EAPSI: Identifying the Transcriptomic Basis of Recently Evolved Host Resistance in a Model Host-Parasite System through Genome Editing with CRISPR/Cas 9

EAPSI：通过 CRISPR/Cas 9 基因组编辑识别模型宿主-寄生虫系统中最近进化的宿主抗性的转录组学基础

• 批准号: 1613505
• 负责人: Brian Lohman
• 金额: $ 0.54万
• 依托单位: Lohman Brian K
• 依托单位国家: 美国
• 项目类别: Fellowship Award
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1613505&HistoricalAwards=false
• 关键词: EAPSI; Identifying; Transcriptomic; Basis; Recently

Under this award the PI will travel to Japan?s National Institute of Genetics to learn cutting-edge methods for genome editing under Dr. Jun Kitano, one of only a few investigators in the world to have implemented such technology in the target study system, the Threespine Stickleback fish. The research will focus on how to make alterations to the stickleback genome in order to establish a causal link between candidate genes and traits. The technology will then be applied to the PI's own work modifying genes as potential mediators of host-parasite interactions. These genes, vital to the host immune system, are involved in the production and regulation of reactive oxygen species, a strong innate immune response. Lohman will share protocols for primary cell culture of stickleback cells, so that the Kitano lab can use cell culture assays in their future work. This collaboration furthers the technical repertoire of both labs, and will advance our understanding of the regulation of the vertebrate immune system. Although modern molecular genetics and statistics have become incredibly effective at identifying candidate genes for virtually any phenotype, genetic mapping is inherently correlational. Gene expression profiling often identifies hundreds of genes as differentially expressed as a function of population or experimental treatment. Establishing causality of candidate genes requires functional testing without perturbing any other genes. Molecular biology offers several solutions to this problem. The CRISPR/Cas9 system enables knockouts of any gene and has been well developed for model systems. However, it remains to be widely applied in many up-and-coming model systems. For example, the threespine stickleback is a powerful model system for studying evolutionary genetics (including evolutionary immunology), but transgenic and knockout tools have rarely been applied to validate inferences from genetic mapping. From a large lab infection experiment, the PI has identified promising candidate genes for host immune phenotypes which mediate stickleback-cestode interactions, including ROS production, using a large scale RNAseq approach (N=99). He proposes to establish the causal relationship between these candidate genes and host ROS production though both CRISPR/Cas9 knock outs and knock ins of regulatory elements, followed by measurements of ROS production. The proposed work not only develops CRISPR for use in stickleback, but will also establish the function of immune genes that appear to control parasite growth.This award under the East Asia and Pacific Summer Institutes program supports summer research by a U.S. graduate student and is funded jointly by NSF and the Japan Society for the Promotion of Science.

在这个奖项下，PI将前往日本？日本国立遗传学研究所在Jun Kitano博士的领导下学习基因组编辑的尖端方法，Jun Kitano博士是世界上为数不多的在目标研究系统Threespine Stickleback鱼中实施此类技术的研究人员之一。这项研究将集中在如何改变棘鱼的基因组，以建立候选基因和性状之间的因果关系。然后，该技术将应用于PI自己的工作，修改基因作为宿主-寄生虫相互作用的潜在介质。这些基因对宿主免疫系统至关重要，参与活性氧的产生和调节，这是一种强烈的先天免疫反应。洛曼将分享刺鱼细胞的原代细胞培养方案，以便北野实验室在未来的工作中使用细胞培养试验。这种合作进一步促进了两个实验室的技术库，并将推进我们对脊椎动物免疫系统调节的理解。虽然现代分子遗传学和统计学在识别几乎任何表型的候选基因方面已经变得非常有效，但遗传图谱具有内在的相关性。基因表达谱通常鉴定出数百个基因作为群体或实验治疗的函数而差异表达。建立候选基因的因果关系需要在不干扰任何其他基因的情况下进行功能测试。分子生物学为这个问题提供了几种解决方案。CRISPR/Cas9系统能够敲除任何基因，并且已经很好地开发用于模型系统。然而，它仍有待于在许多新兴的模型系统中得到广泛应用。例如，三棘鱼是研究进化遗传学（包括进化免疫学）的一个强大的模型系统，但转基因和敲除工具很少被应用于验证遗传作图的推论。从大型实验室感染实验中，PI已经使用大规模RNAseq方法（N=99）鉴定了介导棘鱼-绦虫相互作用（包括ROS产生）的宿主免疫表型的有希望的候选基因。他建议通过CRISPR/Cas9敲除和敲入调控元件，然后测量ROS产生，来建立这些候选基因与宿主ROS产生之间的因果关系。这项研究计划不仅开发用于棘鱼的CRISPR，而且还将建立似乎控制寄生虫生长的免疫基因的功能。该奖项属于东亚和太平洋夏季研究所计划，支持美国研究生的夏季研究，由NSF和日本科学促进会共同资助。