Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Microbial Cell Factories

Research

Florian David¹  Raphael Ferreira¹  Justin D. Smith²  Dushica Arsovska³  Ling Ding³  Michael K. Jensen³  Emil D. Jensen³  Jie Zhang³  Tadas Jakočiūnas³  Jens Nielsen⁴  Jay D. Keasling⁵ 

[1] Department of Biology and Biological Engineering, Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden;Department of Genetics, Stanford University School of Medicine, 94305, Stanford, CA, USA;Stanford Genome Technology Center, 94304, Palo Alto, CA, USA;The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark;The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark;Department of Biology and Biological Engineering, Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 412 96, Gothenburg, Sweden;The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs, Lyngby, Denmark;Joint BioEnergy Institute, Emeryville, CA, USA;Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA;Department of Chemical and Biomolecular Engineering & Department of Bioengineering, University of California, Berkeley, CA, USA;

关键词: dCas9;    gRNA;    VPR;    Mxi1;    scRNA;    Yeast;    Isoprenoids;    Triacylglycerols;    Transcriptional regulation;   

DOI  :  10.1186/s12934-017-0664-2

 received in 2016-12-01, accepted in 2017-03-11,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundTranscriptional 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.ResultsIn 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 ~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.ConclusionTaken 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.

【 授权许可】

CC BY   
© The Author(s) 2017