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<p><i>Magnaporthe oryzae</i>, the causal agent of blast disease in various crops, is a filamentous fungus belonging to the phylum Ascomycota. Its amenability to laboratory culture and genetic manipulation has greatly facilitated studies of genetic interactions with host plants. For decades, gene knockout mutations in <i>M. oryzae</i> have primarily been generated using homologous recombination (HR)-based methods, particularly the split-marker approach. Recently, combining CRISPR/Cas9 with HR has been shown to enhance gene editing efficiency, especially in cases where HR is inefficient. Moreover, this combined strategy can reduce the reliance on sequencing-based validation, thereby lowering overall costs. This report describes gene knockout in <i>M. oryzae</i> using a combined split-marker and CRISPR/Cas9 approach, including construct design, protoplast generation, transformation, and mutant validation. In addition, we discuss the advantages and perspectives of applying this approach to other filamentous species.</p>

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CRISPR/Cas9-Based Gene Knockout in Magnaporthe oryzae in Combination with Homologous Recombination

  • 期刊论文

  • 发表日期：

    2025年11月14日

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  • Yi Qi,
  • Kieu Thi Xuan Vo,
  • Min-Jeong Park,
  • You-Jin Lim,
  • Yong-Hwan Lee,
  • Jong-Seong Jeon

摘要

Magnaporthe oryzae, the causal agent of blast disease in various crops, is a filamentous fungus belonging to the phylum Ascomycota. Its amenability to laboratory culture and genetic manipulation has greatly facilitated studies of genetic interactions with host plants. For decades, gene knockout mutations in M. oryzae have primarily been generated using homologous recombination (HR)-based methods, particularly the split-marker approach. Recently, combining CRISPR/Cas9 with HR has been shown to enhance gene editing efficiency, especially in cases where HR is inefficient. Moreover, this combined strategy can reduce the reliance on sequencing-based validation, thereby lowering overall costs. This report describes gene knockout in M. oryzae using a combined split-marker and CRISPR/Cas9 approach, including construct design, protoplast generation, transformation, and mutant validation. In addition, we discuss the advantages and perspectives of applying this approach to other filamentous species.