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Sporisorium scitamineum  is the causative agent responsible for smut disease of sugarcane worldwide. However, lack of efficient gene manipulation system makes this fungus much behind the type model of the smut fungi in molecular biology. Here, we report the development of a CRISPR/Cas9 and T-DNA based dual vector system that allowed efficient knock-out or knock-in of a gene of interest in the  S. scitamineum  in a site-specific manner. By using  Mfa2 , a key player in the mating event in  S. scitamineum  as a tester gene, site-specific insertions of the introduced fragments were achieved both for  Mfa2  knockout and complementation. Of particular advantage of this system is the simplicity of selection and identification for the desired transformants by using drug resistance coupled with PCR. This system greatly facilitates the gene function study in  S. scitamineum , and could potentially be used for other basidiomycete fungi.

Development of an efficient vector system for gene knock-out and near in-cis gene complementation in the sugarcane smut fungus