Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
Division of Forest Pathology, Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Almas allé 5, 756 51, Uppsala, Sweden.
Sci Rep. 2021 Jan 13;11(1):1118. doi: 10.1038/s41598-020-80864-3.
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.
丝状真菌是生物活性化合物的丰富资源库，这些化合物被应用于从作物保护到医学等多个领域。可用基因组数据的激增表明，真菌仍然是新药物的极好来源。然而，大多数负责生物合成的基因簇在实验室生长条件下转录沉默。因此，需要通用的策略来激活这些簇。在这里，我们提出了一种基于 CRISPR 激活（CRISPRa）方法的丝状真菌无基因组编辑、转录调控工具。在此，Cas9 的一种无核酸酶突变体（dCas9）与一个高效的三部分激活剂 VP64-p65-Rta（VPR）融合，以允许 sgRNA 指导靶向基因调控。将 dCas9-VPR 与易于使用的 sgRNA“即插即用”模块一起引入非整合 AMA1-载体中，该载体与几种丝状真菌物种兼容。为了证明其潜力，该载体用于在 Penicillium rubens 的 penDE 核心启动子的控制下转录激活荧光报告基因。随后，我们通过将 dCas9-VPR 靶向转录因子 macR 的启动子区域，激活了天然的、转录沉默的 P. rubens 巨孢菌素生物合成基因簇。这导致了抗菌巨孢菌素的产生。该 CRISPRa 技术可用于快速方便地激活沉默的真菌生物合成基因簇，从而有助于鉴定新的化合物，如抗菌剂。
