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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

gsdf :

gonadal soma derived factor

SMGT:

sperm-mediated gene transfer

ms:

millisecond

MS222:

tricaine methanesulfonate

GFP:

green fluorescence protein

PBS:

phosphate buffer saline

PBST:

0.1% Tween 20 in PBS

References

  1. Atkins RL, Wang D, Burke R (2000) Localized electroporation: A method for targeting expression of genes in avian embryos. Biotechniques 28(1):94–94+. https://doi.org/10.1023/A:1011191818927

    CAS  Article  PubMed  Google Scholar 

  2. Cerda GA, Thomas JE, Allende ML, Karlstrom RO, Palma V (2006) Electroporation of DNA, RNA, and morpholinos into zebrafish embryos. Methods 39:207–211. https://doi.org/10.1016/j.ymeth.2005.12.009

    CAS  Article  PubMed  Google Scholar 

  3. Chakraborty T, Zhou LY, Chaudhari A, Iguchi T, Nagahama Y (2016) Dmy initiates masculinity by altering Gsdf/Sox9a2/Rspo1 expression in medaka (Oryzias latipes). Sci Rep 6:19480. https://doi.org/10.1038/srep19480

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res 23:465–472. https://doi.org/10.1038/cr.2013.45

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Chen J, Wang W, Tian Z, Dong Y, Dong T, Zhu H, Zhu Z, Hu H, Hu W (2018) Efficient gene transfer and gene editing in sterlet (Acipenser ruthenus). Front Genet 9:117. https://doi.org/10.3389/fgene.2018.00117

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Cui Z, Liu Y, Wang W, Wang Q, Zhang N, Lin F, Wang N, Shao C, Dong Z, Li Y, Yang Y, Hu M, Li H, Gao F, Wei Z, Meng L, Liu Y, Wei M, Zhu Y, Guo H, Cheng CH, Schartl M, Chen S (2017) Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis). Sci Rep 7:42213. https://doi.org/10.1038/srep42213

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Draga M, Pröls F, Scaal M (2018) Double electroporation in two adjacent tissues in chicken embryos. Dev Dynam 247:1211–1216. https://doi.org/10.1002/dvdy.24674

    CAS  Article  Google Scholar 

  8. Edvardsen RB, Leininger S, Kleppe L, Skaftnesmo KO, Wargelius A (2014) Targeted mutagenesis in Atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation. PLos One 9:e108622. https://doi.org/10.1371/journal.pone.0108622

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Goto R, Saito T, Kawakami Y, Kitauchi T, Takagi M, Todo T, Arai K, Yamaha E (2015) Visualization of primordial germ cells in the fertilized pelagic eggs of the barfin flounder Verasper moseri. Int J Dev Biol 59(10–12):465–470. https://doi.org/10.1387/ijdb.150008rg

    CAS  Article  PubMed  Google Scholar 

  10. Hendricks M, Jesuthasan S (2007) Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development. Neural Dev 2:6. https://doi.org/10.1186/1749-8104-2-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Hu P, Zhao XY, Zhang QH, Li WM, Zu Y (2018) Comparison of various nuclear localization signal-fused Cas9 proteins and Cas9 mRNA for genome editing in zebrafish. G3 (Bethesda) 8:823–831. https://doi.org/10.1534/g3.117.300359

    CAS  Article  PubMed Central  Google Scholar 

  12. Hwang WY, Fu Y, Reyon D, Maeder ML, Kaini P, Sander JD, Joung JK, Peterson RT, Yeh JR (2013) Heritable and precise zebrafish genome editing using a CRISPR-Cas system. PLoS One 8:e68708. https://doi.org/10.1371/journal.pone.0068708

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Inoue K, Yamashita S, Hata Ji., Kabeno S, Asada S, Nagahisa E, Fujita T (1990) Electroporation as a new technique for producing transgenic fish. Mech Develop 29:123–128. https://doi.org/10.1016/0922-3371(90)90030-Z

    CAS  Article  Google Scholar 

  14. Jiang DN, Yang HH, Li MH, Shi HJ, Zhang XB, Wang DS (2016) Gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia. Mol Reprod Dev 83:497–508. https://doi.org/10.1002/mrd.22642

  15. Jiao S, Tan XG, Li MJ, Sui YL, Du SJ, You F (2015) The duplicated paired box protein 7 (pax7) genes differentially transcribed during Japanese flounder (Paralichthys olivaceus) embryogenesis. Comp Biochem Physiol B Biochem Mol Biol 189:62–8. https://doi.org/10.1016/j.cbpb.2015.08.003

  16. Kalebic N, Taverna E, Tavano S, Wong FK, Suchold D, Winkler S, Huttner WB, Sarov M (2016) CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo. EMBO Rep 17:338–348. https://doi.org/10.15252/embr.201541715

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Kaneko T, Mashimo T (2015) Simple genome editing of rodent intact embryos by electroporation. PLoS One 10:e0142755. https://doi.org/10.1371/journal.pone.0142755

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Kari W, Zeng F, Zitzelsberger L, Will J, Rothbacher U (2016) Embryo microinjection and electroporation in the Chordate Ciona intestinalis. J Vis Exp 116:e54316. https://doi.org/10.3791/54313

  19. Khoo HW, Ang LH, Lim HB, Wong KY (1992) Sperm cells as vectors for introducing foreign DNA into zebrafish. Aquaculture 107:1–19. https://doi.org/10.1016/0044-8486(92)90046-N

