A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning,Applied Microbiology and Biotechnology

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A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2020-10-02 , DOI: 10.1007/s00253-020-10900-9
Andreas Schüller ₁ , Lisa Wolansky ₂ , Harald Berger ₁ , Lena Studt ₁ , Agnieszka Gacek-Matthews _{1,

3} , Michael Sulyok ₄ , Joseph Strauss ₁

Affiliation

Abstract

Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway.

Keypoints

• An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans.

• Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning.

• The system was successfully applied to activate genes within two silent biosynthetic gene clusters.

中文翻译：

基于诱导型 VPR-dCas9 和核小体图谱引导 sgRNA 定位的新型真菌基因调控系统

抽象的

可编程转录调控是研究基因功能的强大工具。目前选择性调控靶基因的方法主要基于启动子交换或过表达转录激活子。为了扩展发现工具箱，我们为构巢曲霉设计了一种基于 dCas9 的 RNA 引导合成转录激活系统，该系统使用酶促禁用的“死”Cas9 融合到三个连续激活域 (VPR-dCas9)。 dCas9 编码基因受到雌激素响应启动子的控制，以允许诱导定时并避免高活性 VPR 结构域的强组成型表达可能产生的负面影响。特别是在沉默基因组区域，兼性异染色质和严格定位的核小体可能对转录机制构成相关障碍。为了避免这种负面影响并促进 RNA 引导的 VPR-dCas9 最佳定位到目标启动子，我们从活跃生长的细胞和静止培养物中创建了全基因组核小体图谱，以识别同源无核小体区域 (NFR)。基于这些图谱，设计了不同的单引导 RNA (sgRNA)，并测试了它们的靶向和激活潜力。我们的结果表明，该系统可用于并行调节多个基因，并且根据 VPR-dCas9 的定位，表达可以提高到非常高的水平。我们使用该系统打开两个不同的生物合成基因簇（BGC）中的单个基因，这些基因在正常生长条件下是沉默的。该方法还为逐步激活簇中的单个基因以破译相关的生物合成途径提供了机会。