Guangyuan Liang , Baoyue Lu , Shengfei Dai, Minghui Li, Jiawen Yao, Hao Liu, Xiayue Liu, Xingyong Liu, Deshou Wang
{"title":"利用CRISPR/Cas9介导的多基因突变培育无色透明罗非鱼。","authors":"Guangyuan Liang , Baoyue Lu , Shengfei Dai, Minghui Li, Jiawen Yao, Hao Liu, Xiayue Liu, Xingyong Liu, Deshou Wang","doi":"10.1016/j.nbt.2025.07.009","DOIUrl":null,"url":null,"abstract":"<div><div>Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form <em>tyrb;csf1ra</em> double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of <em>pnp4a</em> and <em>tfec</em> using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in <em>pnp4a</em> mutants but not in <em>tfec</em> mutants in the later stage. In addition, we also found that <em>tfec</em> mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of <em>tyrb</em>;<em>csf1ra</em>;<em>pnp4a</em> (named <em>amber</em>) and <em>tyrb</em>;<em>csf1ra</em>;<em>tfec</em> (named <em>ruby</em>), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.</div></div>","PeriodicalId":19190,"journal":{"name":"New biotechnology","volume":"89 ","pages":"Pages 163-176"},"PeriodicalIF":4.9000,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Creation of colorless transparent tilapia using CRISPR/Cas9 mediated multi-gene mutation\",\"authors\":\"Guangyuan Liang , Baoyue Lu , Shengfei Dai, Minghui Li, Jiawen Yao, Hao Liu, Xiayue Liu, Xingyong Liu, Deshou Wang\",\"doi\":\"10.1016/j.nbt.2025.07.009\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form <em>tyrb;csf1ra</em> double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of <em>pnp4a</em> and <em>tfec</em> using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in <em>pnp4a</em> mutants but not in <em>tfec</em> mutants in the later stage. In addition, we also found that <em>tfec</em> mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of <em>tyrb</em>;<em>csf1ra</em>;<em>pnp4a</em> (named <em>amber</em>) and <em>tyrb</em>;<em>csf1ra</em>;<em>tfec</em> (named <em>ruby</em>), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.</div></div>\",\"PeriodicalId\":19190,\"journal\":{\"name\":\"New biotechnology\",\"volume\":\"89 \",\"pages\":\"Pages 163-176\"},\"PeriodicalIF\":4.9000,\"publicationDate\":\"2025-07-31\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"New biotechnology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1871678425000767\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"New biotechnology","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1871678425000767","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
Creation of colorless transparent tilapia using CRISPR/Cas9 mediated multi-gene mutation
Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form tyrb;csf1ra double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of pnp4a and tfec using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in pnp4a mutants but not in tfec mutants in the later stage. In addition, we also found that tfec mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of tyrb;csf1ra;pnp4a (named amber) and tyrb;csf1ra;tfec (named ruby), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.
期刊介绍:
New Biotechnology is the official journal of the European Federation of Biotechnology (EFB) and is published bimonthly. It covers both the science of biotechnology and its surrounding political, business and financial milieu. The journal publishes peer-reviewed basic research papers, authoritative reviews, feature articles and opinions in all areas of biotechnology. It reflects the full diversity of current biotechnology science, particularly those advances in research and practice that open opportunities for exploitation of knowledge, commercially or otherwise, together with news, discussion and comment on broader issues of general interest and concern. The outlook is fully international.
The scope of the journal includes the research, industrial and commercial aspects of biotechnology, in areas such as: Healthcare and Pharmaceuticals; Food and Agriculture; Biofuels; Genetic Engineering and Molecular Biology; Genomics and Synthetic Biology; Nanotechnology; Environment and Biodiversity; Biocatalysis; Bioremediation; Process engineering.