Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0664-2) contains supplementary material, which is available to authorized users.

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…1d, 2b). Likewise, dCas9 can be coupled to activating transcription factor domains, such as the tripartite VP64-p65-Rta (VPR) or the RNAP ω-subunit (rpoZ), which have been characterized as powerful tools for activating genes [4,7,44,91] (Fig. 1d).…”

“…It differs from it by possessing a specific RNA processing domain that allows to process the crRNA into multiple gRNAs [55,69,92,101]. Finally, the gRNA scaffold can be extended to include effector protein recruitment stem-loops, which has been shown to enhance transcriptional regulation [8,44,100] (Fig. 1d).…”

“…on the TSS for strong downregulation or more distanced from it for a medium repression. Thus, optimal gene expression can be elucidated by targeting dCas9 at different positions on the studied promoter [16,17,44]. This feature is subject to several parameters, such as the distance to TSS, condition dependent presence of transcription factors, chromatin accessibility, but the complete understanding of how to obtain precise regulation has yet to be characterized and is most likely dependent on specific promoters [57,91].…”

Methods of yeast genome editing

“…Ген белка Cas9 экспрессировали под контролем конститутивных промо-торов разной силы. Наиболее часто используемый про-мотор -TEF1 Zhang et al, 2014;Bao et al, 2015;Jakočiūnas et al, 2015;Mans et al, 2015;Shi et al, 2016;Jensen et al, 2017;Liu et al, 2017;Ng, Dean, 2017), второй по частоте использования промотор -ADH1 (Gao, Zhao, 2014;Jacobs et al, 2014;Fernandez, Berro, 2016;Reider Apel et al, 2017), далее следуют RNR2 , FBA1 (Horwitz et al, 2015), TDH3 (Laughery et al, 2015), PGK1 (Lee et al, 2015) и др.…”

“…2. Saccharomyces cerevisiae DiCarlo et al, 2013;Gao, Zhao, 2014;Zhang et al, 2014;Bao et al, 2015;Horwitz et al, 2015;Jakočiūnas et al, 2015;Mans et al, 2015;Stovicek et al, 2015;Generoso et al, 2016;Jensen et al, 2017;Liu et al, 2017;Ng, Dean, 2017;Reider Apel et al, 2017; et al Schwartz et al, 2016 Kluyveromyces lactis, Kluyveromyces marxianus Horwitz et al, 2015;NambuNishida et al, 2017 Komagataella phaffii (before Pichia pastoris) Weninger et al, 2016 Schizosaccharomyces pombe Jacobs et al, 2014 Ogataea polymorpha Numamoto et al, 2017 Candida albicans, C. glabrata, C. lusitaniae Vyas et al, 2015;Cen et al, 2017;Norton et al, 2017 Cryptococcus neoformans ГидРНК -это второй важный элемент метода CRISPR/ Cas редактирования генома. Введение и правильная тран скрипция гидРНК имеют наибольшее значение во всем процессе клонирования с использованием системы CRISPR/Cas.…”