Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.
Bibliographical noteFunding Information:
We thank Dr. Benjamin Kotopka for valuable comments on the manuscript. We thank Dr. Christopher W. J. Smith for sharing the TS23D plasmid. We are grateful to Dr. Joseph Puglisi for allowing us to conduct long-read sequencing experiments on his laboratory’s PacBio RS II sequencer and Dr. Elizabeth Tseng for her guidance with long-read sequencing data analysis. Fluorescence microscopy was performed at the Stanford Cell Sciences Imaging Facility. This work was supported by the National Institutes of Health, National Science Foundation, Human Frontiers Science Program (grants to C.D. S.), Stanford Bio-X Institute, National Science Foundation, Siebel Scholars Foundation (graduate student fellowships to M.M.), Stanford Enhancing Diversity through Graduate Education in STEM, Agilent Graduate Fellowship, and the National Institute of General Medical Sciences of the National Institutes of Health [award number T32GM008412] (graduate student fellowships to C.M.K.). Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the National Institute of Standards and Technology, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose.