(CS-1-2) Coordinated expression of multi-gene pathways in plants using the MoonTag programmable transcriptional activator and synthetic trans-activated promoters
Body of Abstract: Programmable transcriptional activators (PTAs) are synthetic transcription factors 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. Cas9-based systems 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. We recently described MoonTag, a second-generation PTA that uses a nanobody-antigen peptide interaction to recruit multiple copies of an activation domain to its target promoters. MoonTag is capable of inducing high levels of transcription in reporter as well as in endogenous genes in monocot and dicotyledonous plants. We leveraged MoonTag PTAs to develop synthetic trans-activated promoters (STAPs) for coordinated and tunable expression of multiple transgenes. STAPs are composed of an array of gRNA binding sites upstream of an inactive minimal promoter. The advantage of the STAPs is that they share minimal sequence similarity, limited to the gRNA binding sites, allowing for the design of multigene vectors with limited duplicated sequences but with robust expression levels. Yet, if needed, the strength of each STAP can be tuned by changing the number of gRNA binding sites in the array. As proof-of-concept, we designed STAPs driving the genes from the betalain biosynthetic pathway. In the presence of MoonTag, these genes were simultaneously activated leading to the accumulation of betalain. We also demonstrate tissue-specific regulation of the synthetic promoters by expressing MoonTag from a seed-specific promoter which lead to the accumulation of betalains exclusively in seeds. Thus, the use of STAPs together with PTAs should allow for the deployment of multigene constructs such as those for metabolic pathways or trait stacking that can be specifically expressed in a particular tissue, organ, developmental time, or in response to external cues.