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-resistant selection marker, and a recipient strain deficient in non-homologous end joining (NHEJ), have since modestly increased the frequency of genome integration and the rate of homologous replacement of the DNA introduced by electroporation. However, electroporation was not the method of choice for transformation until the recent adoption of CRISPR-Cas9 systems. We have developed a Transient CRISPR-Cas9 coupled with Electroporation System (TRACE), which dramatically facilitates targeted mutagenesis in the Cryptococcus species complex. TRACE combines the high transformation efficiency of electroporation with the high rates of DNA integration due to the transiently expressed CRISPR-Cas9. Here, we briefly discussed the history of electroporation for Cryptococcus transformation and provided detailed procedures for electroporation and the cassettes construction of the TRACE system for various genetic manipulations.

新型担子菌隐球菌不仅是临床上重要的病原体，还是研究微生物发病机理和真核生物的模型生物。其作为模型有机体兴起的一个关键因素是其遗传适应性。广泛使用的转化新孢梭菌物种复合物的方法是用于随机插入诱变的农杆菌介导转化（AMT）和用于靶向诱变的生物弹转化。将电穿孔引入新孢子虫在1990年代初期。尽管电穿孔是经济的并且产生大量转化体，但是引入的DNA很少整合到隐球菌基因组中，这限制了它的使用。生物射弹转化虽然成本高昂且效率低下，但却是过去二十年来用于目标诱变的唯一方法。此后进行了一些修改，包括使用带有分裂标记的供体DNA，抗药性选择标记和非同源末端连接（NHEJ）缺陷的受体菌株，从而适度增加了基因组整合的频率和同源率。替换通过电穿孔引入的DNA。然而，直到最近采用CRISPR-Cas9系统，电穿孔才成为转化的选择方法。我们已经开发了带有电穿孔系统（TRACE）的瞬时CRISPR-Cas9，隐球菌物种复杂。由于瞬时表达的CRISPR-Cas9，TRACE将电穿孔的高转化效率与高DNA整合率结合在一起。在这里，我们简要讨论了隐球菌转化电穿孔的历史，并提供了用于电穿孔的详细程序以及TRACE系统用于各种遗传操作的盒式构建。