Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Abstract

The phytopathogenic smut fungus <i>Ustilago maydis</i> is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of <i>U. maydis</i> by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the <i>U. maydis</i> community but can also act as a template for genome editing in other important fungi.