Efficient genome editing in medaka (<italic style="font-style: italic">Oryzias latipes</italic>) using a codon-optimized SaCas9 system

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

doi:10.1631/jzus.B2300899

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Research Article | Updated：2024-12-27

Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system
Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system
基于密码子优化的SaCas9系统高效编辑青鳉（Oryzias latipes）基因组
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) Vol. 25, Issue 12, Pages: 1083-1096(2024)
Affiliations：

1.Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, College of Fisheries, Huazhong Agricultural University, Wuhan430070, 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, Xiamen361021, China
Funds：
DOI：10.1631/jzus.B2300899
CLC：
CSTR：
Published： 15 December 2024 ，

Received： 11 December 2023 ，

Revised： 26 March 2024 ，
Accepted：

蒋月雯,潘启华,王志等.基于密码子优化的SaCas9系统高效编辑青鳉（Oryzias latipes）基因组[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2024,25(12):1083-1096.

YUEWEN JIANG, QIHUA PAN, ZHI WANG, et al. Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system. [J]. Journal of zhejiang university-science b (biomedicine & biotechnology), 2024, 25(12): 1083-1096.
蒋月雯,潘启华,王志等.基于密码子优化的SaCas9系统高效编辑青鳉（Oryzias latipes）基因组[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2024,25(12):1083-1096. DOI： 10.1631/jzus.B2300899.

YUEWEN JIANG, QIHUA PAN, ZHI WANG, et al. Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system. [J]. Journal of zhejiang university-science b (biomedicine & biotechnology), 2024, 25(12): 1083-1096. DOI： 10.1631/jzus.B2300899.

摘要

CRISPR-Cas9系统属于II型CRISPR/Cas系统，作为一种有效的基因编辑工具被广泛应用于不同生物体中，但化脓性链球菌（

Streptococcus pyogenes

）Cas9（SpCas9）体积较大（4.3 kb），因此使用载体传递较为不便。本研究利用密码子优化后的金黄色葡萄球菌（

Staphylococcus aureus

）Cas9（SaCas9）系统进行了青鳉（

Oryzias latipes

）的

tyr

、

oca2

和

pax6.1

基因编辑。SaCas9片段（3.3 kb）要小，所需的原型间隔相邻基序（PAM）序列为5'-NNGRRT-3'。此外，本研究还利用tRNA-sgRNA系统在体内和体外转录表达功能性sgRNA，经SaCas9和tRNA-sgRNA的组合编辑青鳉基因组中的

tyr

基因。实验结果表明，SaCas9/sgRNA和SaCas9/tRNA-sgRNA系统均能有效编辑青鳉基因组，而PAM序列是该系统进行有效编辑的关键部分。此外，tRNA还能使sgRNA受巨细胞病毒等常见启动子的控制，从而提高系统的适应性。本研究还发现，CMV-SaCas9-tRNA-sgRNA-tRNA一体化结构在青鳉基因编辑中同样能发挥作用。综上，经密码子优化的SaCas9系统为编辑青鳉及潜在的其他鱼类基因组提供了一种更便捷的工具。

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 either

in 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.

关键词

金黄色葡萄球菌（Staphylococcus aureus）Cas9（SaCas9）青鳉转运核糖核酸（tRNA）基因编辑酪氨酸酶（tyr）眼皮肤白化病2型（oca2）配对盒基因6.1（pax6.1）

Keywords

Staphylococcus aureus Cas9 (SaCas9)MedakaTransfer RNA(tRNA)Gene editingTyrosinase (tyr)Oculocutaneous albinism II (oca2)Paired box 6.1 (pax6.1)

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Related Institution

Faculty of Law, Universiti Teknologi MARA, Jalan Sarjana 1/2, Shah Alam

Centre for Research in Biotechnology for Agriculture (CEBAR), Level 3, Research Management and Innovation Complex, Universiti Malaya

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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

College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University

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

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

DOI：10.1631/jzus.B2300899

摘要

Abstract

关键词

Keywords

references