{"title":"在养殖的太平洋鲍鱼(Haliotis discus hannai)中进行易于使用的 CRISPR-Cas9 基因组编辑。","authors":"Ruohui Li, Yue Xu, Fucun Wu, Zhangjie Peng, Jiulin Chan, Linlin Zhang","doi":"10.1089/crispr.2023.0070","DOIUrl":null,"url":null,"abstract":"<p><p>The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene <i>β-tubulin</i> and light-sensitive transmembrane protein <i>r-opsin</i> as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of <i>β-tubulin</i> and <i>r-opsin</i> genes from injected larvae identified various genomic long-fragment deletions. <i>In situ</i> hybridization showed gene expression patterns of <i>β-tubulin</i> and <i>r-opsin</i> were significantly altered in the mosaic mutants. Knocking out <i>β-tubulin</i> in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of <i>r-opsin</i> in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 1","pages":"41-52"},"PeriodicalIF":3.7000,"publicationDate":"2024-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (<i>Haliotis discus hannai</i>).\",\"authors\":\"Ruohui Li, Yue Xu, Fucun Wu, Zhangjie Peng, Jiulin Chan, Linlin Zhang\",\"doi\":\"10.1089/crispr.2023.0070\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene <i>β-tubulin</i> and light-sensitive transmembrane protein <i>r-opsin</i> as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of <i>β-tubulin</i> and <i>r-opsin</i> genes from injected larvae identified various genomic long-fragment deletions. <i>In situ</i> hybridization showed gene expression patterns of <i>β-tubulin</i> and <i>r-opsin</i> were significantly altered in the mosaic mutants. Knocking out <i>β-tubulin</i> in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of <i>r-opsin</i> in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.</p>\",\"PeriodicalId\":54232,\"journal\":{\"name\":\"CRISPR Journal\",\"volume\":\"7 1\",\"pages\":\"41-52\"},\"PeriodicalIF\":3.7000,\"publicationDate\":\"2024-02-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"CRISPR Journal\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1089/crispr.2023.0070\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"GENETICS & HEREDITY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"CRISPR Journal","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1089/crispr.2023.0070","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"GENETICS & HEREDITY","Score":null,"Total":0}
Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (Haliotis discus hannai).
The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene β-tubulin and light-sensitive transmembrane protein r-opsin as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of β-tubulin and r-opsin genes from injected larvae identified various genomic long-fragment deletions. In situ hybridization showed gene expression patterns of β-tubulin and r-opsin were significantly altered in the mosaic mutants. Knocking out β-tubulin in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of r-opsin in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.
CRISPR JournalBiochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍:
In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR.
Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.