Implementation of CRISPR based genetic circuits in the yeast S. cerevisiae

Abstract

Many different routes for targeted gene expression are available and with the help of Synthetic Biology (SynBio), more and more controlled expression systems are developed every year aimed at robustly and precisely switching one or more genes on and off (1). Targeted repression and expression are essential for basic research, as well as for many industrial applications. In this context the unicellular eukaryote S. cerevisiae is one of the oldest and most commonly used hosts (2,3). Despite the fact, that S. cerevisiae has been investigated extensively for many years, researchers are still lacking easy-to-implement and well-organized toolboxes (3). In addition, many frequently used regulatory systems influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation (4). Within this work, a modular system for targeted gene expression was designed, implemented and extensively characterized to overcome these limitations. Additionally, it was aimed to contribute to and therefore improve the already existing publicly available yeast toolbox. A scaffold RNA (scRNA) CRISPR/dCas9 system served as a modular expression platform (5). This is based on three components namely dCas9, the RNA-binding protein MCP fused to the transcriptional activation domain VP64 and a scRNA encoding the desired locus and action. A scRNA specifically targeting a tetO binding sequence was used, resulting in Venus reporter gene expression. The functionality of the reporter gene setup was demonstrated by the expression of the reporter gene Venus. A natural switch was integrated based on the well-established Gal10 expression cassette by implementation of a GAL promoter in front of dCas9. High expression levels were achieved, but comparatively high basal expression was observed. Hence, a second switch based on the synthetic LexA-ER-AD heterologous transcription factor was added. A functional AND gate was achieved by implementing four β-estradiol (ES) inducible lexA boxes in front of MCP-VP64. The AND gate was demonstrated to achieve high expression rates in presence of galactose and ES while being tightly regulated in their absence. Additionally, a reporter gene setup was implemented combining a gene of interest (GOI) with the fluorophore tGFP by the ribosomal skipping T2A sequence. This setup allows to adapt the system to any gene of interest without losing reporter function. To extend the possibilities for applications, a positive feedback loop was designed and verified as functional. The modularity of the system was demonstrated by the construction of a second AND gate, in which the scRNA was placed under the control of ES. For this purpose, a ribozyme-scRNA-ribozyme construct was introduced and its functionality verified for both, a constitutive promoter and an ES inducible promoter. The established switches and AND gates were characterized in detail for their expression levels, basal activity and their dose-dependency. A better understanding of the underlying principles and the functioning of the AND gate design was achieved by backing up of the experimental findings with the development of a mathematical model and single-cell analysis. In summary, a predictable and modular, as well as versatile expression control system was developed.