Generation of albino via SLC45a2 gene targeting by CRISPR/Cas9 in the marine medaka Oryzias melastigma

doi:10.1016/j.marpolbul.2020.111038

Marine Pollution Bulletin

Volume 154, May 2020, 111038

https://doi.org/10.1016/j.marpolbul.2020.111038 Get rights and content

Abstract

To produce albinism in the marine medaka Oryzias melastigma, we disrupted the solute carrier family 45 (SLC45a2) gene by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with a single guide RNA (sgRNA). Selected sgRNAs were able to target a SLC45a2 gene as confirmed by genotyping and heteroduplex mobility assay (HMA). Of the survived embryos after injection, 54.2% and 60.0% embryos exhibited albinism phenotype by sgRNA1 and sgRNA2, respectively. Deep sequencing at the on-target sites showed different insertion and deletion (indel) mutation profiles near the DNA cleavage sites, indicating high efficacy of producing SLC45a2 knock-out mutants by this method. Moreover, HMA at the potential off-target sites revealed that off-target activity would be induced at a low rate, or not induced at all. This albino marine medaka will be a good model for marine molecular ecotoxicology in establishment of diverse in vivo endpoints, and the application of this efficient gene targeting method in the marine medaka would be useful tool for mechanistic approaches.

Section snippets

CRediT authorship contribution statement

Chang-Bum Jeong:Investigation, Data curation, Visualization, Writing - original draft, Writing - review & editing.Hye-Min Kang:Investigation, Data curation, Visualization, Writing - original draft, Writing - review & editing.Sung-Ah Hong:Investigation, Data curation.Eunjin Byeon:Investigation, Data curation.Jin-Sol Lee:Investigation, Data curation.Young Hwan Lee:Investigation, Data curation.Ik-Young Choi:Writing - original draft, Writing - review & editing.Sangsu Bae:Writing - original draft,

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Collaborative Genome Program of the Korea Institute of Marine Science and Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (MOF) [No. 20180430] and also supported by a grant of the National Research Foundation of Korea (NRF-2019R1A2C1007963) funded to Hye-Min Kang.

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¹: These authors contributed equally to this work.

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Generation of albino via SLC45a2 gene targeting by CRISPR/Cas9 in the marine medaka Oryzias melastigma

Abstract

Section snippets

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgements

Comp. Biochem. Physiol. D

J. Biol. Chem.

Comp. Biochem. Physiol. A

Aquat. Toxicol.

Aquat. Toxicol.

Fish Shellfish Immunol

Mech. Dev.

Comp. Biochem. Physiol. C

Aquat. Toxicol.

Mar. Genomics

Mar. Environ. Res.

Aquat. Toxicol.

Aquat. Toxicol.

Environ. Pollut.

J. Biomed. Sci.

Aquat. Toxicol.

Drug Discov. Today Technol.

Mar. Environ. Res.

Methods Cell Biol.

Aquat. Toxicol.

Mar. Pollut. Bull.

Fish Shellfish Immunol.

Targeted mutagenesis using CRISPR/Cas system in medaka

Biol Open

Design, evaluation, and screening methods for efficient targeted mutagenesis with transcription activator-like effector nucleases in medaka

Develop. Growth Differ.

Microhomology-based choice of Cas9 nuclease target sites

Nat. Methods

Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

Bioinformatics

Hepatic proteomic responses in marine medaka (Oryzias melastigma) chronically exposed to antifouling compound butenolide [5-octylfuran-2(5H)-one] or 4,5-dichloro-2-N-octyl-4-isothiazolin-3-one (DCOIT)

Environ. Sci. Technol.