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© Copyright 2018 William E. Voje © Copyright 2018 William E. Voje Jr. Design of gRNA Expression Systems and Characterization of CRISPR Transcriptional Networks in Saccharomyces cerevisiae. William E. Voje Jr. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2018 Reading Committee: James Carothers, Chair Mary Lidstrom Jesse Zalatan Program Authorized to Offer Degree: Chemical Engineering University of Washington Abstract Design of gRNA Expression Systems and Characterization of CRISPR Transcriptional Networks in Saccharomyces cerevisiae. William E. Voje Jr. Chair of the Supervisory Committee: Assistant Professor James Carothers Chemical Engineering 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. TABLE OF CONTENTS LIST OF FIGURES.......................................................................................................................iv LIST OF TABLES.........................................................................................................................vi Chapter 1. Introduction...................................................................................................................1 1.1 Motivation............................................................................................................................1 1.2 RNA as a Functional Molecule............................................................................................2 1.3 CRISPR-Cas.........................................................................................................................2 1.4 Genetic circuits....................................................................................................................4 1.5 Organization of thesis..........................................................................................................5 Chapter 2. Computational RNA design..........................................................................................8 2.1 Introduction..........................................................................................................................8 2.1.1 RNA function is derived from its sequence and structure.........................................8 2.1.2 Computational methods for RNA structure prediction............................................11 2.1.3 Examples of computational RNA design.................................................................12 2.1.4 Challenges that still exist.........................................................................................12 2.2 Pyrfold a python package for RNA design........................................................................13 2.2.1 RNA part design......................................................................................................13 2.2.2 RNA simulation.......................................................................................................15 2.3 Conclusions........................................................................................................................17 Chapter 3. Digital logic circuits in yeast with CRISPR-dCas9 NOR gates..................................18 3.1 Attribution..........................................................................................................................18 3.2 Abstract..............................................................................................................................18 3.3 Introduction........................................................................................................................19 3.4 Results................................................................................................................................21 3.4.1 NOR gate architecture..............................................................................................21 3.4.2 Input stage promoter design.....................................................................................25 3.4.3 Output stage RNA design........................................................................................25 3.4.4 Logic circuits...........................................................................................................26 3.4.5 Cascades...................................................................................................................28 i 3.4.6 Mathematical modeling...........................................................................................30 3.5 Discussion..........................................................................................................................33 3.6 Methods..............................................................................................................................36 3.6.1 Construction of yeast strains....................................................................................36 3.6.2 RNA design..............................................................................................................36 3.6.3 Cytometry................................................................................................................37 3.6.4 Data collection for orthogonality matrix..................................................................38 3.6.5 Data collection for logic circuits and static cascades...............................................38 3.6.6 Model description....................................................................................................38 3.6.7 Fitting procedure......................................................................................................39 3.6.8 Model predictions....................................................................................................40 Chapter 4. Engineering predictable CRISPRi transcriptional networks with ribozyme- and aptazyme-regulated gRNA expression..........................................................................................42 4.1 Abstract..............................................................................................................................42 4.2 Introduction........................................................................................................................43 4.3 Methods..............................................................................................................................46 4.3.1 Plasmid construction................................................................................................46 4.3.2 Creation of stable yeast strains................................................................................46 4.3.3 Yeast growth condition............................................................................................47 4.3.4 Testing RG, GR, iRGR, RGiR devices....................................................................47 4.3.5 Testing RGA one-layer cascades.............................................................................47 4.3.6 Flow cytometry........................................................................................................48 4.3.7 Calibrating and normalizing experiments................................................................48 4.3.8 Fitting system response with a Hill-equation...........................................................49 4.3.9 Quantifying relative sensitivity and sensitivity ratio...............................................49 4.3.10 Simulating CRISPRi systems................................................................................49 4.3.11 Computational design of insulated ribozyme devices...........................................50 4.3.12 In vitro ribozyme cleavage rates............................................................................52 4.4 Results................................................................................................................................53 ii 4.4.1 Quantifying system sensitivity to gRNA inputs expressed from pol II promoters..53 4.4.2 RGRs can be engineered from diverse ribozymes...................................................58
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