Improving resistance to infectious salmon anaemia using genome editing: Novel approaches to tackling viral disease in aquaculture

Farmed salmon is a major source of high quality protein and fatty acids essential for human health. Salmon aquaculture is worth approximately £1Bn to the UK economy, and supports many rural and coastal communities. However, disease outbreaks have a major negative effect on salmon production and animal welfare. Infectious salmon anaemia (ISA) is one such disease, and is sometimes dubbed 'salmon flu' because it is caused by a virus (ISAV) that is similar in to influenza. At present, ISA is a notifiable disease in the UK, meaning farmers are obliged to cull their stock in the event of an outbreak. Vaccination and biosecurity cannot fully prevent outbreaks, and developing disease resistant strains of salmon is high priority.

Selective breeding can result in moderate improvements in disease resistance of salmon stocks and may take many generations. However, a revolutionary approach known as genome editing has potential to rapidly increase the rate at which disease resistant salmon can be produced. Genome editing involves the use of "gene scissors" to precisely cut the genome at a specific location, leading to small-scale targeted changes in the DNA sequence. In this proposal, genome editing technology will be used to investigate genes underlying resistance to ISAV, and potentially to produce a disease-resistant salmon.

The first stage of the project is to identify target genes that will be edited. This will be achieved by measuring the ISAV resistance in a selective breeding program. Genetic markers dispersed throughout the salmon genome will then be used to map individual genes that contribute to variation in resistance in the population. Salmon from resistant and susceptible families will also be sequenced and to identify candidate genes and mutations causing this genetic effect on resistance to ISAV.

In parallel to the 'forward genetic' approach described above, a 'reverse genetic' approach to identifying ISAV resistance candidates will be employed using cell culture models. A genome editing method known as CRISPR-Cas9 will be applied to destroy the function of key candidate ISAV resistance genes in the cell lines. Two methods of choosing candidate genes will be used. The first is based on prior knowledge of the biology of the interaction between the virus and the host cell, partly harnessing extensive research which has been performed on ISAV's close relative influenza. The second is to use the genes affecting natural resistance identified in the forward genetic screen described above. These edited cell lines will be infected with ISAV, and the impact of the edited gene on ISAV resistance and cellular response to infection will be assessed. This will build on an ongoing project to develop genome editing for salmon cell lines.

Finally, genome editing will be used in Atlantic salmon embryos to test the highest priority ISAV resistance genes, especially where knockout of the gene has an impact on resistance in cell culture. Targeted editing of the genes will be performed by microinjecting newly fertilised embryos, which will be reared until the freshwater fry stage. These edited embryos, and unedited controls from the same family, will be challenged with ISAV. The nature and frequency of the edited genes in the resistant and susceptible salmon will be measured.

This proposal has potential to create Atlantic salmon with resistance to a problematic viral disease (ISA) using a novel breeding technology. As such, it could have major animal welfare and economic impacts via prevention of outbreaks and subsequent culling of stocks. The approaches will be directly relevant to other viral disease in fish aquaculture. While the regulatory landscape for application of edited animals in food production is uncertain, a successful outcome of this proposal will provide a high profile example of the power of this technology to understand biology and to improve food security and animal health.

Salmon aquaculture is worth approximately £1 billion per annum to the UK economy and is the primary source of employment in many rural and coastal communities. Infectious disease epidemics constrain sustainability and expansion, and cause major negative economic and animal welfare impact. Infectious Salmon Anaemia Virus (ISAV) is a notifiable disease that requires culling of stocks for control. Improving genetic resistance of salmon stocks is a critical component of tackling ISAV. Genome editing raises the possibility of creating targeted and informed de novo disease resistance alleles (e.g. CD163 deletions for PRRS virus in pigs), and to test the causality of naturally-occurring variants, and potentially increase genetic gain in breeding programs. ISAV is a close relative of influenza, and knowledge of the conserved function of viral binding, internalisation and replication across orthomyxoviridae is one route to identifying target genes for editing.

This proposal will use forward and reverse genetics to identify genome editing targets for resistance to ISAV in Atlantic salmon. Harnessing large scale disease challenge and pedigree data provided by the industrial partner Benchmark, the team will map and characterise genes underpinning host resistance. Genome editing will be applied in cell culture to screen putative functional resistance candidate genes, with targets including 'naturally-occurring' variants, and de novo targets with a predicted key role in the ISAV host-cell interaction. These approaches will deliver high priority candidates for in vivo genome editing of salmon embryos, followed by an ISAV challenge to assess the resistance of the edited fish. The outcomes will include fundamental knowledge of mechanisms of genetic resistance to ISAV, and potential novel strategies to combat viral diseases impacting on salmon aquaculture. As such, it is aligned to the BBSRC priorities on 'Sustainably enhancing agricultural production' and 'Animal health'.

This proposal is applying novel and innovative methods with potential to develop an Atlantic salmon with resistance to ISAV as a result of genome editing. This outcome, and the research that leads to it, is likely to have high impact for academic, industry, government and societal groups.

It is likely that the major impact of the proposal will be medium and long term, due to uncertainty over the regulatory landscape and public appetite for genome editing technologies. However, high profile success stories are key, and this proposal has high potential to deliver such success stories. Use of genome editing to produce a disease resistant food fish has animal welfare, environmental and economic benefits.

It should also be noted that intermediate impacts of the research will include improved methods of genomic selection for disease resistance in aquaculture breeding, improved knowledge of host-pathogen interaction for ISAV, and optimised methods of using CRISPR-Cas9 for animal bioscience research. The following groups can expect positive impact from the proposal:

(i) UK and global aquaculture industry: ISAV has a major negative impact on aquaculture production, and is a notifiable disease in the UK. This proposal has potential for developing disease-resistant strains of salmon within a relatively short timeframe, and major breeding companies such as Benchmark view genome editing as a high priority R&D activity. While the regulatory landscape for genome editing is uncertain at present, achievement of the aims of this proposal is likely to be a driver to incorporate this technology to benefit food production.

(ii) Animal breeding industry: The improved genomic selection arising from the identification of functional genomic variants will have short term impact on selective breeding programmes. In the longer term, aquaculture species such as salmon are amenable to genome editing due to their high fecundity and easily visible embryos, and may be a good model for other species.

(iii) UK economy: This project has potential for long term impact for the UK economy via improved sustainable production of a high quality food product with reduced environmental impact. There will be direct contribution to the UK treasury via improved market share for the project partner Benchmark, and downstream impacts on fish farming companies, and the communities that depend on these industries.

(iv) Academic community: The impact for academic groups will include improved knowledge of the genetic basis of resistance to ISAV in salmon, specific genes and mutations impacting resistance, and improvement in methods of effective genome editing of cell lines and embryos.

(v) UK science capacity. This project will enable capacity and expertise for application of genome editing technology to address (applied) biological questions via research programs in academia and industry. This should lead to the UK taking a leading global position in the application of this technology for animal research and production.

(vi) Political and regulatory bodies. The regulatory landscape of genome editing currently precludes its use for food production in the short term. Successful achievement of the objectives of this project will influence ethical and regulatory frameworks to encourage full exploitation of genome editing in an informed and responsible manner.

(vii) General public and society. This project has potential to influence societal attitudes to genome editing in aquaculture and, more broadly, in food production animals. In the longer term, there will be direct benefits to society via improved economic stability and reduced environmental impact of the aquaculture industry.