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Targeted mutagenesis in the olive flounder (Paralichthys olivaceus) using the CRISPR/Cas9 system with electroporation


As a new breeding technology, genome editing becomes a powerful tool owing to its high efficiency of gene targeting. In CRISPR/Cas9 system, how to efficiently transfer gRNA and Cas9 mRNA into embryos is an important step. Though microinjection is the most common method for operating on fish embryos, it is not easy to inject the RNA into pelagic and telolecithal eggs with hard egg chorion, such as the olive flounder (Paralichthys olivaceus) eggs. Therefore, an efficient and simple technology is urgently needed for this kind of study. In the present study, we used the electroporation method to introduce foreign gene into the flounder eggs. The results showed that the proper electroporation condition was 3 pulses for 1 millisecond (ms), 50 ms interval, at 25 V with high survival rate. Under this condition, the effect of CRISPR/Cas9 system on genome editing by using two different genes, myomaker and gonadal soma derived factor (gsdf) was investigated. Around 12% and 7% of the electroporated embryos for myomaker and gsdf hatched, respectively. The mutation sites including insert and deletion mutations at the candidate sites were visible for both targeted genes in the hatched larvae. The checked frame-shift and start codon deletion mutations would lead to complete destruction of these genes’ structure. Above results implied that CRISPR/Cas9 system could work well in marine fish with pelagic eggs by using electroporation, and genome editing could be achieved on a large scale which may be useful for study of gene function in marine fish.

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gsdf :

gonadal soma derived factor


sperm-mediated gene transfer




tricaine methanesulfonate


green fluorescence protein


phosphate buffer saline


0.1% Tween 20 in PBS


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We would like to thank HuaYue Enterprise Holdings Limited Company for their kindly supplying Electroporator NEPA 21. The study was supported by the projects from the National Key R&D Program of China (No.2018YFD0900202) and the National Sciences of Foundation of China (Nos.31672636 and 31772834).

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Authors and Affiliations



Xungang TAN, Ling WANG, and Feng YOU conceived and designed the experiment. Ling WANG, Xungang TAN, Lijuan WANG, and Zhihao WU performed the experiments. Ling WANG, Xungang TAN, Shuang JIAO, and Yuxia ZOU performed data analyses. Guanglei JI provided ideal experiment materials. Ling WANG, Xungang TAN, and Feng YOU wrote the manuscript. Xungang TAN and Feng YOU approved the manuscript. Feng YOU supervised the study.

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Correspondence to Xungang Tan or Feng You.

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The authors declare that they have no conflict of interest. 

Ethicals approval

All experiments were performed according to the regulation of local and central government of China, and the protocol was approved by the Institutional Animal Care and Use Committee of Institute of Oceanology, Chinese Academy of Sciences.

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Wang, L., Tan, X., Wu, Z. et al. Targeted mutagenesis in the olive flounder (Paralichthys olivaceus) using the CRISPR/Cas9 system with electroporation. Biologia 76, 1297–1304 (2021).

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