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

Mozsik, Laszlo and Hoekzema, Mirthe and de Kok, Niels A. W. and Bovenberg, Roel A. L. and Nygard, Yvonne and Driessen, Arnold J. M. (2021). CRISPR-based transcriptional activation tool for silent genes in filamentous fungi. Scientific Reports. 11 , 1118
[Research article]

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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.

Authors/Creators:Mozsik, Laszlo and Hoekzema, Mirthe and de Kok, Niels A. W. and Bovenberg, Roel A. L. and Nygard, Yvonne and Driessen, Arnold J. M.
Title:CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
Series Name/Journal:Scientific Reports
Year of publishing :2021
Volume:11
Article number:1118
Number of Pages:13
Publisher:NATURE RESEARCH
ISSN:2045-2322
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 3 Medical and Health Sciences > 304 Medical Biotechnology > Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
URN:NBN:urn:nbn:se:slu:epsilon-p-111289
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-111289
Additional ID:
Type of IDID
DOI10.1038/s41598-020-80864-3
Web of Science (WoS)000621766600012
ID Code:23002
Faculty:S - Faculty of Forest Sciences
Department:(NL, NJ) > Dept. of Forest Mycology and Plant Pathology
(S) > Dept. of Forest Mycology and Plant Pathology
Deposited By: SLUpub Connector
Deposited On:07 Apr 2021 15:03
Metadata Last Modified:07 Apr 2021 15:11

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