Programmable control of small non-coding RNA expression in mammalian cells with synthetic RNA polymerase III transactivators.

  Speaker: Matt Demelo Doctoral Candidate Thesis Defense Department: BioSciences-Biochemistry and Cell Biology Location: Keck Hall 102 Reliable control of gene expression via synthetic genetic circuits is essential to develop next-generation therapies, to facilitate efficient bioindustrial processes, and to enable advanced methods in basic cell biology. Synthetic control of small non-coding RNAs (sncRNAs) is of immense interest due to their important roles in development and disease in mammals, as well as their central role in technologies such as CRISPR/Cas gene editing and RNA interference. RNA polymerase III (RNAP3) is the canonical polymerase for sncRNA expression, but few tools for regulated RNAP3 expression exist, so genetic circuits requiring regulated sncRNA expression must use RNA polymerase II (RNAP2) promoters. While there has been extensive development of synthetic regulatory methods for expressing protein-coding RNAs and large non-coding RNAs via RNAP2, these methods are often ineffective for robust expression of functional sncRNAs and often require post-transcriptional processing for sncRNAs to achieve functionality, impairing the feasibility of translating sncRNA technologies into medical and industrial applications. Here, we develop a toolkit capable of highly programmable and multiplexable expression of synthetic sncRNAs using Synthetic RNAP3 Transcription Factors (Syn3TFs), achieving greater than 10-fold improvement in efficiency compared to analogous RNAP2-mediated methods. Additionally, we outline concise design rules for the construction of minimal RNAP3 promoters and complementary Syn3TFs, facilitating seamless construction of orthogonal RNAP3 circuits with customizable outputs. Finally, we demonstrate the utility of Syn3TFs in performing complex genetic logic and acting as efficient sensors for continuous molecular recording of cellular events. This platform represents a crucial first step to expanding the arsenal of circuit construction strategies for sncRNA expression and provides a strong foundation for continued development of RNAP3-specific synthetic regulators.   (Department : BioSciences-Biochemistry and Cell Biology)