57 A comprehensive overview: Evolution of TETR/TETO System toward Orthogonal Transcriptional Repression

Faculty Mentor: Justin G. English (Biochemistry, University of Utah)

The adverse consequences caused by misregulation of gene expression programs and their transcriptional regulators are incredibly varied, meaning that much of the mechanisms of these programs are unknown and understudied, especially in the scope of cancer and disease treatment.

To more deeply understand the underlying factors influencing gene expression misregulation and corresponding cell signaling cascades, we are exploiting the Tet-On system as a switch-like tool to explore the limits of flexible exogenous gene expression in mammals. Tet-On allows the expression of any gene to be reversibly, specifically, and differentially controlled on command with the addition and removal of a miniscule dose of a tetracycline-class antibiotic from the system. In this system, a dimer of the tetracycline repressor (TetR) binds the tetracycline operator (TetO), impeding transcription of any downstream gene embedded by the researcher. The wild-type TetR-TetO pair of the Tet-On system offers one of the strongest and most precise controls over target gene expression, where TetR can bind TetO with a remarkably high affinity; this interaction can block transcription even downstream of some of the strongest virus-mediated promoters and remain faithfully functional even in the most sensitive tissues. However, the Tet-On system only exists as a singular wild-type circuit. To expand upon its diverse utility, we are in pursuit of developing novel TetR-TetO orthologous pairs that don’t interfere with this wild-type circuit for use in parallel expression regulation. As a first step to generating TetR-TetO orthologs, we are mapping the usage of TetO by TetR in a massively-parallel reporter assay (MPRA) by engineering an extensive library of mutated TetOs paired with a unique molecular identifier (UMI) sequence to accurately quantify the resulting range of TetR regulation through fluorescent reporter gene expression. From this screen, we will identify candidate TetO mutants with varying levels of detectable wild-type TetR binding activity, and use them to persuade Sindbis virus-mediated evolution of the wild-type TetR toward complementary states to those selected TetO mutant sequences. Finally, to showcase the zero cross-reactivity between our mutant Tet-On circuits and the wild-type-circuit, we plan to engineer a synthetic genetic circuit in mammalian cells, representative of complex circuits and patterns of synchronized cell signaling we observe in naturally-occurring systems. By generating a novel suite of TetR-TetO mutant pairs, we create a toolbox of expression “off-switches” for selective and dependable control over modified gene expression programs with unknown implications in the pursuit of disease prevention and treatment.