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

在这个奖项下，PI将前往日本？北野俊（Jun Kitano）博士是世界上为数不多的几个在目标研究系统——三刺鱼（Threespine Stickleback）身上实施了这种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，以便在候选基因和性状之间建立因果关系。这项技术随后将被应用于PI自己的工作，将基因修改为宿主-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。Lohman将分享棘鱼细胞原代培养的方案，这样北野实验室就可以在他们未来的工作中使用细胞培养分析。这次合作进一步提高了两个实验室的技术水平，并将促进我们对脊椎动物免疫系统调节的理解。尽管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，但遗传作图本质上是相关的。基因表达谱通常识别出数百个基因作为群体或实验治疗的功能差异表达。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系统可以敲除任何基因，并且已经为模型系统开发得很好。然而，它仍然在许多新兴的模型系统中得到广泛的应用。例如，三棘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，但转基因和基因敲除工具很少被应用于验证遗传作图的推论。通过一项大型实验室感染实验，PI利用大规模RNAseq方法确定了宿主免疫表型的有希望的候选基因，这些基因介导棘鱼与鱼的相互作用，包括ROS的产生（N=99）。他建议通过CRISPR/Cas9敲除和敲入调控元件，然后测量ROS的产生，来建立这些候选基因与宿主ROS产生之间的因果关系。这项提议的工作不仅开发了用于棘鱼的CRISPR，而且还将建立似乎控制寄生虫生长的免疫基因的功能。该奖项隶属于东亚和太平洋暑期研究所项目，由美国国家科学基金会和日本科学促进会共同资助，支持一名美国研究生进行暑期研究。

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.