Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster
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 electropo...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Marine Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmars.2022.912409/full |
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| author | Jiulin Chan Jiulin Chan Wei Zhang Wei Zhang Wei Zhang Yue Xu Yue Xu Yu Xue Yu Xue Yu Xue Linlin Zhang Linlin Zhang Linlin Zhang |
| author_facet | Jiulin Chan Jiulin Chan Wei Zhang Wei Zhang Wei Zhang Yue Xu Yue Xu Yu Xue Yu Xue Yu Xue Linlin Zhang Linlin Zhang Linlin Zhang |
| author_sort | Jiulin Chan |
| collection | DOAJ |
| description | 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 β-tubulin and built a highly efficient genome editing system in Crassostrea gigas angulate. We identified the β-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 Cgβ-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 Cgβ-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 β-tubulin is sufficient to mediate cilia development and swimming behavior in oyster larvae. By applying Cgβ-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. |
| first_indexed | 2024-04-12T10:59:42Z |
| format | Article |
| id | doaj.art-08fa0f954d3b408087dbb04ed4fa0513 |
| institution | Directory Open Access Journal |
| issn | 2296-7745 |
| language | English |
| last_indexed | 2024-04-12T10:59:42Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Marine Science |
| spelling | doaj.art-08fa0f954d3b408087dbb04ed4fa05132022-12-22T03:35:59ZengFrontiers Media S.A.Frontiers in Marine Science2296-77452022-05-01910.3389/fmars.2022.912409912409Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured OysterJiulin Chan0Jiulin Chan1Wei Zhang2Wei Zhang3Wei Zhang4Yue Xu5Yue Xu6Yu Xue7Yu Xue8Yu Xue9Linlin Zhang10Linlin Zhang11Linlin Zhang12Chinese Academy of Sciences (CAS) and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, ChinaLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, ChinaChinese Academy of Sciences (CAS) and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, ChinaLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, ChinaCollege of Life Sciences, Qingdao Agricultural University, Qingdao, ChinaChinese Academy of Sciences (CAS) and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, ChinaLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, ChinaChinese Academy of Sciences (CAS) and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, ChinaLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, ChinaCollege of Life Sciences, Qingdao Agricultural University, Qingdao, ChinaChinese Academy of Sciences (CAS) and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, ChinaLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, ChinaCollege of Marine Science, University of Chinese Academy of Sciences, Beijing, ChinaGenome 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 β-tubulin and built a highly efficient genome editing system in Crassostrea gigas angulate. We identified the β-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 Cgβ-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 Cgβ-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 β-tubulin is sufficient to mediate cilia development and swimming behavior in oyster larvae. By applying Cgβ-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.https://www.frontiersin.org/articles/10.3389/fmars.2022.912409/fullmosaic mutagenesisCRISPR/Cas9long deletiongene editinggene knockoutaquaculture breeding |
| spellingShingle | Jiulin Chan Jiulin Chan Wei Zhang Wei Zhang Wei Zhang Yue Xu Yue Xu Yu Xue Yu Xue Yu Xue Linlin Zhang Linlin Zhang Linlin Zhang Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster Frontiers in Marine Science mosaic mutagenesis CRISPR/Cas9 long deletion gene editing gene knockout aquaculture breeding |
| title | Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster |
| title_full | Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster |
| title_fullStr | Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster |
| title_full_unstemmed | Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster |
| title_short | Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of β-Tubulin in the Cultured Oyster |
| title_sort | electroporation based crispr cas9 mosaic mutagenesis of β tubulin in the cultured oyster |
| topic | mosaic mutagenesis CRISPR/Cas9 long deletion gene editing gene knockout aquaculture breeding |
| url | https://www.frontiersin.org/articles/10.3389/fmars.2022.912409/full |
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