional activator.

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 worked 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.

Characterization of Programmable Transcription Activators in the Model Monocot Setaria viridis Via Protoplast Transfection.

Recent advances in DNA synthesis and assembly allow for genetic constructs to be designed and constructed in high throughput. Characterizing large numbers of variant genetic designs is not feasible with low-throughput and time-consuming plant transformation workflows. Protoplast transformation offers a rapid, high-throughput compatible alternative for testing genetic constructs in plant-relevant molecular environments. Here, we describe a protocol for protoplast transformation using a recent experiment in genetic optimization of dCas9-based programmable transcription activators as an example.

Fast-TrACC: A Rapid Method for Delivering and Testing Gene Editing Reagents in Somatic Plant Cells.

The production of transgenic or gene edited plants requires considerable time and effort. It is of value to know at the onset of a project whether the transgenes or gene editing reagents are functioning as predicted. To test molecular reagents transiently, we implemented an improved, Agrobacterium tumefaciens-based co-culture method called Fast-TrACC (Fast Treated Agrobacterium Co-Culture). Fast-TrACC delivers reagents to seedlings, allowing high throughput, and uses a luciferase reporter to monitor and calibrate the efficiency of reagent delivery. We demonstrate the use of Fast-TrACC in multiple solanaceous species and apply the method to test promoter activity and the effectiveness of gene editing reagents.

CRISPR-Cas Activators for Engineering Gene Expression in Higher Eukaryotes.

CRISPR-Cas-based transcriptional activators allow genetic engineers to specifically induce expression of one or many target genes in trans. Here we review the many design variations of these versatile tools and compare their effectiveness in different eukaryotic systems. Lastly, we highlight several applications of programmable transcriptional activation to interrogate and engineer complex biological processes.