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

养殖鲑鱼是人体健康所必需的高质量蛋白质和脂肪酸的主要来源。鲑鱼养殖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，疾病的爆发对鲑鱼生产和动物福利产生了重大的负面影响。传染性鲑鱼贫血（伊萨）就是这样一种疾病，有时被称为“鲑鱼流感”，因为它是由一种类似于流感的病毒（ISAV）引起的。目前，伊萨在英国是一种必须报告的疾病，这意味着农民有义务在爆发时扑杀他们的股票。疫苗接种和生物安全不能完全预防疫情，开发鲑鱼抗病品系是当务之急。选择性育种可以适度提高鲑鱼种群的抗病能力，可能需要许多代。然而，一种被称为基因组编辑的革命性方法有可能快速提高抗病鲑鱼的生产速度。基因组编辑涉及使用“基因剪刀”在特定位置精确切割基因组，导致DNA序列的小规模靶向变化。在该提案中，基因组编辑技术将用于研究抗ISAV的基因，并有可能产生抗病鲑鱼。该项目的第一阶段是确定将被编辑的靶基因。这将通过在选择性育种计划中测量ISAV抗性来实现。然后，分散在鲑鱼基因组中的遗传标记将用于绘制有助于种群抗性变化的个体基因。也将对来自抗性和易感家族的鲑鱼进行测序，以鉴定引起对ISAV抗性的这种遗传效应的候选基因和突变。与上述“正向遗传”方法平行，将使用细胞培养模型采用“反向遗传”方法来鉴定ISAV抗性候选者。一种被称为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的近亲流感进行的广泛研究。第二种方法是使用在上述正向遗传筛选中鉴定的影响天然抗性的基因。将用ISAV感染这些编辑的细胞系，并评估编辑的基因对ISAV抗性和细胞对感染的反应的影响。这将建立在一个正在进行的项目，以开发鲑鱼细胞系的基因组编辑。最后，将在大西洋鲑鱼胚胎中使用基因组编辑来测试最优先的ISAV抗性基因，特别是在基因敲除对细胞培养中的抗性有影响的情况下。基因的定向编辑将通过显微注射新受精的胚胎进行，这些胚胎将被饲养到淡水鱼苗阶段。这些编辑的胚胎和来自同一家族的未编辑对照将用ISAV进行攻毒。将测量抗性和易感鲑鱼中编辑基因的性质和频率。这项提议有可能使用一种新的育种技术创造出对有问题的病毒性疾病（伊萨）具有抗性的大西洋鲑鱼。因此，它可能通过预防疫情爆发和随后的扑杀种群产生重大的动物福利和经济影响。这些方法将与鱼类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编辑动物的监管前景尚不确定，但该提案的成功结果将提供一个引人注目的例子，说明该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.