Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Additional file 3: Figure S2. Time-dependent regulation of reporter gene expression. Data were obtained with a BioLector from the same cultures as used in Supplementary Fig. S1. Indicated strains were targeted at pHMG1 or pOLE1 as in Fig. 1C with controls (ctrl) expressing dCas9 and no gRNA. Data were collected for ~47 hrs and are presented as the average of three biological replicates. A. MFI from strains targeted with the constitutive system is presented per DCW/L (dry cell weight per liter) as a function of time. Blue; control. Green; activation (MCP-VPR). Red; repression (PCP-Mxi1). B. MFI from cultures added 250 ng/mL aTc to activate the inducible system is presented per OD (OD600) over time. Blue; control. Green; activation (dCas9-VPR). Red; repression (dCas9-Mxi1)

Modular 5′-UTR hexamers for context-independent tuning of protein expression in eukaryotes

Functional characterization of regulatory DNA elements in broad genetic contexts is a prerequisite for forward engineering of biological systems. Translation initiation site (TIS) sequences are attractive to use for regulating gene activity and metabolic pathway fluxes because the genetic changes are minimal. However, limited knowledge is available on tuning gene outputs by varying TISs in different genetic and environmental contexts. Here, we created TIS hexamer libraries in baker's yeast Saccharomyces cerevisiae directly 5' end of a reporter gene in various promoter contexts and measured gene activity distributions for each library. Next, selected TIS sequences, resulted in almost 10-fold changes in reporter outputs, were experimentally characterized in various environmental and genetic contexts in both yeast and mammalian cells. From our analyses, we observed strong linear correlations (R2 = 0.75-0.98) between all pairwise combinations of TIS order and gene activity. Finally, our analysis enabled the identification of a TIS with almost 50% stronger output than a commonly used TIS for protein expression in mammalian cells, and selected TISs were also used to tune gene activities in yeast at a metabolic branch point in order to prototype fitness and carotenoid production landscapes. Taken together, the characterized TISs support reliable context-independent forward engineering of translation initiation in eukaryotes