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

养殖三文鱼是人类健康所必需的优质蛋白质和脂肪酸的主要来源。三文鱼水产养殖业对英国经济的价值约为10亿英镑，并支持许多农村和沿海社区。然而，疾病暴发对三文鱼生产和动物福利产生了重大负面影响。传染性鲑鱼贫血(ISA)就是这样一种疾病，有时被称为“鲑鱼流感”，因为它是由一种与流感相似的病毒(ISAV)引起的。目前，ISA在英国是一种应通报的疾病，这意味着一旦爆发，农民有义务扑杀他们的牲畜。疫苗接种和生物安全不能完全防止暴发，开发抗病品系的鲑鱼是当务之急。选择性养殖可以适度提高鲑鱼种群的抗病能力，可能需要许多世代。然而，一种名为基因组编辑的革命性方法有可能迅速提高抗病三文鱼的生产速度。基因组编辑涉及到使用“基因剪刀”在特定位置精确切割基因组，从而导致DNA序列发生小规模的靶向改变。在这项计划中，基因组编辑技术将被用来研究对ISAV的抗性基因，并有可能生产出抗病的鲑鱼。该项目的第一阶段是确定将被编辑的目标基因。这将通过在选择育种计划中测量ISAV抗性来实现。然后，散布在鲑鱼基因组中的遗传标记将被用来定位导致种群抗药性变异的个别基因。来自抗性和易感家庭的三文鱼也将被测序，以确定导致对ISAV抗性产生这种遗传效应的候选基因和突变。与上文所述的正向遗传方法类似，将使用细胞培养模型来识别ISAV抗性候选的“反向遗传”方法。一种名为CRISPR-Cas9的基因组编辑方法将被应用于破坏细胞系中关键候选ISAV抗性基因的功能。将使用两种选择候选基因的方法。第一个是基于对病毒和宿主细胞之间相互作用的生物学的先验知识，部分利用了对ISAV近亲流感进行的广泛研究。第二种方法是利用上述正向遗传筛选中确定的影响自然抗性的基因。这些编辑后的细胞株将被ISAV感染，并将评估编辑后的基因对ISAV抗性和细胞感染反应的影响。这将建立在一个正在进行的为鲑鱼细胞系开发基因组编辑的项目上。最后，基因组编辑将用于大西洋鲑鱼胚胎，以测试最优先的ISAV抗性基因，特别是在细胞培养中基因敲除对耐药性有影响的情况下。基因的定向编辑将通过显微注射新受精的胚胎来进行，这些胚胎将被饲养到淡水鱼苗阶段。这些经过编辑的胚胎和来自同一家族的未经编辑的对照将受到ISAV的挑战。将测量抗性和易感三文鱼中编辑基因的性质和频率。这项提议有可能利用一种新的育种技术创造出对一种有问题的病毒病(ISA)具有抵抗力的大西洋鲑鱼。因此，它可以通过预防暴发和随后扑杀种群来产生重大的动物福利和经济影响。这些方法将与鱼类水产养殖中的其他病毒性疾病直接相关。虽然在食品生产中应用转基因动物的监管环境尚不确定，但这项提案的成功结果将提供一个引人注目的例子，说明这项技术在理解生物学和改善食品安全和动物健康方面的力量。

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.