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<p>The advent of CRISPR/Cas technology has revolutionized genome editing, offering simplicity, precision, and cost-effectiveness. While its application in biological control fungi has been limited, including the cosmopolitan fungus <i>Metarhizium anisopliae</i>, recent advancements show promise. However, integrating <i>cas9</i> and selection-marker genes into fungal genomes poses challenges, including reduced efficiency, toxicity, and off-target effects. Besides, marker-free genetic engineering through a CRISPR recyclable system presents a viable solution, enabling efficient mutant generation without compromising fitness and virulence. This study pioneers the construction of marker-free strains of <i>M. anisopliae</i> using a CRISPR/Cas9 recyclable system. Precise deletion of <i>albA</i> and <i>ku70</i>, alongside <i>gfp</i> cassette insertion, confirms the system efficiency. This innovative approach holds significant potential for facilitating in-depth molecular studies, understanding their ecological roles in agricultural systems, and enhancing biocontrol efficacy against insect pests through genetic improvement.</p>

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Metarhizium anisopliae engineering mediated by a CRISPR/Cas9 recyclable system

  • 期刊论文

  • 发表日期：

    2025年2月21日

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  • Maria Letícia de Siqueira Virgílio,
  • Eliane Dias Quintela,
  • Liriel Helen Rodrigues Maciel,
  • Gabriela Souza Silva Goulart,
  • José Francisco Arruda e Silva,
  • Marcio Vinicius de Carvalho Barros Cortes

摘要

The advent of CRISPR/Cas technology has revolutionized genome editing, offering simplicity, precision, and cost-effectiveness. While its application in biological control fungi has been limited, including the cosmopolitan fungus Metarhizium anisopliae, recent advancements show promise. However, integrating cas9 and selection-marker genes into fungal genomes poses challenges, including reduced efficiency, toxicity, and off-target effects. Besides, marker-free genetic engineering through a CRISPR recyclable system presents a viable solution, enabling efficient mutant generation without compromising fitness and virulence. This study pioneers the construction of marker-free strains of M. anisopliae using a CRISPR/Cas9 recyclable system. Precise deletion of albA and ku70, alongside gfp cassette insertion, confirms the system efficiency. This innovative approach holds significant potential for facilitating in-depth molecular studies, understanding their ecological roles in agricultural systems, and enhancing biocontrol efficacy against insect pests through genetic improvement.