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

Andreas Schüller,Lisa Wolansky,Agnieszka Gacek-Matthews,Harald Berger,Michael Sulyok,Joseph Strauss,Lena Studt

doi:10.1007/s00253-020-10900-9

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.Graphical abstractKeypoints• 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.

Highlights

Filamentous fungi produce a plethora of metabolites and enzymes which are essential components of their response to environmental, nutritional or developmental signals
The expression of VPR-dead” Cas9 (dCas9) can be induced by the addition of estrogens (i.e. DES) which leads to the activation of hER, its subsequent dimerisation, binding to estrogen response elements (EREs; i.e. promoter region of VPR-dCas9) and activation of respective genes (Pachlinger et al 2005)
Programmable transcriptional regulation is a powerful tool for the study of gene functions in general and for the activation of silent fungal biosynthetic gene clusters (BGCs) in particular

Summary

Introduction

Filamentous fungi produce a plethora of metabolites and enzymes which are essential components of their response to environmental, nutritional or developmental signals. The individual genes within a given BGC are transcriptionally co-regulated, not expressed by default and activated only in response to a “proprietary” expression signal. For most of these clusters, standard laboratory conditions do not generate this critical signal and their cognate products cannot be identified (Bachleitner et al 2019; Chujo and Scott 2014; Connolly et al 2013; Gacek-Matthews et al 2016; Gacek and Strauss 2012; Reyes-Dominguez et al 2010; Studt et al 2016). The absence of a pathway-specific TF complicates the targeted activation of a specific cluster (Brakhage 2013; Macheleidt et al 2016; Bergh and Brakhage 1998; Tilburn et al 1995)

Methods

Results

Conclusion