Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

Area of Science:

• Mycology
• Molecular Biology
• Plant Pathology

Background:

• The smut fungus *Ustilago maydis* is a key model organism for studying plant diseases, fungal genetics, and molecular cell biology.
• Efficient genome manipulation tools are crucial for advancing research in *U. maydis*.

Purpose of the Study:

• To establish and present novel CRISPR/Cas9-based genome editing strategies for *Ustilago maydis*.
• To demonstrate the versatility of these techniques for various genetic modifications.

Main Methods:

• CRISPR/Cas9 technology was employed for genome editing.
• Techniques included targeted gene deletion using homologous recombination with short double-stranded oligonucleotides.
• Introduction of point mutations, heterologous complementation, and endogenous N-terminal tagging with mCherry were performed.

Main Results:

• Successful gene deletion, point mutation introduction, and gene tagging were achieved in *U. maydis*.
• All genome editing applications were performed using transient expression of Cas9hf and sgRNA, eliminating the need for permanent selectable markers.
• The developed methods are efficient and versatile for genetic manipulation.

Conclusions:

• The presented CRISPR/Cas9 strategies significantly enhance the genome editing toolkit for *Ustilago maydis*.
• These techniques facilitate faster research progress in fungal genetics and plant pathology.
• The methods serve as a valuable template for genome editing in other fungal species.