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.
Department of Biological Science, College of Science, Sungkyunkwan University, Suwon 16419, South Korea.
Mar Pollut Bull. 2020 May;154:111038. doi: 10.1016/j.marpolbul.2020.111038. Epub 2020 Mar 12.
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.
为了在海洋青鳉中产生白化病，我们使用了成簇规律间隔短回文重复（CRISPR）/Cas9 与单个向导 RNA（sgRNA）对溶质载体家族 45（SLC45a2）基因进行了干扰。通过基因分型和异源双链迁移分析（HMA）证实，所选 sgRNA 能够靶向 SLC45a2 基因。在注射后的存活胚胎中，sgRNA1 和 sgRNA2 分别使 54.2%和 60.0%的胚胎表现出白化病表型。在靶位点的深度测序显示，在 DNA 切割位点附近存在不同的插入和缺失（indel）突变谱，表明该方法产生 SLC45a2 敲除突变体的效率很高。此外，在潜在的脱靶位点的 HMA 表明，脱靶活性的诱导率很低，或者根本不诱导。这种白化海洋青鳉将成为建立各种体内终点的海洋分子生态毒理学的良好模型，并且这种高效的基因靶向方法在海洋青鳉中的应用将是一种用于机制研究的有用工具。
