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.

Agronomy Postgraduate Program Federal University of Goiás Goiás GO 74690 900 Brazil

Embrapa Rice and Beans Brazilian Agricultural Research Corporation Santo Antônio de Goiás GO 75375 000 Brazil

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