Modular, Synthetic Boolean Logic Gates Enabled in Saccharomyces cerevisiae through T7 Polymerases/CRISPR dCas9 Designs

Abstract

Synthetic control of gene expression, whether simply promoter selection or higher-order Boolean-style logic, is an important tool for metabolic engineering and synthetic biology. This work develops a suite of orthogonal T7 RNA polymerase systems capable of exerting AND/OR switchlike control over transcription in the yeastSaccharomyces cerevisiae. When linked with CRISPR dCas9-based regulation systems, more complex circuitry is possible including AND/OR/NAND/NOR style control in response to combinations of extracellular copper and galactose. Additionally, we demonstrate that these T7 system designs are modular and can accommodate alternative stimuli sensing as demonstrated through blue light induction. These designs should greatly reduce the time and labor necessary for developing Boolean gene circuits in yeast with novel applications including metabolic pathway control in the future.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools

“…The CUP1 promoter may decrease the editing efficiency when promoting the expression of cas9 for gene editing, but the combination of the CUP1 and Gal1 promoters can achieve complex transcriptional regulation. For example, when connected to the CRISPR/dCas9-based regulatory system, more complicated circuits with AND/OR/NAND/NOR type controls could be introduced to respond to the combination of extracellular galactose and copper . In addition, some chemical molecules (e.g., β-estradiol and plant growth hormone ABA) or various synthetic switches can be used for precise regulation to achieve CRISPR/Cas-mediated control of translational dynamics. , Recently a colocalization-dependent protein switch, Co-LOCK, has been developed that activates when binding to the DNA target site.…”

“…In order to enhance transcriptional activity, a tripartite activator of VPR (VP64+p65+Rta) was constructed through a combination of these activation domains. − For example, in order to activate gene transcription by targeting the HED1 and GAL7 promoters of S. cerevisiae , dCas9-VP64 could introduce 8-fold and 14-fold upregulation, respectively, compared with 40-fold and 100-fold upregulation introduced by dCas9-VPR . In recent years, VPR has been widely used for transcriptional activation in S. cerevisiae . − …”

“…Under the action of various inducers, the inducible promoters can introduce precise spatiotemporal regulation of the CRISPR/dCas9 system. The most commonly used inducible promoters in S. cerevisiae are P Gal1/7/10 , induced by galactose and P CUP1 , induced by copper ions. The CUP1 promoter may decrease the editing efficiency when promoting the expression of cas9 for gene editing, but the combination of the CUP1 and Gal1 promoters can achieve complex transcriptional regulation.…”