F L Almeida, K O Skaftnesmo, E Andersson, L Kleppe, R B Edvardsen, B Norberg, P G Fjelldal, T J Hansen, R W Schulz, A Wargelius
Research Group Reproduction and Developmental Biology, Institute of Marine Research, Bergen, Norway.
Embrapa Amazonia Ocidental, Manaus, Brazil.
Front Cell Dev Biol. 2022 Sep 19;10:977779. doi: 10.3389/fcell.2022.977779. eCollection 2022.
Genetic introgression of farmed salmon into wild populations can damage the genetic integrity of wild stocks and is therefore considered as an environmental threat. One possible solution is to induce sterility in farmed salmon. We have searched for proteins potentially essential for germline survival in Atlantic salmon. One of these is the argonaute protein Piwil1, known to be required for germ cell survival. To examine Piwil1 function in salmon, we induced indels in the N domain by CRISPR-Cas9. The encoded domain is present in all vertebrate Piwi proteins and has been linked to Tdrd1 protein interaction and PAZ lobe structure. The F0 founder generation of crispant males and females displayed a mosaic pattern of mutations, exhibiting highly mutated alleles (53%-97%) in their fin gDNA samples. In general, crispants carried germ cells, went through puberty and became fertile, although a transient and partial germ cell loss and delays during the spermatogenic process were observed in many male crispants, suggesting that Piwil1 functions during salmon spermatogenesis. By crossing highly mutated F0 founders, we produced F1 fish with a mixture of: loss-of-function alleles (); functional in frame mutated alleles ( ) and wt alleles (). In F1, all fish lacked germ cells, while siblings showed normal ovaries and testes. Yet, most juvenile F1 males and females displayed an intermediate phenotype with a higher somatic/germ cell ratio without an increase in germ cell apoptosis, suggestive of a gene dose effect on the number of germ cells and/or insufficient replacement of lost germ cells in heterozygous fish. Interestingly, the two longest in-frame indels in the N domain also ensured germ cell loss. Hence, the loss of 4-6 aa in this region - may result in crucial changes of the protein structure, potentially affecting piRNA binding of the PAZ lobe, and/or affecting the binding of Piwil1 interacting proteins such as Tdrd protein, with critical consequences for the survival of primordial germ cells. In conclusion, we show that loss of leads to loss of germ cells in salmon and that part of the N domain of Piwil1 is crucial for its function.
养殖三文鱼基因渗入野生种群会损害野生种群的遗传完整性，因此被视为一种环境威胁。一种可能的解决办法是诱导养殖三文鱼不育。我们在大西洋三文鱼中寻找对生殖系存活可能至关重要的蛋白质。其中一种是AGO蛋白Piwil1，已知它是生殖细胞存活所必需的。为了研究Piwil1在三文鱼中的功能，我们通过CRISPR-Cas9在N结构域诱导插入缺失。编码的结构域存在于所有脊椎动物的Piwi蛋白中，并且与Tdrd1蛋白相互作用和PAZ叶结构有关。F0代的crispant雄性和雌性呈现出突变的嵌合模式，其鳍部基因组DNA样本中显示出高度突变的等位基因（53%-97%）。一般来说，crispant携带生殖细胞，经历青春期并变得可育，尽管在许多雄性crispant中观察到生殖细胞有短暂和部分损失以及精子发生过程中的延迟，这表明Piwil1在三文鱼精子发生过程中发挥作用。通过杂交高度突变的F0代亲本，我们产生了具有以下混合类型的F1代鱼：功能缺失等位基因（）、框内功能突变等位基因（）和野生型等位基因（）。在F1代中，所有 鱼都缺乏生殖细胞，而 同胞显示出正常的卵巢和睾丸。然而，大多数幼年F1代雄性和雌性呈现出中间表型，体细胞/生殖细胞比例较高，且生殖细胞凋亡没有增加，这表明基因剂量对生殖细胞数量有影响，和/或杂合鱼中丢失的生殖细胞没有得到充分补充。有趣的是，N结构域中两个最长的框内插入缺失也确保了生殖细胞的丢失。因此，该区域4-6个氨基酸的缺失——可能导致蛋白质结构的关键变化，潜在地影响PAZ叶的piRNA结合，和/或影响Piwil1相互作用蛋白（如Tdrd蛋白）的结合，对原始生殖细胞的存活产生关键影响。总之，我们表明 的缺失导致三文鱼生殖细胞的丢失，并且Piwil1的N结构域的一部分对其功能至关重要。
