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

Qihua Pan; Junzhi Luo; Yuewen Jiang; Zhi Wang; Ke LU; Tiansheng Chen

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

Author: Qihua PAN ¹ ; Junzhi LUO ² ; Yuewen JIANG ² ; Zhi WANG ² ; Ke LU ² ; Tiansheng CHEN ³
Author Information

1. 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.
2. College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China.
3. 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. tiansheng.chen@jmu.edu.cn.
Publication Type:Journal Article
Keywords: Embryos; Fish cells; Gene editing; Medaka (Oryzias latipes); Poly-tRNA-gRNA
MeSH: Animals; CRISPR-Cas Systems; Cell Line; Gene Editing; Oryzias/genetics*; RNA, Guide/genetics*; RNA, Transfer/genetics*
From: Journal of Zhejiang University. Science. B 2022;23(1):74-83
CountryChina
Language:English
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.