Efficient Single-Gene and Gene Family Editing in the Apicomplexan Parasite Eimeria tenella Using CRISPR-Cas9
Efficient Single-Gene and Gene Family Editing in the Apicomplexan Parasite Eimeria tenella Using CRISPR-Cas9
复制标题
使用 CRISPR-Cas9 对顶复门寄生虫艾美耳球虫进行高效单基因和基因家族编辑
DOI：
10.3389/fbioe.2020.00128
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发表时间：
2020-02-25
影响因子：
5.7
通讯作者：
Liu, Xianyong
中科院分区：
文献类型：
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作者：
Hu, Dandan;Tang, Xinming;Liu, Xianyong
艾美耳球虫属致病原虫，宿主范围广泛，是引起家禽球虫病的原因，给养禽业造成巨大的经济损失。这些寄生虫编码大约 8000 个基因的基因组，控制着无性复制、性分化、传播和毒力的高度协调的生命周期。然而，大多数这些基因的功能和生理重要性仍然未知，这主要是由于缺乏对基因功能进行系统分析的工具。在这里，我们报告了 CRISPR-Cas9 基因编辑技术在柔嫩艾美耳球虫中的首次应用，用于单基因水平的基因功能分析以及整个基因家族的系统功能分析。使用组成型表达 Cas9 的转基因系，我们通过非同源末端连接以及引导同源重组证明了成功且有效的功能丧失。应用这种方法来研究 EtGRA9 的定位表明，该基因编码一种分泌蛋白，其细胞分布在生命周期中发生变化。使用这种方法对 ApiAp2 转录因子基因家族进行系统性破坏表明，这种寄生虫表达的 33 个因子中有 23 个对于宿主的发育和生存至关重要。因此，我们的数据使 CRISPR-Cas9 成为艾美耳球虫基因编辑的强大技术，并将为其基因组的系统功能分析奠定基础，以了解其生物学和发病机制，并使识别和验证球虫病治疗的新靶点成为可能。
Eimeria species are pathogenic protozoa with a wide range of hosts and the cause of poultry coccidiosis, which results in huge economic losses to the poultry industry. These parasites encode a genome of similar to 8000 genes that control a highly coordinated life cycle of asexual replication and sexual differentiation, transmission, and virulence. However, the function and physiological importance of the large majority of these genes remain unknown mostly due to the lack of tools for systematic analysis of gene functions. Here, we report the first application of CRISPR-Cas9 gene editing technology in Eimeria tenella for analysis of gene function at a single gene level as well as for systematic functional analysis of an entire gene family. Using a transgenic line constitutively expressing Cas9, we demonstrated successful and efficient loss of function through non-homologous end joining as well as guided homologous recombination. Application of this approach to the study of the localization of EtGRA9 revealed that the gene encodes a secreted protein whose cellular distribution varied during the life cycle. Systematic disruption of the ApiAp2 transcription factor gene family using this approach revealed that 23 of the 33 factors expressed by this parasite are essential for development and survival in the host. Our data thus establish CRISPR-Cas9 as a powerful technology for gene editing in Eimeria and will set the stage for systematic functional analysis of its genome to understand its biology and pathogenesis, and will make it possible to identify and validate new targets for coccidiosis therapy.