Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of beta-Tubulin in the Cultured Oyster

doi:10.3389/fmars.2022.912409

IOCAS-IR > 实验海洋生物学重点实验室

	Electroporation-Based CRISPR/Cas9 Mosaic Mutagenesis of beta-Tubulin in the Cultured Oyster
	Chan, Jiulin 1,2,5,6; Zhang, Wei 1,2,3,5,6; Xu, Yue 1,2,5,6; Xue, Yu 1,2,3,5,6; Zhang, Linlin 1,2,4,5,6
	2022-05-26
发表期刊	FRONTIERS IN MARINE SCIENCE
卷号	9 页码:12
通讯作者	Zhang, Linlin(linlinzhang@qdio.ac.cn)
摘要	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.
关键词	mosaic mutagenesis CRISPR Cas9 long deletion gene editing gene knockout aquaculture breeding
DOI	10.3389/fmars.2022.912409
收录类别	SCI
语种	英语
资助项目	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研究方向	Environmental Sciences & Ecology ; Marine & Freshwater Biology
WOS类目	Environmental Sciences ; Marine & Freshwater Biology
WOS记录号	WOS:000811826900001
出版者	FRONTIERS MEDIA SA
引用统计	被引频次：2[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/179495
专题	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
通讯作者	Zhang, Linlin
作者单位	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
第一作者单位	深海极端环境与生命过程研究中心
通讯作者单位	深海极端环境与生命过程研究中心
推荐引用方式 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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