Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of eggs at the one-cell stage was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 50% of injected eggs. Further analysis of the subcloned mutant gene sequences revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in G0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful resource for future experimental studies that aim to elucidate gene function in anemonefishes and reef fishes in general.

中文翻译：

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CRISPR / Cas9介导的双等位基因G0 anemonefish（Amphiprion ocellaris）突变体的产生

基因组操纵是阐明发育，生理学和行为各方面的分子途径的有用方法。但是，缺乏适用于礁鱼的基因编辑工具，这意味着许多独特特征的遗传基础尚待研究。一组非常适合应用此技术的标志性鱼类是海葵鱼（Amphiprioninae），因为它们与海葵，顺序雌雄同体，复杂的社会等级制度，皮肤模式发展和视力共生而​​受到广泛研究，并且在水族箱中相对容易繁殖。在这项研究中，我们开发了一种基因编辑协议，可将CRISPR / Cas9系统应用于假小丑海葵鱼，双锯鱼ocellaris。在一个细胞阶段显微注射卵用于证明我们的CRISPR / Cas9方法在两个不同的靶位点上的成功使用：视力涉及的视紫红质样2B视蛋白编码基因（RH2B）和酪氨酸酶产生基因（tyr）参与黑色素的生产。通过分析球孢曲霉胚中测序的靶基因区域，发现摄取量高达注射卵的50％。对亚克隆突变体基因序列的进一步分析表明，我们的方法在ocellaris胚胎中产生双等位基因突变的效率为75％至100％。此外，我们清楚地显示出轮胎功能丧失突变体胚胎表现出典型的低黑素表型。该协议旨在为将来的实验研究提供有用的资源，这些实验旨在阐明海葵鱼和暗礁鱼的基因功能。