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。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2024-02-01 DOI:10.1089/crispr.2023.0070
Ruohui Li, Yue Xu, Fucun Wu, Zhangjie Peng, Jiulin Chan, Linlin Zhang
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引用次数: 0

摘要

太平洋鲍鱼是一种重要的水产养殖贝类,也是基础生物学研究的重要模型。然而,由于缺乏高效易用的基因编辑工具,对鲍鱼的研究受到了限制。在本文中,我们展示了利用 CRISPR-Cas9 在太平洋鲍鱼中进行高效基因敲除的方法。我们开发了一种高效的显微注射方法,将受精卵嵌套在低浓度琼脂糖凝胶中。我们确定了纤毛发育基因β-tubulin和光敏跨膜蛋白r-opsin为靶基因,并设计了高度特异性的sgRNA来修饰它们的基因组序列。对注射幼虫的β-tubulin和r-opsin基因的基因组区域进行了Sanger测序,发现了不同的基因组长片段缺失。原位杂交显示,在镶嵌突变体中,β-tubulin 和 r-opsin 的基因表达模式发生了显著变化。敲除鲍胚胎中的β-微管蛋白可有效影响纤毛的发育。扫描电子显微镜和游泳行为分析表明纤毛有缺陷,运动能力下降。此外,在鲍鱼胚胎中敲除 r-opsin 能有效影响眼点的表达和发育。总之,这项研究开发了一种易于使用的鲍鱼镶嵌基因敲除方案,这将使研究人员能够利用CRISPR-Cas9方法来研究未开发的鲍鱼生物学,并将为这一水产养殖物种带来新的育种方法。
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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.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, 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.
期刊最新文献
Engineering CjCas9 for Efficient Base Editing and Prime Editing. CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus. Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods. CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans. Genome Editing in Apicomplexan Parasites: Current Status, Challenges, and Future Possibilities.
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