CRISPR-Cas13d系统下调青鳉tyr基因表达的研究
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S917.4

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国家自然科学基金(32273127,31771648);集美大学科研基金项目(ZQ2020003)


Application of Cas13d system in the down-regulation of tyr gene expression in medaka (Oryzias latipes
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    摘要:

    CRISPR-Cas13d蛋白通过crRNA介导结合靶向RNA底物,利用其核糖核酸内切酶活性实现精准切割,为RNA水平的基因调控提供非基因组编辑策略。本研究基于青鳉(Oryzias latipes)密码子偏好性优化Cas13d基因序列,成功构建原核表达系统并纯化获得分子量为115 ku的重组Cas13d蛋白。进一步通过显微注射将体外转录的Cas13d mRNA与靶向酪氨酸酶基因(tyr)的crRNA复合物导入青鳉胚胎,同时利用Cas13d蛋白/crRNA核糖核蛋白(RNP)系统进行平行实验。qRT-PCR及测序分析表明,两种递送方式均能显著降低青鳉胚胎内tyr基因的RNA水平(P<0.01),且基因测序证实靶位点DNA序列未发生突变。本研究在青鳉中验证了Cas13d系统通过RNA编辑实现基因沉默的有效性与特异性,不仅为鱼类基因探索提供新型调控工具,也为构建基于RNA靶向的Cas13d抗病毒转基因鱼类品系奠定了理论基础。

    Abstract:

    The CRISPR-Cas13d protein targets RNA substrates through crRNA-mediated sequence-specific binding and achieves precise cleavage via its ribonuclease activity, offering a non-genome-editing strategy for RNA-level gene regulation. In this study, we optimized the Cas13d gene sequence based on the codon preference of medaka (Oryzias latipes), successfully constructed a prokaryotic expression system, and purified recombinant Cas13d protein with a molecular weight of 115 ku. By microinjecting in vitro-transcribed Cas13d mRNA complexed with crRNA targeting the tyrosinase gene (tyr) into medaka embryos, alongside parallel experiments using Cas13d protein/crRNA ribonucleoprotein (RNP) complexes, we systematically evaluated RNA-editing efficiency. qRT-PCR and sequencing analyses demonstrated that both delivery methods significantly reduced tyr RNA levels in embryos (P<0.01), while DNA sequencing confirmed the absence of mutations in the target DNA sequences. This study validates the effectiveness and specificity of Cas13d-mediated gene silencing through RNA editing in medaka, not only providing a novel regulatory tool for fish genetic research but also establishing a theoretical foundation for developing RNA-targeted Cas13d antiviral transgenic fish lines.

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许朝然,夏必琳,蒋月雯,邓菊,陈若雪,梁晶婕,陈天圣. CRISPR-Cas13d系统下调青鳉tyr基因表达的研究[J].上海海洋大学学报,2025,34(6):1193-1202.
XU Zhaoran, XIA Bilin, JIANG Yuewen, DENG Ju, CHEN Ruoxue, LIANG JingJie, CHEN Tiansheng. Application of Cas13d system in the down-regulation of tyr gene expression in medaka (Oryzias latipes)[J]. Journal of Shanghai Ocean University,2025,34(6):1193-1202.

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  • 收稿日期:2024-11-07
  • 最后修改日期:2025-05-19
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  • 在线发布日期: 2025-12-06
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