Department of Microbiology, University of Georgia, Athens 30602, GA, USA.
Department of Microbiology, University of Georgia, Athens 30602, GA, USA.
Fungal Genet Biol. 2020 Nov;144:103464. doi: 10.1016/j.fgb.2020.103464. Epub 2020 Sep 15.
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，也是研究真核微生物学和微生物发病机制的模式生物。新型隐球菌种复合体分为 A 型、D 型和 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。为了确定所研究的信号网络的多样化或保守性，通常在 A 型和 D 型参考菌株中进行重要途径的遗传研究。已经为 A 型参考菌株 H99 开发了许多新型隐球菌遗传工具，包括基于基因组特征鉴定的用于 DNA 整合的无基因“安全港”(SH)区域。然而，在 D 型菌株中尚未发现这种基因组安全港区域。在这里，我们利用现有的基因组、转录组和染色质数据，在 D 型参考菌株 JEC21 和 XL280 中鉴定了一个名为 SH3 的基因间区域。我们还设计了一个 sgRNA 和一个载体，通过 CRISPR-Cas9 系统促进任何外源基因整合到 SH3 中。我们发现，插入该区域的基因可以互补相应的基因缺失突变体。插入 SH3 的荧光报告基因也可以有效地表达。插入 SH3 本身不会改变相邻基因的表达，也不会影响新型隐球菌的生长或交配。因此，SH3 为 D 型菌株的遗传操作提供了资源，并将促进该种复合体中基因功能的比较分析。此外，在安全港区域的选择中纳入多组学数据可以帮助其他生物体的类似研究。
