Efficient multiple gene knockout in Colletotrichum higginsianum via CRISPR/C as9 ribonucleoprotein and URA3 ‐based marker recycling

Abstract Colletotrichum higginsianum is a hemibiotrophic pathogen that causes anthracnose disease on crucifer hosts, including Arabidopsis thaliana . Despite the availability of genomic and transcriptomic information and the ability to transform both organisms, identifying C. higginsianum genes involved in virulence has been challenging due to recalcitrance to gene targeting and redundancy of virulence factors. To overcome these obstacles, we developed an efficient method for multiple gene disruption in C. higginsianum by combining CRISPR/Cas9 and a URA3 ‐based marker recycling system. Our method significantly increased the efficiency of gene knockout via homologous recombination by introducing genomic DNA double‐strand breaks. We demonstrated the applicability of the URA3 ‐based marker recycling system for multiple gene targeting in the same strain. Using our technology, we successfully targeted two melanin biosynthesis genes, SCD1 and PKS1 , which resulted in deficiency in melanization and loss of pathogenicity in the mutants. Our findings demonstrate the effectiveness of our methods in analysing virulence factors in C. higginsianum , thus accelerating research on plant–fungus interactions.