Plant morphology is a key determinant of their resilience and productivity and has been a focal point of crop improvement. However, predictably engineering developmental outcomes such as organ size, which tend to be multigenic, cross-regulated and dependent on spatiotemporal coordination of expression, remains a major challenge. The Gibberellin (GA) phytohormone signaling pathway is a major determinant of organ size through the control of cell expansion and is highly conserved across plant species. We are developing methods to fine-tune expression of GA signaling genes using Cas9-based synthetic transcription factors (SynTFs). These tools enable targeted regulation of multiple genes, simultaneously, thanks to the programmability of Cas9. We have utilized a viral vector-based method for rapid in planta prototyping of different control system architectures used to implement dwarfing through the layering of additional regulation on GA signaling genes. We have also used transient expression assays to explore how SynTFs can be designed both to titrate expression across a broad range, and to respond to agro-chemicals to facilitate inducible control of expression. We are now investigating the generality of these design rules in stable transgenic lines. We hypothesize the spatiotemporal control these tools provide will enable circumventing negative side-effects typically associated with constitutively altered expression of GA signaling genes, such as delayed germination. The design rules we uncover through this work will have immediate relevance for crop engineering and create a general framework for how complex multigenic traits could be engineered in multicellular organisms.
