Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
| Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of beta-Tubulin in the Cultured Oyster | |
| Chan, Jiulin1,2,5,6; Zhang, Wei1,2,3,5,6; Xu, Yue1,2,5,6; Xue, Yu1,2,3,5,6; Zhang, Linlin1,2,4,5,6 | |
| 2022-05-26 | |
| Source Publication | FRONTIERS IN MARINE SCIENCE
 |
| Volume | 9Pages:12 |
| Corresponding Author | Zhang, Linlin(linlinzhang@qdio.ac.cn) |
| Abstract | Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is enabling genetics improvement of productive traits in aquaculture. Previous studies have proven CRISPR/Cas9 to be feasible in oyster, one of the most cultured shellfish species. Here, we applied electroporation-based CRISPR/Cas9 knockout of beta-tubulin and built a highly efficient genome editing system in Crassostrea gigas angulate. We identified the beta-tubulin gene in the oyster genome and showed its spatiotemporal expression patterns by analyzing RNA-seq data and larval in situ hybridization. We further designed multiple highly specific guide RNAs (sgRNAs) for its coding sequences. Long fragment deletions were detected in the mutants by agarose gel electrophoresis screening and further verified by Sanger sequencing. In addition, the expression patterns of Cg beta-tubulin in the trochophore peritroch and intestinal cilia cells were altered in the mutants. Scanning electron microscopy represented shortened and almost complete depleted cilia at the positions of peritroch and the posterior cilium ring in Cg beta-tubulin mosaic knockout trochophores. Moreover, the larval swimming behavior in the mutants was detected to be significantly decreased by motility assay. These results demonstrate that beta-tubulin is sufficient to mediate cilia development and swimming behavior in oyster larvae. By applying Cg beta-tubulin as a marker gene, our study established CRISPR/Cas9-mediated mosaic mutagenesis technology based on electroporation, providing an efficient tool for gene function validation in the oyster. Moreover, our research also set up an example that can be used in genetic engineering breeding and productive traits improvement in oysters and other aquaculture species. |
| Keyword | mosaic mutagenesis CRISPR Cas9 long deletion gene editing gene knockout aquaculture breeding |
| DOI | 10.3389/fmars.2022.912409 |
| Indexed By | SCI |
| Language | 英语 |
| Funding Project | National Natural Science Foundation of China[41976088] ; Strategic Priority Research Program of the Chinese Academy of Sciences[XDB42000000] ; Key Development Project of Centre for Ocean Mega-Research of Science, Chinese Academy of Science[COMS2019R01] |
| WOS Research Area | Environmental Sciences & Ecology ; Marine & Freshwater Biology |
| WOS Subject | Environmental Sciences ; Marine & Freshwater Biology |
| WOS ID | WOS:000811826900001 |
| Publisher | FRONTIERS MEDIA SA |
| Citation statistics | |
| Document Type | 期刊论文 |
| Identifier | http://ir.qdio.ac.cn/handle/337002/179495 |
| Collection | 实验海洋生物学重点实验室 深海极端环境与生命过程研究中心 |
| Corresponding Author | Zhang, Linlin |
| Affiliation | 1.Chinese Acad Sci, Shandong Prov Key Lab Expt Marine Biol, Qingdao, Peoples R China 2.Qingdao Natl Lab Marine Sci & Technol, Lab Marine Biol & Biotechnol, Qingdao, Peoples R China 3.Qingdao Agr Univ, Coll Life Sci, Qingdao, Peoples R China 4.Univ Chinese Acad Sci, Coll Marine Sci, Beijing, Peoples R China 5.Chinese Acad Sci, Qingdao, Peoples R China 6.Chinese Acad Sci, Inst Oceanol, Ctr Deep Sea Res, Ctr Ocean Mega Sci, Qingdao, Peoples R China |
| First Author Affilication | Chinese Acad Sci, Inst Oceanol, Ctr Deep Sea Res |
| Corresponding Author Affilication | Chinese Acad Sci, Inst Oceanol, Ctr Deep Sea Res |
| Recommended Citation GB/T 7714 | Chan, Jiulin,Zhang, Wei,Xu, Yue,et al. Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of beta-Tubulin in the Cultured Oyster[J]. FRONTIERS IN MARINE SCIENCE,2022,9:12. |
| APA | Chan, Jiulin,Zhang, Wei,Xu, Yue,Xue, Yu,&Zhang, Linlin.(2022).Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of beta-Tubulin in the Cultured Oyster.FRONTIERS IN MARINE SCIENCE,9,12. |
| MLA | Chan, Jiulin,et al."Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of beta-Tubulin in the Cultured Oyster".FRONTIERS IN MARINE SCIENCE 9(2022):12. |
| Files in This Item: | ||||||
| File Name/Size | DocType | Version | Access | License | ||
| fmars-09-912409.pdf（11733KB） | 期刊论文 | 出版稿 | 延迟开放 | CC BY-NC-SA | View 2023-7-1后可获取 | |
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.
Edit Comment