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

Chang-Bum Jeong; Hye-Min Kang; Sung-Ah Hong; Eunjin Byeon; Jin-Sol Lee; Young Hwan Lee; Ik-Young Choi; Sangsu Bae; Jae-Seong Lee

doi:10.1016/j.marpolbul.2020.111038

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

Mar Pollut Bull. 2020 May:154:111038. doi: 10.1016/j.marpolbul.2020.111038. Epub 2020 Mar 12.

Authors

Chang-Bum Jeong¹, Hye-Min Kang², Sung-Ah Hong³, Eunjin Byeon², Jin-Sol Lee², Young Hwan Lee², Ik-Young Choi⁴, Sangsu Bae³, Jae-Seong Lee⁵

Affiliations

¹ Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea; Department of Marine Science, College of Nature Science, Incheon National University, Incheon 22012, South Korea.
² Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea.
³ Department of Chemistry, College of Nature Sciences, Hanyang University, Seoul 04763, South Korea.
⁴ Department of Agriculture and Life Industry, Kangwon National University, Chuncheon 24341, South Korea.
⁵ Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea. Electronic address: jslee2@skku.edu.

PMID: 32174491
DOI: 10.1016/j.marpolbul.2020.111038

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.

Keywords: Albinism; CRISPR/Cas9; Marine medaka; SLC45a2.

MeSH terms

Albinism*
Animals
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Targeting
Oryzias*