(75f) Design and Characterization of Orthogonal Pol II gRNA Expression Systems for dCas9 Transcriptional Repression Networks in S. Cerevisiae

Robust, modular, and orthogonal genetic control systems have applications in metabolic pathway regulation and optimization, the creation of whole cell biosensors, and cell-fate decisions for therapeutics and tissue engineering. These systems require an interoperable biological framework, a library of orthogonal characterized parts, and an understanding of inherent retroactivity. In recent work [Gander, M. W. et al. Nat. Commun. 8, 15459 doi:10.1038/ncomms15459 (2017)] we adapted the nuclease-inactive bacterial CRISPR/dCas9 system to create a functionally complete genetic NOR logic gate where a single output may be targeted to one of two inputs using only gRNA. The NOR gate input stage is a pol II promoter with two unique target sites (pGRR) which drives the expression of computationally-designed self-cleaving-ribozyme flanked gRNA (RGRs). These ribozymes mediate post-transcriptional processing to remove the 5’ cap and poly-A tail nuclear export signals. With minimal characterization these NOR gates were composed to create five two input boolean logic functions and a six layer repression cascade. Despite the overall success, impedance mismatching of uncharacterized components and strong retroactive effects resulted in failed logic circuits and unexpected signal gain in longer cascades. To characterize these components and investigate sources of retroactivity, we have developed titratable 1 layer (gRNA repressing - reporter protein) and 2 layer (gRNA repressing - gRNA repressing - reporter promoter) repression cascade framework. We have found that improper ribozyme processing leads to a degradation of signal with increasing cascade size and that gRNA input sensitivity decreases with increasing cascade size. Surprisingly, gRNA input sensitivity is recovered with decreased dCas9 expression - implying that a major source of retroactivity is caused by differences in gRNA binding affinity for the dCas9. In this presentation, we will discuss the computational methods used to design functional RGR devices at the level of RNA, computational methods for selecting orthogonal components, how retroactivity affect the behavior of concurrent gRNA expression, and ongoing experiments to harness gRNA expression as a platform for building biosensors and dynamically responsive genetic control systems.