Introduction

Controlling gene expression is a fundamental goal of basic and synthetic biology because it allows insight into cellular function and control of cellular activity. We explored the possibility of generating an optogenetic repressor of gene expression in the model organism Saccharomyces cerevisiae by using light to control the nuclear localization of nuclease-dead Cas9, dCas9.

Methods

The dCas9 protein acts as a repressor for a gene of interest when localized to the nucleus in the presence of an appropriate guide RNA (sgRNA). We engineered dCas9, the mammalian transcriptional repressor Mxi1, and an optogenetic tool to control nuclear localization (LINuS) as parts in an existing yeast optogenetic toolkit. This allowed expression cassettes containing novel dCas9 repressor configurations and guide RNAs to be rapidly constructed and integrated into yeast.

Results

Our library of repressors displays a range of basal repression without the need for inducers or promoter modification. Populations of cells containing these repressors can be combined to generate a heterogeneous population of yeast with a 100-fold expression range. We find that repression can be dialed modestly in a light dose- and intensity-dependent manner. We used this library to repress expression of the lanosterol 14-alpha-demethylase Erg11, generating yeast with a range of sensitivity to the important antifungal drug fluconazole.

Conclusions

This toolkit will be useful for spatiotemporal perturbation of gene expression in Saccharomyces cerevisiae. Additionally, we believe that the simplicity of our scheme will allow these repressors to be easily modified to control gene expression in medically relevant fungi, such as pathogenic yeasts.

中文翻译：

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使用光控核定位的酵母基因表达的光遗传学抑制子。

 介绍

控制基因表达是基础生物学和合成生物学的基本目标，因为它可以深入了解细胞功能和细胞活动的控制。我们探索了通过使用光控制核酸酶死亡 Cas9（dCas9）的核定位，在模式生物酿酒酵母中产生基因表达的光遗传学抑制子的可能性。

 方法

当 dCas9 蛋白在适当的引导 RNA (sgRNA) 存在的情况下定位到细胞核时，可充当感兴趣基因的阻遏物。我们设计了 dCas9、哺乳动物转录抑制子 Mxi1 和控制核定位 (LINuS) 的光遗传学工具，作为现有酵母光遗传学工具包的一部分。这使得包含新型 dCas9 阻遏物配置和引导 RNA 的表达盒能够快速构建并整合到酵母中。

 结果

我们的阻遏蛋白库显示了一系列基础阻抑，无需诱导剂或启动子修饰。含有这些阻遏物的细胞群可以组合起来产生具有 100 倍表达范围的异质酵母群。我们发现，抑制可以以剂量和强度依赖的方式适度调节。我们使用这个库来抑制羊毛甾醇 14-α-去甲基酶 Erg11 的表达，产生对重要的抗真菌药物氟康唑具有一系列敏感性的酵母。

 结论

该工具包可用于酿酒酵母基因表达的时空扰动。此外，我们相信我们方案的简单性将使这些阻遏物能够轻松修改以控制医学相关真菌（例如病原酵母）中的基因表达。