In the present chapter, we present the protocols and guidelines to facilitate implementation of CRISPR-Cas9 technology in fungi where few or no genetic tools are in place. Hence, we firstly explain how to identify dominant markers for genetic transformation. Secondly, we provide a guide for construction of Cas9/sgRNA episomal expression vectors. Thirdly, we present how to mutagenize reporter genes to explore the efficiency of CRISPR-Cas9 in the relevant fungus and to ease subsequent CRISPR-mediated genetic engineering. Lastly, we describe how to make CRISPR-mediated marker-dependent and marker-free gene targeting.