Golden Gate Assembly for Genome Editing in Ustilago maydis.

Golden Gate cloning has become a key strategy for genetic manipulation of Ustilago maydis. Originally established for this organism by Terfrüchte et al. (2014), the approach uses type IIS restriction enzyme BsaI to assemble homology arms and integration module in a single reaction, yielding a plasmid from which a linear DNA fragment for transformation can be excised. In this chapter, the Golden Gate method for U. maydis is presented, with a focus on primer design and the DNA assembly workflow, taking practical considerations into account. By structuring the method into a step-by-step guide, it is made accessible for researchers new to U. maydis aiming to generate genome engineered mutants in this intriguing fungus.