Programming Protein Function with Synthetic RNA Splicing Devices

Nature offers enormous functional diversity that is largely untapped in engineered biological systems. Given its roots in bacterial transcriptional gene networks, synthetic biology has yet to develop a comprehensive set of tools that harness the complexity and biological regulation prevalent in mammalian cells. A critically important mechanism in mammalian cells, alternative RNA splicing, increases the coding capacity of genomes to generate several proteins from an individual gene. Few engineered genetic devices utilizing alternative splicing mechanisms have been described, and existing approaches are limited to turning on or off the expression of a gene coupled to a splicing regulatory component. A novel alternative splicing platform has the potential to broaden the current regulatory capabilities of genetic devices by providing a strategy to program protein function. We describe the design and implementation of an intron framework for controlling the mutually exclusive alternative splicing of exons in a four exon-three intron system, wherein the exon sequences can be recoded. The coding capacity of the genetic device is increased by the mutually exclusive nature of the alternative splicing event, as one of two internal exons is incorporated into a mature mRNA, thereby resulting in multiple isoforms. RNA profiling and protein expression assays established that these devices effectively spliced to produce the expected dominant isoform. Systematic tuning of consensus sequence element activity in the intron framework revealed the ability to modify isoform profiles and favor alternate isoform production by a device. Furthermore, we conducted an exploratory study of intronic regulatory elements for controlling and predicting mutually exclusive alternative splicing events within the context of the intron framework. Finally, we applied this intron framework to program transcription factor activity by encoding domains of modular transcription factors in synthetic RNA splicing devices, modifying domain functionality through alternative splicing, and producing precise transcription factor profiles that control gene expression patterns. Our work highlights the first demonstration of an intron framework design that leverages a complex mode of alternative splicing to program protein function with synthetic RNA splicing devices. Continued development of such foundational frameworks in mammalian synthetic biology will enable increasingly sophisticated devices that expand the capabilities of genetic engineering for tackling critical global challenges in health and medicine.