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The basidiomycete Cryptococcus neoformans is not only a clinically important pathogen, but also a model organism for studying microbial pathogenesis and eukaryotic biology. One key factor behind its rise as a model organism is its genetic amenability. The widely used methods for transforming the C. neoformans species complex are Agrobacterium-mediated transformation (AMT) for random insertional mutagenesis and biolistic transformation for targeted mutagenesis. Electroporation was introduced to C. neoformans in early 1990s. Although electroporation is economic and yields a large number of transformants, introduced DNA rarely integrates into cryptococcal genome, which limits its use. Biolistic transformation, although costly and inefficient, has been the only method used in targeted mutagenesis in the past two decades. Several modifications, including the use of a donor DNA with split markers, a drug …