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Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein (RNP) complexes

Qiang Wang: Auburn University

<div>The <em>Fusarium oxysporum</em> species complex (FOSC) is an important group of filamentous fungi that are able to cause disease on over 120 genera of plants. Based on its economic importance, <em>F. oxysporum</em> has become an important microbe for studying the genetics and pathogenicity of a plant pathogen; however, efficient reverse genetic methods including gene disruption/deletion strategies are limited. To aid in the study of these fungi, we developed a gene editing system using a <em>F. oxysporum</em>-optimized Cas9 RNP transformation method. The optimized Cas9 protein and sgRNA can be assembled to form a stable ribonucleotideprotein <em>in vitro</em> and this complex and/or template donor DNA can be transferred into fungal protoplasts for gene editing by PEG-mediated transformation. This procedure was developed using Cas9 RNPs that were transferred to fungal protoplasts to disrupt two genes (<em>URA5</em> and <em>URA3</em>), generating uracil auxotroph mutants and are resistant to 5-FOA. Another gene, <em>bik</em>1, was used to further optimize this system where the maximum efficiency of disruption for this gene was able to reach ~50%. Further analysis of the <em>bik</em>1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment bikaverin. All mutants in this study demonstrated strong expected phenotypes, which suggests this <em>F. oxysporum</em>-optimized CRISPR/Cas9 system is stable and can be efficiently used to disrupt the gene(s) of interest.</div>