JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B

Accepted manuscript available online (unedited version)

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

Author(s): Yuewen JIANG, Qihua PAN, Zhi WANG, Ke LU, Bilin XIA, Tiansheng CHEN
Affiliation(s): College of Fisheries, Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China; more
Corresponding email(s): tiansheng.chen@jmu.edu.cn
Key Words: SaCas9; Medaka; tRNA; gene editing; tyr; oca2; pax6.1

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Yuewen JIANG, Qihua PAN, Zhi WANG, Ke LU, Bilin XIA, Tiansheng CHEN. Efficient genome editing in Medaka (Oryzias latipes) using a codon optimized SaCas9 system[J]. Journal of Zhejiang University Science B,in press.Frontiers of Information Technology & Electronic Engineering,in press.https://doi.org/10.1631/jzus.B2300899

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author="Yuewen JIANG, Qihua PAN, Zhi WANG, Ke LU, Bilin XIA, Tiansheng CHEN",
journal="Journal of Zhejiang University Science B",
year="in press",
publisher="Zhejiang University Press & Springer",
doi="https://doi.org/10.1631/jzus.B2300899"
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%A Ke LU
%A Bilin XIA
%A Tiansheng CHEN
%J Journal of Zhejiang University SCIENCE B
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doi="https://doi.org/10.1631/jzus.B2300899"

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A1 - Zhi WANG
A1 - Ke LU
A1 - Bilin XIA
A1 - Tiansheng CHEN
J0 - Journal of Zhejiang University Science B
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Y1 - in press
PB - Zhejiang University Press & Springer
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doi="https://doi.org/10.1631/jzus.B2300899"

Abstract
Chinese Summary
Academic Network
Reviewer Comment

Abstract: The CRISPR-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 SpCas9 is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized SaCas9 system to edit the tyr, oca2, and 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 prototype spacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a tRNA-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 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.

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