CRISPR-Mediated Activation of Biosynthetic Gene Clusters for Bioactive Molecule Discovery in Filamentous Fungi

Abstract

Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGC could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multi-gene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone.Additionally, we demonstrated the utility of the variant dLbCas12a D156R -VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas..

Total Mycosynthesis: Rational Bioconstruction and Bioengineering of Fungal Natural Products

“…At present, host organisms used for pathway expression are closely related to wild-type fungi, which possess very many of their own biosynthetic pathways which can interfere in the desired mycosyntheses. However, new, rapid and reliable genetic methods such as CRISPR/Cas (which is highly effective in fungi) 109 are likely to be deployed to systematically improve fungal hosts. Finally, it should be noted that total mycosynthesis should not necessarily be regarded as being in direct competition with chemical total synthesis, which has significant advantages in terms of the range and scope of known chemical transformations.…”

The emerging role of biosynthetic gene clusters in plant defense and plant interactions

“…Two prominent chromatin marks, H2A.Z and H3K27me3, are associated with activation and repression of plant BGCs, respectively [ 6 ], thus manipulation of cluster regulation at this level could potentially be achieved by selectively interfering with chromatin remodeling at the cluster locus. Locus-specific epigenetic editing for gene activation/repression with the CRISPR-Cas9 system has already been demonstrated by several studies in mammalian cells via coupling of dCas9 with chromatin-modifying enzymes [ 63 ], and BGC activation in filamentous fungi using CRISPR-Cas9 has also recently been reported [ 64 ].…”

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering

“…Based on knowledge obtained from the work on enAsCas12a (Kleinstiver et al 2019), an improved ''temperature-tolerant'' LbCas12a (ttLbCas12a) which harbors a D156R point mutation, was developed which had significantly higher editing efficiency in plants in comparison to the wild type enzyme (Huang et al 2021;. Later on, higher editing efficiencies were also reported in human cells and fungi using this D156R mutated variant (Roux et al 2020;Tóth et al 2020). Another engineering strategy is the combination of mutations from different Cas variants.…”