Tools for Genetic Control and Viral Delivery of Multi-Part Gene Cassettes

Controlling genes with light is a powerful tool for studying biology, and a system that is both reversible and can penetrate tissues is optimal for spatiotemporal and in vivo gene control. The highly sensitive red/far-red light “PhytochromeB gene switch” has those characteristics. Yet past attempts to genetically encode this system in non-plant eukaryotic cells have been limited by the inability to produce the necessary chromophore. We found that matching the species of the Ferredoxin enzyme system with the metabolic pathway is required for electron transport to synthesize the chromophore efficiently, enabling us to genetically encode the entire system in mammalian cells (Kyriakakis et al., 2018)-(WO2017192920A1). This species matching concept is now being used by many others to engineer metabolic pathways to produce fertilizer in yeast and in crops (Allen et al., 2017) (Burén and Rubio, 2018).  Because of the large size of the genes encoding these metabolic pathways, my current efforts focus on developing tools for efficient DNA assembly and gene delivery. Using a new method that allows for recursive assembly of DNA, uLoop, we are constructing a library of parts (promoters, PolyA, optogenetics, fluorescent reporters, etc.) to assemble genetic systems quickly. To deliver these large constructs in vivo, we are also developing several nontoxic viral vectors to be compatible with our uLoop DNA parts. Combining uLoop assembly with large payload viral vectors and optogenetics, we are building tools to study development/disease in mouse models of hearing loss and for neural circuit tracing.