    CAS  Article  Google Scholar 

  20. Kim J, Cho JY, Kim JW, Kim HC, Noh JK, Kim YO, Hwang HK, Kim WJ, Yeo SY, An CM, Park JY, Kong HJ (2019) CRISPR/Cas9-mediated myostatin disruption enhances muscle mass in the olive flounder Paralichthys olivaceus. Aquaculture 512:734336. https://doi.org/10.1016/j.aquaculture.2019.734336

    CAS  Article  Google Scholar 

  21. Kishimoto K, Washio Y, Yoshiura Y, Toyoda A, Ueno T, Fukuyama H, Kato K, Kinoshita M (2018) Production of a breed of red sea bream Pagrus major with an increase of skeletal muscle mass and reduced body length by genome editing with CRISPR/Cas9. Aquaculture 495:415–27. https://doi.org/10.1016/j.aquaculture.2018.05.055

  22. Landemaine A, Rescan PY, Gabillard JC (2014) Myomaker mediates fusion of fast myocytes in zebrafish embryos. Biochem Biophys Res Commun 451:480–484. https://doi.org/10.1016/j.bbrc.2014.07.093

    CAS  Article  PubMed  Google Scholar 

  23. Liu D, Wang ZX, Xiao A, Zhang YT, Li WY, Zu Y, Yao SH, Lin S, Zhang B (2014) Efficient gene targeting in zebrafish mediated by a zebrafish-codon-optimized cas9 and evaluation of off-targeting effect. J Genet Genomics 41:43–46. https://doi.org/10.1016/j.jgg.2013.11.004

    CAS  Article  PubMed  Google Scholar 

  24. Monti JM, Jantos H (2018) The effects of local microinjection of selective dopamine D1 and D2 receptor agonists and antagonists into the dorsal raphe nucleus on sleep and wakefulness in the rat. Behav Brain Res 339:11–18. https://doi.org/10.1016/j.bbr.2017.11.006

    CAS  Article  PubMed  Google Scholar 

  25. Qin WN, Wang HY (2019) Delivery of CRISPR-Cas9 into mouse zygotes by electroporation. Methods Mol Biol (Clifton NJ) 1874:179–190. https://doi.org/10.1007/978-1-4939-8831-0_10

    CAS  Article  Google Scholar 

  26. Su JG, Zhu ZY, Wang YP, Xiong F, Zou J (2008) The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo. Mar Biotechnol 10:262–269. https://doi.org/10.1007/s10126-007-9059-4

    CAS  Article  Google Scholar 

  27. Tan XG, Du SJ (2002) Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream (Sparus aurata). Dev Genes Evol 212:207–217. https://doi.org/10.1007/s00427-002-0224-5

    CAS  Article  PubMed  Google Scholar 

  28. Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, Takemoto T, Nakai M, Fuchimoto DI, Otoi T (2019) Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation. Anim Sci J 90:55–61. https://doi.org/10.1111/asj.13129

    CAS  Article  PubMed  Google Scholar 

  29. Thomas JK, Janz DM (2016) Embryo microinjection of selenomethionine reduces hatchability and modifies oxidant responsive gene expression in zebrafish. Sci Rep 6:26520. https://doi.org/10.1038/srep26520

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Tsai HJ, Tseng FS (1994) Electroporation of a foreign gene into black porgy Acanthopagrus schlegeli embryos. Fish Sci 60(6):787–788. https://doi.org/10.1016/0165-7836(94)90091-4

  31. Wang Q, Tan XG, Jiao S, You F, Zhang PJ (2014) Analyzing cold tolerance mechanism in transgenic zebrafish (Danio rerio). PLoS ONE 9(7):e102492. https://doi.org/10.1371/journal.pone.0102492

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Weng SD, You F, Fan ZF, Wang LJ, Wu ZH, Zou YX (2016) Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus). Fish Physiol Biochem 42:1167–1176. https://doi.org/10.1007/s10695-016-0206-6

    CAS  Article  PubMed  Google Scholar 

  33. Xin N, Liu TT, Zhao HT, Wang ZW, Liu JX, Zhang QQ, Qi J (2014) The impact of exogenous DNA on the structure of sperm of olive flounder (Paralichthys olivaceus). Anim Reprod Sci 149:305–310. https://doi.org/10.1016/j.anireprosci.2014.06.029

    CAS  Article  PubMed  Google Scholar 

  34. Zhang CQ, Ren ZH, Gong ZY (2020) Transgenic expression and genome editing by electroporation of zebrafish embryos. Mar Biotechnol (NY) 22:644–650. https://doi.org/10.1007/s10126-020-09985-0

    CAS  Article  Google Scholar 

  35. Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR (2014) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73. https://doi.org/10.1038/nbt.3081

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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).

Author information

Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Xungang Tan or Feng You.

Ethics declarations

Conflict of interest

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.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, L., Tan, X., Wu, Z. et al. Targeted mutagenesis in the olive flounder (Paralichthys olivaceus) using the CRISPR/Cas9 system with electroporation. Biologia (2021). https://doi.org/10.2478/s11756-020-00677-7

Download citation

Keywords

  • Genome editing
  • CRISPR/Cas9 system
  • Electroporation
  • Myomaker and gsdf
  • Olive flounder (Paralichthys olivaceus)