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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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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).
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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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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). https://doi.org/10.2478/s11756-020-00677-7
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DOI: https://doi.org/10.2478/s11756-020-00677-7