Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies
Journal article, 2017

Background: Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site. Results: In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker's yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to similar to threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG. Conclusion: Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.\






Transcriptional regulation





E. D. Jensen

Technical University of Denmark (DTU)

Raphael Ferreira

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

T. Jakociunas

Technical University of Denmark (DTU)

D. Arsovska

Technical University of Denmark (DTU)

J. Zhang

Technical University of Denmark (DTU)

L. Ding

Technical University of Denmark (DTU)

J. D. Smith

Stanford University

Florian David

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Jens B Nielsen

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

M. K. Jensen

Technical University of Denmark (DTU)

J.D. Keasling

Lawrence Berkeley National Laboratory

University of California

Joint BioEnergy Institute, California

Technical University of Denmark (DTU)

Microbial Cell Factories

14752859 (eISSN)

Vol. 16 1 46- 46

Subject Categories

Industrial Biotechnology


Chalmers Infrastructure for Mass spectrometry



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