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SPECIAL SESSION: Current Trends and Directions in CRISPR/Cas9 Technology for Filamentous Fungi and Oomycetes

CRISPR/Cas9 gene editing in Fusarium oxysporum
Jeffrey Coleman - Auburn University.

Members of the Fusarium oxysporum species complex collectively have a broad host range, in part due to supernumerary chromosomes that exist in these fungi. To gain further insight into the molecular mechanisms by which these fungi cause disease on agriculturally relevant crops, a CRISPR/Cas9 gene editing procedure was developed via direct transformation with ribonucleoprotein complexes (RNPs). As a proof of concept, auxotroph mutants of URA3 and URA5 were generated and this method was used to insert a selectable marker into the ortholog of the polyketide synthase encoding gene BIK1 demonstrating this gene it is responsible for synthesis of the secondary metabolite bikaverin. The CRISPR/Cas9 RNPs were also used as a tool to generate gene fusions/tagging utilizing two methods, 1) homology-independent targeted integration and 2) homology-dependent recombination integration. As the nuclear localization sequence of this Cas9 protein is conserved among a broad range of Ascomycetes, this CRISPR/Cas9 tool has the potential for applications in many phytopathogenic fungi.