Efficient genome editing in medaka (<i>Oryzias latipes</i>) using a codon-optimized SaCas9 system.

Yuewen JIANG; Qihua PAN; Zhi WANG; Ke LU; Bilin XIA; Tiansheng CHEN

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Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system.

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

1. Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China.
2. State Key Laboratory of Mariculture Breeding, Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, Fisheries College of Jimei University, Xiamen 361021, China.
3. Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China. tiansheng.chen@jmu.edu.cn.
Publication Type:Journal Article
Keywords: Gene editing; Medaka; Oculocutaneous albinism II (oca2); Paired box 6.1 (pax6.1); Staphylococcus aureus Cas9 (SaCas9); Transfer RNA(tRNA); Tyrosinase (tyr)
MeSH: Animals; Oryzias/genetics*; Gene Editing/methods*; CRISPR-Cas Systems; Codon; RNA, Guide, CRISPR-Cas Systems/genetics*; Monophenol Monooxygenase/genetics*; CRISPR-Associated Protein 9/genetics*; RNA, Transfer/genetics*; Staphylococcus aureus/genetics*; PAX6 Transcription Factor/genetics*
From: Journal of Zhejiang University. Science. B 2024;25(12):1083-1096
CountryChina
Language:English
Abstract: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)‍-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.