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On-line Access: 2022-01-12

Received: 2021-04-11

Revision Accepted: 2021-06-09

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Tiansheng CHEN

https://orcid.org/0000-0003-4763-2307

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

http://doi.org/10.1631/jzus.B2100343


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.

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author="Qihua PAN, Junzhi LUO, Yuewen JIANG, Zhi WANG, Ke LU, Tiansheng CHEN",
journal="Journal of Zhejiang University Science B",
volume="23",
number="1",
pages="74-83",
year="2022",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2100343"
}

%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 Zhi WANG
%A Ke LU
%A Tiansheng CHEN
%J Journal of Zhejiang University SCIENCE B
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%@ 1673-1581
%D 2022
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2100343

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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


Abstract: 
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

目的:探究巨细胞病毒(CMV)启动子驱动转运RNA-导向RNA(tRNA-gRNA)的表达是否能够在青鳉细胞系及胚胎中产生功能性的gRNA并诱导基因的突变。
创新点:利用CMV启动子驱动tRNA-gRNA的表达在青鳉细胞系及胚胎中产生功能性的gRNA,结合Cas9系统诱导了青鳉胚胎和细胞系中的基因突变,为在鱼类胚胎和细胞系中进行基因编辑提供了一种可行的方案。
方法:将tRNA-tyrgRNA(酪氨酸酶基因,tyr)或tyrgRNA与Cas9mRNA注射到青鳉1胞期的胚胎中观察胚胎表型并通过聚合酶链式反应(PCR)和测序检测tRNA-tyrgRNA在青鳉胚胎中的功能。将pCS2-tRNA-tyr和pCS2-tRNA-PT质粒分别与Cas9mRNA注射到青鳉1胞期的胚胎中观察胚胎表型,并通过PCR和测序检测CMV启动子驱动的tRNA-gRNA在胚胎中的功能。将pCMV-tRNA-PT-zCas9-puro以及1:1比例的pCVpf或pCS2-tRNA-tyr与pCV-zCas9-puro的混合物分别转染SG3细胞,通过PCR和测序检测CMV启动子驱动的tRNA-gRNA在青鳉细胞系中的功能。
结论:tRNA-tyrgRNA与tyrgRNA一样能够引起青鳉胚胎中tyr基因的突变。CMV启动子驱动tRNA-gRNA的表达在青鳉细胞系及胚胎中能够产生功能性的gRNA,结合Cas9系统诱导青鳉胚胎和细胞系中基因的突变。

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

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