CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
Journal article, 2021

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.

Author

László Mózsik

University of Groningen

Mirthe Hoekzema

Swedish University of Agricultural Sciences (SLU)

University of Groningen

Niels A.W. de Kok

University of Groningen

R. A. L. Bovenberg

DSM Biotechnology Centre

University of Groningen

Yvonne Nygård

DSM Biotechnology Centre

Chalmers, Biology and Biological Engineering, Industrial Biotechnology

University of Groningen

Arnold J.M. Driessen

University of Groningen

Scientific Reports

2045-2322 (ISSN)

Vol. 11 1 1118

Subject Categories

Microbiology

Bioinformatics and Systems Biology

Genetics

DOI

10.1038/s41598-020-80864-3

PubMed

33441979

More information

Latest update

2/1/2021 5