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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 PublicationFRONTIERS IN MARINE SCIENCE
Volume9Pages:12
Corresponding AuthorZhang, Linlin(linlinzhang@qdio.ac.cn)
AbstractGenome 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.
Keywordmosaic mutagenesis CRISPR Cas9 long deletion gene editing gene knockout aquaculture breeding
DOI10.3389/fmars.2022.912409
Indexed BySCI
Language英语
Funding ProjectNational 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 AreaEnvironmental Sciences & Ecology ; Marine & Freshwater Biology
WOS SubjectEnvironmental Sciences ; Marine & Freshwater Biology
WOS IDWOS:000811826900001
PublisherFRONTIERS MEDIA SA
Citation statistics
Document Type期刊论文
Identifierhttp://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 AuthorZhang, Linlin
Affiliation1.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 AffilicationChinese Acad Sci, Inst Oceanol, Ctr Deep Sea Res
Corresponding Author AffilicationChinese 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.
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