Construction of an all-in-one doxycycline-inducible Cre/loxP marker recycling system in Aspergillus species

Aspergillus features numerous species with diverse characteristics, and efforts to identify the genes responsible for these traits are ongoing worldwide. Advanced multi-gene knockout technologies are required to analyze the functions of these genes. Although CRISPR/Cas9-based gene disruption methods have become widely used in many organisms, conventional homologous recombination remains the most reliable method for multiple-gene disruption in Aspergillus species. Here, we report the development of a novel marker recycling cassette for Aspergillus spp. This system enables highly efficient gene disruption and marker recycling by combining a doxycycline-inducible Cre/ loxP system with a uracil biosynthesis pathway and counterselection using uracil analogs. Using the model filamentous fungi Aspergillus nidulans and the human pathogen Aspergillus fumigatus , we disrupted genes involved in conidial pigment biosynthesis and amino acid and vitamin biosynthesis. This system is expected to facilitate and accelerate functional genomic analyses in Aspergillus spp.