Metarhizium anisopliae engineering mediated by a CRISPR/Cas9 recyclable system.

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.