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 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 an 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.

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

“…2021; Wilson and Harrison 2021), and CRISPRa has recently been optimized for use in the filamentous fungi Aspergillus nidulans and Penicillium rubens (Roux et al . 2020; Mózsik et al . 2021).…”

“…These CRISPRa systems were applied to activate transcriptionally-silent biosynthetic gene clusters in these filamentous fungi, with important applications for the discovery of novel bioactive molecules (Roux et al . 2020; Mózsik et al . 2021).…”

“…CRISPR techniques have already been adapted to numerous industrially-relevant fungi (Nødvig et al 2015;Kwon et al 2019;Jiang et al 2021;Wilson and Harrison 2021), and CRISPRa has recently been optimized for use in the filamentous fungi Aspergillus nidulans and Penicillium rubens (Roux et al 2020;Mózsik et al 2021). These CRISPRa systems were applied to activate transcriptionally-silent biosynthetic gene clusters in these filamentous fungi, with important applications for the discovery of novel bioactive molecules (Roux et al 2020;Mózsik et al 2021). As many Candida species play important industrial roles in manufacturing metabolites for food and pharmaceutical industries, as well as remediating wastewater via hydrocarbon degradation (Joo et al 2008;Gargouri et al 2015;Kieliszek et al 2017;Payen and Thompson 2019;García-Béjar et al 2020), species-specific adaptations to the CRISPRa tool presented here could produce optimized Candida strains for valuable biomanufacturing and bioremediation applications.…”

“…Genetic overexpression systems also have many useful applications for industrially-important fungi, as targeted genetic overexpression can be exploited for metabolic engineering to enhance the production of commercially-important metabolites (Zhao et al 2018;Donohoue et al 2018;Ouedraogo and Tsang 2020;Jiang et al 2021). CRISPR techniques have already been adapted to numerous industrially-relevant fungi (Nødvig et al 2015;Kwon et al 2019;Jiang et al 2021;Wilson and Harrison 2021), and CRISPRa has recently been optimized for use in the filamentous fungi Aspergillus nidulans and Penicillium rubens (Roux et al 2020;Mózsik et al 2021). These CRISPRa systems were applied to activate transcriptionally-silent biosynthetic gene clusters in these filamentous fungi, with important applications for the discovery of novel bioactive molecules (Roux et al 2020;Mózsik et al 2021).…”

Identification of a conserved N‐terminal domain in the first module of ACV synthetases

“…The plasmid pLM‐AMA15.0 (AddGene ID #138944) was constructed using Modular Cloning (Weber et al, 2011) and Gibson Assembly (Gibson et al, 2009), as described previously (Mózsik et al, 2020). The sequence encoding Sp Cas9‐NLS (nuclear localization sequence) was amplified from pYTK036 (yeast MoClo toolkit, AddGene ID # 65143).…”