Full Text:   <2711>

Summary:  <705>

Suppl. Mater.: 

CLC number: 

On-line Access: 2022-01-12

Received: 2021-04-11

Revision Accepted: 2021-06-09

Crosschecked: 0000-00-00

Cited: 0

Clicked: 4189

Citations:  Bibtex RefMan EndNote GB/T7714


Tiansheng CHEN


-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2022 Vol.23 No.1 P.74-83


Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA

Author(s):  Qihua PAN, Junzhi LUO, Yuewen JIANG, Zhi WANG, Ke LU, Tiansheng CHEN

Affiliation(s):  Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Fisheries College, Jimei University, Xiamen 361021, China; more

Corresponding email(s):   tiansheng.chen@jmu.edu.cn

Key Words:  Medaka (Oryzias latipes), Gene editing, Poly-tRNA-gRNA, Embryos, Fish cells

Qihua PAN, Junzhi LUO, Yuewen JIANG, Zhi WANG, Ke LU, Tiansheng CHEN. Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA[J]. Journal of Zhejiang University Science B, 2022, 23(1): 74-83.

@article{title="Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA",
author="Qihua PAN, Junzhi LUO, Yuewen JIANG, Zhi WANG, Ke LU, Tiansheng CHEN",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA
%A Qihua PAN
%A Junzhi LUO
%A Yuewen JIANG
%A Ke LU
%A Tiansheng CHEN
%J Journal of Zhejiang University SCIENCE B
%V 23
%N 1
%P 74-83
%@ 1673-1581
%D 2022
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2100343

T1 - Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA
A1 - Qihua PAN
A1 - Junzhi LUO
A1 - Yuewen JIANG
A1 - Zhi WANG
A1 - Ke LU
A1 - Tiansheng CHEN
J0 - Journal of Zhejiang University Science B
VL - 23
IS - 1
SP - 74
EP - 83
%@ 1673-1581
Y1 - 2022
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2100343

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase (tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

利用化脓链球菌成簇的规则间隔短回文重复序列相关蛋白9(spCas9)/转运RNA(tRNA)-导向RNA(gRNA)(SpCas9/tRNA-gRNA)在青鳉(Oryzias latipes)细胞系和胚胎中进行高效基因编辑


关键词:青鳉(Oryzias latipes);基因编辑;Poly-tRNA-gRNA;胚胎;鱼类细胞系

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]Avis JM, Conn GL, Walker SC, 2012. Cis-acting ribozymes for the production of RNA in vitro transcripts with defined 5' and 3' ends. Methods Mol Biol, 941:83-98.

[2]Čermák T, Curtin SJ, Gil-Humanes J, et al., 2017. A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell, 29(6):1196-1217.

[3]Chen J, Du YN, He XY, et al., 2017. A convenient Cas9-based conditional knockout strategy for simultaneously targeting multiple genes in mouse. Sci Rep, 7:517.

[4]Chen TS, Cavari B, Schartl M, et al., 2017. Identification and expression of conserved and novel RNA variants of medaka pax6b gene. J Exp Zool B (Mol Dev Evol), 328(5): 412-422.

[5]Dong FP, Xie KB, Chen YY, et al., 2017. Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells. Biochem Biophys Res Commun, 482(4):889-895.

[6]Fang J, Chen TS, Pan QH, et al., 2018. Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9. J Exp Zool B (Mol Dev Evol), 330(4):242-246.

[7]Feng Y, Liu S, Chen R, et al., 2021. Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(1):73-86.

[8]Ferreira R, Skrekas C, Nielsen J, et al., 2018. Multiplexed CRISPR/Cas9 genome editing and gene regulation using Csy4 in Saccharomyces cerevisiae. ACS Synth Biol, 7(1):10-15.

[9]Forster AC, Altman S, 1990. External guide sequences for an RNA enzyme. Science, 249(4970):783-786.

[10]Fujimura N, Klimova L, Antosova B, et al., 2015. Genetic interaction between Pax6 and β-catenin in the developing retinal pigment epithelium. Dev Genes Evol, 225(2):121-128.

[11]He YB, Zhang T, Yang N, et al., 2017. Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing. J Genet Genomics, 44(9):469-472.

