Gene editing for elucidating gene function and refining genomic selection for CMS resistance in Atlantic salmon

Summary of microinjection for the main trial To validate a candidate gene potentially involved in increased resistance to piscine myocarditis virus (PMCV), a gene knock-out experiment was conducted using CRISPR/Cas9 gene editing technology on Atlantic salmon eggs. Approximately 6,000 eggs and milt were provided by Mowi Genetics, and the experiment was carried out at Nofima’s research station in Sunndalsøra. Immediately after fertilization, the eggs were incubated at 6°C until the first cell division was visible. At the 1-cell stage, around 3,000 eggs were microinjected with a CRISPR/Cas9 construct (IDT) targeting the PMCV resistance gene. For visual screening purposes, a second construct targeting a pigmentation gene in Atlantic salmon was co-injected. The remaining eggs served as controls, divided into two groups: Control Group 1 consisted of eggs homozygous for the CMS-strong allele, while Control Group 2 included a mix of eggs homozygous and heterozygous for the CMS-weak allele. When the fish reached approximately 20 grams, tissue samples were collected for sequencing to assess CRISPR editing efficiency. Sequencing confirmed the presence of individuals with varying degrees of gene edits, and those with higher editing levels were retained. Once the fish reach an average body weight of 90 grams, a PMCV challenge test will be conducted at Veso Aqualab.

Gene editing technologies are developing at a faster pace than ever and open new opportunities for breeding to improve production efficiency and diseases resistance. Cardiomyopathy syndrome (CMS) is an important disease for the aquaculture industry in Norway because of economic loss and welfare implications. To mitigate the impact of the disease selective breeding is practiced, however, it is challenged by veterinary restrictions and expensive challenge tests. Genomic studies for the trait have located quantitative trait loci (QTL) with strong signal on chromosome 27. The use of state-of-the-art gene editing technologies for improvement of CSM resistance is likely to be highly beneficial in this case. The project will identify potential genes and causative mutations affecting CMS resistance, and evaluates their function by in-vitro cardiomyocyte cell culture. Further, the proposal will develop breeding strategies that effectively employ gene editing to develop CMS resistant salmon, and a special scheme to deliver edited genes to multipliers tiers, which produces fingerlings that are grown for production, will be designed. The ethical, social and legal implications of developing CMS resistant salmon using gene editing will be determined. Dynamic RRI (responsible research and innovation) processes, in particular active stakeholder involvement, are used to promote an active attitude towards RRI in gene editing. The project will create a knowledge base and example for the industry regarding disease management through state-of-the-art gene editing, this will greatly improve the bioeconomy and welfare of Atlantic salmon production.