Expanding the CRISPR activation toolbox for secondary metabolite discovery in filamentous fungi Clara Woodcraft, Indra Roux, YitHeng Chooi University of Western Australia, Australia Filamentous fungi are prolific producers of small bioactive molecules known as secondary metabolites that serve as an important source of drug and agrochemical leads. Genome sequencing has enabled the mass identification of fungal biosynthetic gene clusters (BGCs) likely to encode novel secondary metabolites. However, the study of these BGCs is hindered by the fact that many are transcriptionally silent under laboratory growth conditions. A dCas12a-VPR based CRISPR activation (CRISPRa) system has previously been established in our lab in Aspergillus nidulans towards facilitating efficient and scalable BGC activation for metabolite discovery. Here we build on this system and work towards improving on some of its limitations in fungi. As the temperature range required for Cas12a based tools is incompatible with the growth requirements for some species of fungi, we constructed and tested a temperature tolerant version of the dCas12a-VPR protein by introducing a D156R mutation. Additionally, we have worked towards further characterising the potential of this system by examining more BGC targets. The successful refinement of CRISPRa in filamentous fungi has the potential to accelerate secondary metabolite discovery for fuelling drug and agrochemical leads.
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