Design of gRNA Expression Systems and Characterization of CRISPR Transcriptional Networks in Saccharomyces cerevisiae

Abstract

The incredibly complex, nuanced, and robust processes happening within cells allow them to adapt to their environment, proliferate, and communicate with their neighbors. These processes are mediated by a network of bio-molecules that regulate each others’ activity and expression. The sophistication of these natural systems make them intractable to recapitulate, let alone fully understand. An engineerable platform for the creation of predictable in vivo regulatory networks would make it significantly easier to access the potential biology has for human health, chemical manufacturing, and biosensing. This thesis discusses advances in engineering interconnected regulatory gene networks in Saccharomyces cerevisiae. These networks employ a version of CRISPR/Cas9, isolated from Streptococcus pyogenes, that has been optimized for transcriptional repression. This work develops a modular genetic unit composed of an engineered gRNA responsive promoters and a computationally designed gRNA expression platform. This thesis first discusses the computational methods used for RNA design. Then, how the CRISPR/Cas9 platform is used to build large networks composed of upwards of seven gRNA interactions. Finally, this work details how the platform was used to quantitatively characterize CRISPR/Cas9 systems, gRNA expression platforms, and to develop a novel ligand sensitive gRNA expression platform. The advances and insights gained in this work have impacts in transcriptional network design, Pol II gRNA expression and multi-gRNA CRISPR applications.