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

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 t...

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Autores principales:	László Mózsik, Mirthe Hoekzema, Niels A. W. de Kok, Roel A. L. Bovenberg, Yvonne Nygård, Arnold J. M. Driessen
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4561eafae88d41fcb7ce654ae2c2f5db
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spelling	oai:doaj.org-article:4561eafae88d41fcb7ce654ae2c2f5db2021-12-02T14:12:46ZCRISPR-based transcriptional activation tool for silent genes in filamentous fungi10.1038/s41598-020-80864-32045-2322https://doaj.org/article/4561eafae88d41fcb7ce654ae2c2f5db2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80864-3https://doaj.org/toc/2045-2322Abstract 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.László MózsikMirthe HoekzemaNiels A. W. de KokRoel A. L. BovenbergYvonne NygårdArnold J. M. DriessenNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q László Mózsik Mirthe Hoekzema Niels A. W. de Kok Roel A. L. Bovenberg Yvonne Nygård Arnold J. M. Driessen CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
description	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.
format	article
author	László Mózsik Mirthe Hoekzema Niels A. W. de Kok Roel A. L. Bovenberg Yvonne Nygård Arnold J. M. Driessen
author_facet	László Mózsik Mirthe Hoekzema Niels A. W. de Kok Roel A. L. Bovenberg Yvonne Nygård Arnold J. M. Driessen
author_sort	László Mózsik
title	CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
title_short	CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
title_full	CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
title_fullStr	CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
title_full_unstemmed	CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
title_sort	crispr-based transcriptional activation tool for silent genes in filamentous fungi
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/4561eafae88d41fcb7ce654ae2c2f5db
work_keys_str_mv	AT laszlomozsik crisprbasedtranscriptionalactivationtoolforsilentgenesinfilamentousfungi AT mirthehoekzema crisprbasedtranscriptionalactivationtoolforsilentgenesinfilamentousfungi AT nielsawdekok crisprbasedtranscriptionalactivationtoolforsilentgenesinfilamentousfungi AT roelalbovenberg crisprbasedtranscriptionalactivationtoolforsilentgenesinfilamentousfungi AT yvonnenygard crisprbasedtranscriptionalactivationtoolforsilentgenesinfilamentousfungi AT arnoldjmdriessen crisprbasedtranscriptionalactivationtoolforsilentgenesinfilamentousfungi
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CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

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