Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Shandong Provincial Maternal and Child Health Care Hospital, 250014, Jinan, China.
Department of Biological Sciences, National University of Singapore, 14 Sciences Drive 4, 117558, Singapore, Singapore.
Mar Biotechnol (NY). 2023 Apr;25(2):281-290. doi: 10.1007/s10126-023-10204-9. Epub 2023 Mar 14.
Albinism is the most common color variation described in fish and is a fascinating trait of some ornamental fish species. Albino mutants can be generated by knocking out core genes affecting melanin synthesis like slc45a2 in several fish species. However, genetic mutation remains challenging for species with unknown genome information. In this study, we generated albino mutants in two selected ornamental fish species, royal farlowella (Sturisoma panamense), and redhead cichlid (Vieja melanura). For this purpose, we carried out phylogenetic analyses of fish slc45a2 sequences and identified a highly conserved region among different fish species. A pair of degenerate primers spanning this region was designed and used to amplify a conserved slc45a2 fragment of 340 bp from the two fish species. Based on the amplified sequences, a target site in the 6 exon was used for designing guide RNA and this targeted site was first verified by the CRISPR/Cas9 system in the zebrafish (Danio rerio) model for the effectiveness. Then, specific guide RNAs were designed for the two ornamental fish species and tested. Most of the injected larvae completely lost black pigment over the whole body and eyes. DNA sequencing confirmed a high degree of mutation at the targeted site. Overall, we described a fast and efficient method to generate albino phenotype in fish species by targeting the conserved 6 exon of slc45a2 gene for genome editing via CRISPR/Cas9 and this approach could be a new genetic tool to generate desirable albino ornamental fish.
白化病是鱼类中最常见的颜色变异，是一些观赏鱼类的迷人特征。通过敲除影响黑色素合成的核心基因，如 slc45a2，可以在几种鱼类中产生白化突变体。然而，对于基因组信息未知的物种，遗传突变仍然具有挑战性。在这项研究中，我们在两种选定的观赏鱼中生成了白化突变体，即皇家副丽鱼（Sturisoma panamense）和红头丽鱼（Vieja melanura）。为此，我们对鱼类 slc45a2 序列进行了系统发育分析，并在不同鱼类中鉴定出一个高度保守的区域。设计了一对跨越该区域的简并引物，用于从这两种鱼类中扩增 340bp 的保守 slc45a2 片段。基于扩增序列，在 6 号外显子中使用靶位点设计 guide RNA，并首先在斑马鱼（Danio rerio）模型中的 CRISPR/Cas9 系统中验证该靶位点的有效性。然后，针对这两种观赏鱼设计了特定的 guide RNA 并进行了测试。大多数注射的幼虫全身和眼睛完全失去了黑色色素。DNA 测序证实靶位点的突变程度很高。总的来说，我们描述了一种通过靶向 slc45a2 基因的保守 6 号外显子，利用 CRISPR/Cas9 进行基因组编辑，在鱼类中产生白化表型的快速有效的方法，这种方法可以成为一种新的遗传工具，用于产生理想的白化观赏鱼。
