An intergenic "safe haven" region in Cryptococcus neoformans serotype D genomes.
An intergenic "safe haven" region in Cryptococcus neoformans serotype D genomes.
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DOI：
10.1016/j.fgb.2020.103464
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发表时间：
2020-11
期刊：
影响因子：
--
通讯作者：
Lin X
中科院分区：
文献类型：
--
作者：
Fan Y;Lin X
新型隐球菌是一种机会性人类真菌病原体，是研究真核微生物学和微生物致病性的模式生物。新型隐球菌物种复合体分为血清型A、血清型D和AD杂交型，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性或保守性，通常在血清型A和D参考菌株中对重要途径进行遗传学研究。已经为新型隐球菌血清型A参考菌株H99开发了许多遗传工具，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，在血清型D菌株中尚未鉴定出这样的基因组安全港区域。在此，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能够互补相应的基因缺失突变体。插入SH3的荧光报告基因也能高效表达。SH3自身的插入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为血清型D菌株的遗传操作提供了一种资源，并将促进该物种复合体中基因功能的比较分析。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。
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 express 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.
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影响因子：
3.3
作者：
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通讯作者：
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