[12]Hong YH, Liu TM, Zhao HB, et al., 2004. Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. Proc Natl Acad Sci USA, 101(21): 8011-8016.

[13]Jao LE, Wente SR, Chen WB, 2013. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA, 110(34):13904-13909.

[14]Jiang WY, Bikard D, Cox D, et al., 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 31(3):233-239.

[15]Jinek M, Chylinski K, Fonfara I, et al., 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337(6096):816-821.

[16]Knapp DJHF, Michaels YS, Jamilly M, et al., 2019. Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression. Nat Commun, 10:1490.

[17]Li C, Brant E, Budak H, et al., 2021. CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(4):253-284.

[18]Liu QZ, Yuan YM, Zhu F, et al., 2018. Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells. Biol Open, 7(8):bio035170.

[19]Ma J, Fan YD, Zhou Y, et al., 2018. Efficient resistance to grass carp reovirus infection in JAM-A knockout cells using CRISPR/Cas9. Fish Shellfish Immunol, 76:206-215.

[20]Mali P, Esvelt KM, Church GM, 2013. Cas9 as a versatile tool for engineering biology. Nat Methods, 10(10):957-963.

[21]Mefferd AL, Kornepati AVR, Bogerd HP, et al., 2015. Expression of CRISPR/Cas single guide RNAs using small tRNA promoters. RNA, 21(9):1683-1689.

[22]Merenda A, Andersson-Rolf A, Mustata RC, et al., 2017. A protocol for multiple gene knockout in mouse small intestinal organoids using a CRISPR-concatemer. J Vis Exp, (125):e55916.

[23]Nissim L, Perli SD, Fridkin A, et al., 2014. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell, 54(4):698-710.

[24]Platt RJ, Chen SD, Zhou Y, et al., 2014. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 159(2):440-455.

[25]Port F, Bullock SL, 2016. Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nat Methods, 13(10):852-854.

[26]Qi WW, Zhu T, Tian ZR, et al., 2016. High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol, 16:58.

[27]Qin W, Liang F, Feng Y, et al., 2015. Expansion of CRISPR/Cas9 genome targeting sites in zebrafish by Csy4-based RNA processing. Cell Res, 25(9):1074-1077.

[28]Ran FA, Hsu PD, Wright J, et al., 2013. Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8(11):2281-2308.

[29]Schiffer S, Rösch S, Marchfelder A, 2002. Assigning a function to a conserved group of proteins: the tRNA 3'-processing enzymes. EMBO J, 21(11):2769-2777.

[30]Shiraki T, Kawakami K, 2018. A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish. Sci Rep, 8:13366.

[31]Tan JT, Zhao YC, Wang B, et al., 2020. Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining. Plant Biotechnol J, 18(11):2161-2163.

[32]Tan YY, Du H, Wu X, et al., 2020. Gene editing: an instrument for practical application of gene biology to plant breeding. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 21(6):460-473.

[33]Tang YD, Liu JT, Wang TY, et al., 2017. CRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication. Arch Virol, 162(12):3881-3886.

[34]Wang ZW, Zhu HP, Wang D, et al., 2011. A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp. BMC Res Notes, 4:82.

[35]Wefers B, Bashir S, Rossius J, et al., 2017. Gene editing in mouse zygotes using the CRISPR/Cas9 system. Methods, 121-122:55-67.

[36]Xie KB, Minkenberg B, Yang YN, 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA, 112(11): 3570-3575.

[37]Xu AT, Qin C, Lang Y, et al., 2015. A simple and rapid approach to manipulate pseudorabies virus genome by CRISPR/Cas9 system. Biotechnol Lett, 37(6):1265-1272.

[38]Xu L, Zhao LX, Gao YD, et al., 2017. Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA. Nucleic Acids Res, 45(5):e28.

[39]Xue T, Wang YZ, Pan QH, et al., 2018. Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus. J Fish Dis, 41(2):365-374.

[40]Yin LL, Maddison LA, Li MY, et al., 2015. Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs. Genetics, 200(2):431-441.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2024 Journal of Zhejiang University-SCIENCE