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,...
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Published in | Biológia Vol. 76; no. 4; pp. 1297 - 1304 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
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Springer International Publishing
01.04.2021
Springer Nature B.V |
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Abstract | 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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AbstractList | 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. 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. |
Author | Zou, Yuxia Ji, Guanglei You, Feng Wu, Zhihao Tan, Xungang Wang, Ling Wang, Lijuan Jiao, Shuang |
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Cites_doi | 10.1016/j.bbrc.2014.07.093 10.1007/s10126-020-09985-0 10.1038/cr.2013.45 10.1371/journal.pone.0068708 10.1016/j.aquaculture.2019.734336 10.15252/embr.201541715 10.1016/j.bbr.2017.11.006 10.1007/s00427-002-0224-5 10.1038/srep42213 10.1016/j.jgg.2013.11.004 10.1371/journal.pone.0108622 10.1534/g3.117.300359 10.1387/ijdb.150008rg 10.1038/srep26520 10.1023/A:1011191818927 10.1038/srep19480 10.3389/fgene.2018.00117 10.1111/asj.13129 10.1038/nbt.3081 10.1002/dvdy.24674 10.1371/journal.pone.0142755 10.1016/j.anireprosci.2014.06.029 10.1016/0922-3371(90)90030-Z 10.1007/978-1-4939-8831-0_10 10.1371/journal.pone.0102492 10.1016/j.ymeth.2005.12.009 10.1007/s10126-007-9059-4 10.1016/0044-8486(92)90046-N 10.1007/s10695-016-0206-6 10.1186/1749-8104-2-6 10.1002/mrd.22642 10.1016/j.aquaculture.2018.05.055 10.1016/j.cbpb.2015.08.003 10.3791/54313 10.1016/0165-7836(94)90091-4 |
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Snippet | 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... |
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SubjectTerms | Biomedical and Life Sciences Cell Biology Chorion CRISPR Editing Eggs Electroporation Embryos Fish Fish eggs Gene deletion Gene targeting Genes Genome editing Genomes gRNA Larvae Life Sciences Marine fish Microbiology Microinjection mRNA Mutation Original Article Paralichthys olivaceus Plant Sciences Site-directed mutagenesis Survival Zoology |
Title | Targeted mutagenesis in the olive flounder (Paralichthys olivaceus) using the CRISPR/Cas9 system with electroporation |
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