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

Lohman Brian K, Austin TX

Investigators

Abstract

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.