The MoonTag Programmable Transcription Activator and Synthetic Promoters: Tools for Engineering Oil Biosynthesis in Camelina and Pennycress
J. Armando Casas Mollano* (firstname.lastname@example.org), Jonathan Cors, and Michael J. Smanski
University of Minnesota–Saint Paul
Leverage the use of sequence-programmable transcriptional activators (PTAs) and synthetic promoters to coordinately fine tune the expression of multiple endogenous genes and/or transgenes to facilitate targeted lipid production.
Programmable transcriptional activators (PTAs) are synthetic tools that enable tunable regulation of the expression of endogenous genes and transgenes in eukaryotic and prokaryotic organisms. PTAs are composed of activation domains fused to a dead Cas9 enzyme (dCas9) that lacks nuclease activity but can still bind DNA in a sequence-specific manner (Casas-Mollano et al. 2020). The main advantages of Cas9-based systems are that they can achieve high levels of gene activation and are very easy to program via base-pairing between the guide RNA (gRNA) and the DNA target strand (Casas-Mollano et al. 2020). The PTA described here, called MoonTag, is a second-generation system that uses a nanobody-antigen peptide interaction to recruit multiple copies of an activation domain to its target promoters (Casas-Mollano et al. 2023). MoonTag is capable of inducing high levels of transcription in reporter as well as in endogenous genes in the monocot model plant Setaria. MoonTag is also able to efficiently activate genes in eudicot species such as Arabidopsis and tomato (Casas-Mollano et al. 2023). In addition to its activation capabilities, MoonTag components are expressed in transgenic plants to high levels without any deleterious effects resulting from the expressed components being incompatible with plants cells. Thus, MoonTag is a new activator that could be used to regulate the transcription of endogenous genes in many plant species.
The team has also created a set of synthetic promoters that could be used together with MoonTag to coordinate the expression of multiple genes. The synthetic promoters were designed using a modular approach. They comprise a minimal promoter (the region immediately upstream the transcriptional initiation site where the transcription pre-initiation complex and RNA pol II binds) and a trans-activation (TA) region upstream containing binding sequences for transcription factors that stimulate or repress transcription (Belcher et al. 2020). The TA region was designed to contain six gRNA binding sites for a Cas9-based PTA such as MoonTag. The designed synthetic promoters can be strongly activated by MoonTag and other Cas9-based activators. One advantage of these synthetic promoters is that they share a minimal amount of duplicated sequences, limited to the gRNA binding sites, allowing for the design of multigene vectors with limited sequence homology but with robust expression levels.
As proof-of-concept, researchers designed synthetic promoters driving the three genes from the betalain biosynthetic pathway (He et al. 2020). In the presence of the MoonTag PTA, these genes were simultaneously activated leading to the accumulation of betalains. Researchers also demonstrate tissue-specific regulation of the synthetic promoters by expressing MoonTag from a seed-specific promoter that led to the accumulation of betalains exclusively in the seeds. Thus, the use of synthetic promoters together with PTAs should allow for the deployment of multigene constructs such as those for metabolic pathways or trait stacking that can be directed to specifically be expressed in a particular tissue, organ, developmental time, or in response to external cues.
In the context of the B5 (Bigger Better Brassicaceae Biofuels and Bioproducts) project the team will use the MoonTag activator and the synthetic promoters to achieve the precise coordination of gene expression of transgenes as well as endogenous genes in order to facilitate oilseed engineering in pennycress and camelina. Overexpression of endogenous genes in seeds will be achieved by expressing MoonTag driven by seed-specific promoters together with gRNAs targeting the promoters of the genes of interest. When necessary, transgenes driven by synthetic promoters activated by MoonTag will be used so that both transgenes and endogenous genes, can be coordinately regulated in a particular seed developmental stage by where MoonTag is expressed. For overexpression researchers will initially target the FatB gene to boost palmitic acid (16:0) content and WRI1, DGAT1, GDP1 to increase oil content. Oil seed engineering will also require repression of lipid biosynthetic genes. To target genes for repression team members will generate MoonTag PTRs (programmable transcriptional repressors) bearing the repressive SRDX domain (Hiratsu et al. 2003). Alternatively, researchers will use the synthetic promoters to drive expression of double-stranded RNAs that target endogenous genes for downregulation through RNA interference.
Belcher, M. S., et al. 2020. “Design of Orthogonal Regulatory Systems for Modulating Gene Expression in Plants,” Nat Chem Biol 16, 857–65.
Casas-Mollano, J. A., et al. 2020. “CRISPR-Cas Activators for Engineering Gene Expression in Higher Eukaryotes,” CRISPR J. 3, 350–64.
Casas Mollano, J. A., et al. 2023. “Efficient Gene Activation in Plants by the MoonTag Programmable Transcriptional Activator,” bioRxiv 2023.02.15.528671.
He, Y., et al. 2020. “A Reporter for Noninvasively Monitoring Gene Expression and Plant Transformation,” Hortic Res. 7, 152.
Hiratsu, K., et al. 2003. “Dominant Repression of Target Genes by Chimeric Repressors that Include the EAR Motif, a Repression Domain, in Arabidopsis,” Plant J. 34, 733–39.
This research is supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0023142.