Cryptococcus neoformans is an opportunistic human fungal pathogen and serves as a model organism for studies of eukaryotic microbiology and microbial pathogenesis. C. neoformans species complex is classified into serotype A, serotype D, and AD hybrids, which are currently considered different subspecies. Different serotype strains display varied phenotypes, virulence, and gene regulation. Genetic investigation of important pathways is often performed in both serotype A and D reference strains in order to identify diversification or conservation of the interrogated signaling network. Many genetic tools have been developed for C. neoformans serotype A reference strain H99, including the gene free “safe haven” (SH) regions for DNA integration identified based on genomic features. However, no such a genomic safe haven region has been identified in serotype D strains. Here, capitalizing on the available genomic, transcriptomic, and chromatin data, we identified an intergenic region named as SH3 for the serotype D reference strains JEC21 and XL280. We also designed a sgRNA and a vector facilitating any alien gene integration into SH3 through a CRISPR-Cas9 system. We found that gene inserted in this region complemented the corresponding gene deletion mutant. Fluorescent reporter gene inserted in SH3 can also be expressed efficiently. Insertion in SH3 itself did not alter the expression of adjacent genes and did not affect the growth or mating of C. neoformans. Thus, SH3 provides a resource for genetic manipulations in serotype D strains and will facilitate comparative analyses of gene functions in this species complex. In addition, the incorporation of the multi-omic data in our selection of the safe haven region could help similar studies in other organisms.

新型隐球菌是一种机会性人类真菌病原体，可作为真核微生物学和微生物发病机制研究的模型生物。新型隐球菌物种复合体分为血清型A、血清型D和AD杂种，目前被认为是不同的亚种。不同血清型菌株表现出不同的表型、毒力和基因调控。重要途径的遗传研究通常在血清型 A 和 D 参考菌株中进行，以便识别所询问的信号网络的多样化或保守性。已经针对新型隐球菌A 血清型参考菌株 H99开发了许多遗传工具，包括根据基因组特征识别的用于 DNA 整合的无基因“安全港”( SH ) 区域。然而，在 D 血清型菌株中尚未发现这样的基因组安全港区域。在这里，利用现有的基因组、转录组和染色质数据，我们为血清型 D 参考菌株 JEC21 和 XL280确定了一个名为SH3的基因间区域。我们还设计了 sgRNA 和载体，通过 CRISPR-Cas9 系统促进任何外来基因整合到SH3中。我们发现插入该区域的基因与相应的基因缺失突变体互补。插入SH3的荧光报告基因也能高效表达。插入SH3本身并不会改变邻近基因的表达，也不会影响新型隐球菌的生长或交配。因此，SH3为血清型 D 菌株的遗传操作提供了资源，并将有助于对该物种复合体中的基因功能进行比较分析。此外，在我们选择安全港区域时纳入多组学数据可能有助于在其他生物体中进行类似的研究。