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CRISPR-based transc...
CRISPR-based transcriptional activation tool for silent genes in filamentous fungi
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- Mózsik, László (författare)
- Rijksuniversiteit Groningen,University of Groningen
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- Hoekzema, Mirthe (författare)
- Swedish University of Agricultural Sciences,Sveriges lantbruksuniversitet,Department of Forest Mycology and Plant Pathology,Sveriges lantbruksuniversitet (SLU),Swedish University of Agricultural Sciences (SLU),Rijksuniversiteit Groningen,University of Groningen
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- de Kok, Niels A.W. (författare)
- Rijksuniversiteit Groningen,University of Groningen
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- Bovenberg, R. A. L. (författare)
- DSM Biotechnology Centre,Rijksuniversiteit Groningen,University of Groningen
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- Nygård, Yvonne, 1986 (författare)
- DSM Biotechnology Centre,Chalmers tekniska högskola,Chalmers University of Technology,Rijksuniversiteit Groningen,University of Groningen
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- Driessen, Arnold J.M. (författare)
- Rijksuniversiteit Groningen,University of Groningen
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(creator_code:org_t)
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- 2021-01-13
- 2021
- Engelska.
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 11:1
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Mikrobiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Microbiology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Bioinformatik och systembiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Bioinformatics and Systems Biology (hsv//eng)
- NATURVETENSKAP -- Biologi -- Genetik (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Genetics (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
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