Skip to main content
Research article - Peer-reviewed, 2021

CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

Mozsik, Laszlo; Hoekzema, Mirthe; de Kok, Niels A. W.; Bovenberg, Roel A. L.; Nygard, Yvonne; Driessen, Arnold J. M.

Abstract

Filamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA "plug-and-play" module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

Published in

Scientific Reports
2021, volume: 11, number: 1, article number: 1118
Publisher: NATURE RESEARCH

Authors' information

Mozsik, Laszlo
University of Groningen
Swedish University of Agricultural Sciences, Department of Forest Mycology and Plant Pathology
de Kok, Niels A. W.
University of Groningen
Bovenberg, Roel A. L.
DSM NV
Bovenberg, Roel A. L.
University of Groningen
Nygard, Yvonne
DSM NV
Nygard, Yvonne
University of Groningen
Nygard, Yvonne
Chalmers University of Technology
Driessen, Arnold J. M.
University of Groningen

Associated SLU-program

SLU Network Plant Protection

UKÄ Subject classification

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Publication Identifiers

DOI: https://doi.org/10.1038/s41598-020-80864-3

URI (permanent link to this page)

https://res.slu.se/id/publ/111289