IU: CRISPR/Cas9 editing to test and control genes implicated in influencing Aeromonas disease resistance in carp and salmon

Carp are by far the most highly produced species in aquaculture around the world and rohu carp is an important source of nutrition in India. Zebrafish are suitable model species to use for carp genetic research as they belong to the same family. Atlantic salmon production is an important source of jobs and income for Norway. But bacterial diseases have severe effects on the bio-economy of aquaculture in India and Norway. The objective of this project was to develop methods and knowledge that can be used to effectively boost the disease resistance to the most economically significant bacterial disease challenge affecting rohu carp in India. A closely related bacterial disease exists in Norway and vaccination of Atlantic salmon has reduced the problem. Both Aeromonas hydrophila and Aeromonas salmonicida affect fish health and cause mortality. The partners in the project have already collaborated to identify genes that putatively influence the disease resistance of rohu carp to A. hydrophila causing aeromoniasis. This project tested whether the expression of these genes influences the resistance of rohu carp to the disease. We also began work to determine whether the same genes were associated with disease resistance of Atlantic salmon to A. salmonicida which causes furunculosis. The research used gene editing technology to regulate the expression of the genes of interest to test whether they influenced the resistance of rohu carp and Atlantic salmon to Aeromonas sp. infection and to aeromoniasis or furunculosis. In doing so the project is generating knowledge that could be applied to boost selective breeding efforts or used to directly steer the disease resistance phenotype of particular individuals. The project also filled a gap in our knowledge about how to most effectively implement such genomic and gene editing technologies in selective breeding to have the greatest impact on the blue bioeconomy of both countries.

Output: Fish farmers, fish breeders, government and researchers in India and Norway are being informed about the potential for the use of gene editing to boost Aeromonas resistance through our publications, conference presentations and through CIFA’s work with rural communities in India. All knowledge, methodologies and tools generated from the project is being made publicly available through scientific journal publications, presentations at international conferences and aquaculture magazines. Detailed outputs include: -Methodologies established for using zebrafish as a model for studying Aeromonas disease resistance in rohu carp giving a foundation for future research and development of solutions. -Full characterisation of genes of interest giving the foundation for boosting gene effects on Aeromonas resistance through gene editing -Rohu and zebrafish gene knockout CRISPR fish produced. These fish will be challenge tested in 2025 with the disease to see if these edits boost Aeromonas resistance. -Earlier research on the role of these genes in giving Aeromonas resistance validated supporting the potential of using gene editing to benefit the survival of these fish. -Characterisation of homologous genes in Atlantic salmon completed laying a foundation for future research investigating the role of these genes in providing resistance to furunculosis in Atlantic salmon. -Evaluation by computer simulation of different methodologies for implementing gene editing giving important knowledge about ways of implementing gene editing to maximise the distribution of benefits to farmers. -Framework for the assessment of the application of gene editing to aquaculture developed and published so that future projects can weigh the risks and benefits of using this technology (on a case-by-case basis). Outcomes Impact: Possible future impact 1 - Survival rates and productivity in the presence of Aeromonas disease are greatly improved. Possible future impact 2 - Mass mortality due to the disease is eliminated among populations of gene edited fish.The future distribution and demonstration of improved stock occurs through state-run hatcheries and existing Self-Help Groups (consisting of rural women), so that the flow of benefits is focussed on women and their families in small rural communities, contributing to improved social and gender equity in the aquaculture industry. This distribution of the genetically improved stock supports efforts to combat food insecurity.

Carp are by far the most highly produced species in aquaculture around the world and rohu carp is an important source of nutrition in India. Zebrafish are a suitable model species to use for carp genetic research as they belong to the same family. Atlantic salmon production is an important source of jobs and income for Norway. But bacterial diseases have severe effects on the bio-economy of aquaculture in India and Norway. The objective of this project is to develop methods and knowledge that can be used to effectively boost the disease resistance to the most economically significant bacterial disease challenges affecting rohu carp in India and Atlantic salmon in Norway, Aeromonas hydrophila and Aeromonas salmonicida. The partners in the project have already collaborated to identify genes (including perforin and mucin) that putatively influence the disease resistance of rohu carp to A. hydrophila causing aeromoniasis. This project will test whether the expression of these genes influences the resistance of rohu carp to the disease. We will also begin work to determine whether the same genes are associated with disease resistance of Atlantic salmon to A. salmonicida which causes furunculosis. The proposed research will use the CRISPR/Cas9 gene editing technology to regulate the expression of perforin and mucin genes to test whether these genes influence the resistance of rohu carp and Atlantic salmon to Aeromonas sp. infection and to determine whether this can make fish more or less susceptible or resistant to aeromoniasis or furunculosis. In doing so the project will generate knowledge that can be applied to boost selective breeding efforts or could be used to directly steer the disease resistance phenotype of particular individuals. The project will also fill a gap in our knowledge about how to most effectively implement such genomic and gene editing technologies in selective breeding to have the greatest impact on the blue bioeconomy of both countries.