| Description |
Recent gene therapy advancements have progressed the treatment of a spectrum of disorders and diseases caused by dysregulation of gene expression programs and their transcriptional regulators. However, existing genetic therapies largely lack tunable control of exogenous gene expression. Here, we describe our exploitation of the Tet-On system as a switch-like tool to explore the limits of flexible exogenous gene expression in mammalian cells. The preexisting Tet-On system allows the on-command expression of any gene to be reversibly, specifically, and differentially controlled. Although extremely practical in simple synthetic control over the expression of any gene, Tet-On is difficult to apply to complex expression systems as it exists as a solidary tool of its kind. Because of this system's diverse but under optimized utility, we present a venture into developing novel TetR-TetO orthologous pairs that can be used to regulate gene expression in parallel to the wild-type circuit with perfect specificity to their own circuits. We first discovered that a single TetR-TetO2 protein-DNA pair has the capacity to repress transcription of a gene downstream of a strong constitutive promoter to simplify Tet-On into our streamlined TetR/TetO system. By use of a massively parallel reporter assay (MPRA) with an extensive library of TetO2 variants, we mapped the usage of TetO2 by TetR, generating a range of percent repression of reporter gene expression. From this screen, we identified three candidate palindromic TetO2 variants (TetO5CG, TetO5GC, and TetO2GC) that nearly nullify TetR repression. These minimally mutated TetO2 candidates hold the ability to direct the construction of orthogonal iii expression circuits to the TetR/TetO system, where they can be used to pinpoint TetR proteins that can restore the binding interaction with each variant with complete specificity. In addition to these TetO2 variants, our array of TetR binding capacities extends from these near-zero levels through and beyond the upper bounds observed in the wild-type TetO2 interaction. Thereby, we have generated a novel suite of foundational tools that can be utilized to both universally understand the specificity underlying protein-DNA interactions and to develop dynamic control switches for selective and reliable synthetic control over modified gene expression towards complex disease treatment. |
