PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae

Abstract

Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.

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

“…The N-terminal of CIB1 was fused to the two terminals of dCas9 to provide a light-activated CRISPR/Cas9 effector of LACE, which could increase the transcription levels of the reporter genes . Recently, a programmable switch PhiReX 2.0 was developed from the plant-originated optical dimer PIF3 and PhyB, which can activate gene expression in S. cerevisiae by 6.2-fold under red light stimulation by rewiring of metabolic fluxes through targeting promoter sequences with sgRNAs . In addition, another photocaging strategy has been developed for optical control of CRISPR/Cas9 gene editing, which can introduce light-activated Cas9 through the installation of caged lysine .…”

An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

“…Therefore, computer-aided design or other auxiliary tools for the desired gRNA that are appropriate for a variety of Cas proteins are required for efficient genome editing and multiplex gene integration ( Bourgeois et al, 2018 ; Li et al, 2020 ). For example, the programmable red light switch PhiReX 2.0 has been developed to rewire metabolic fluxes by targeting endogenous promoter sequences through sgRNAs, which leads to 6-fold upregulation of a native promoter gene ( Machens et al, 2023 ).…”