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

Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, , 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 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 1 as an example, we demonstrated that PhiReX 2.0 can upregulate 1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.