Lebret Veronique, Duret Cecile, Herpin Amaury, Rescan Pierre-Yves
LPGP Fish Physiology and Genomics, Institut National de Recherche pour l'Agriculture l'Alimentation et l'Environnement Centre Bretagne-Normandie, Rennes, Brittany, 3542, France.
Open Res Eur. 2025 Feb 25;4:235. doi: 10.12688/openreseurope.18223.2. eCollection 2024.
Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals.
Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA.
Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele.
These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).
基因编辑技术为改善水产养殖中的重要性状提供了新机遇。鱼肉的致敏性是水产养殖中的一个主要问题。小清蛋白（Parv）是最常见的鱼类过敏原。例如，在鲑科鱼类中，单一的小清蛋白β-1蛋白（parvb1）已被确定为特定患者的过敏原。因此，培育携带两个从致敏肽编码区域缺失的parvb1等位基因的鳟鱼，可以预防这些敏感个体发生过敏反应。
在此，我们描述了Crispr/cas9系统的应用，试图删除编码致敏肽的parvb1外显子2，或者用不编码致敏肽的小清蛋白β-2蛋白（parvb2）的外显子2替换parvb1的外显子2。通过使用专门设计的双链供体DNA的微同源性介导的末端连接（MMEJ）敲入来实现外显子跳跃和交换。
对大约200尾源自注射了用于外显子2跳跃的供体DNA的鱼卵的F0幼鱼进行基因分型，仅鉴定出一条携带缺失外显子2的等位基因的鱼。对大约150尾源自注射了用于外显子2交换的供体DNA的鱼卵的幼鱼进行基因分型，未得到任何携带预期修饰等位基因的鳟鱼。
这些初步结果表明在养殖鱼类中进行MMEJ KI实验存在潜在困难。最后，在欧洲议会就可能修订现行法律以确定经基因组编辑改造的养殖动物的法律地位展开激烈辩论的背景下，进一步讨论了水产养殖中的新基因组技术。基因编辑、微同源性介导的末端连接敲入、小清蛋白、致敏性、鳟鱼和转基因生物（GMOs）。
