文章摘要
利用CRISPR/Cas9基因编辑技术敲除团头鲂SOCS1重复基因的研究
Knoct-out analysis of duplicated socs1 gene using CRISPR/Cas9 in Megalobrama amblycephala
投稿时间:2019-03-11  修订日期:2019-05-17
DOI:
中文关键词: 团头鲂  CRISPR/Cas9  SOCS1  基因编辑
英文关键词: Megalobrama amblycephala  CRISPR/Cas9  SOCS1  gene editing
基金项目:国家自然科学基金(31572220) ;国家科技支撑计划项目(2012BAD26B00);上海高校知识服务平台(ZF1206)
作者单位E-mail
赵心愉 上海海洋大学 13122313863@163.com 
刘娟 上海海洋大学  
邹曙明 上海海洋大学 smzou@shou.edu.cn 
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中文摘要:
      细胞因子信号传导抑制蛋白1(SOCS1)是一种重要的反馈调节因子,广泛参与调控多种细胞因子介导的信号通路,对于免疫系统的调节和生物体的生长发育都发挥着至关重要的作用。本实验以已获得的团头鲂SOCS1a和SOCS1b基因为编辑对象,在线分析筛选出合适的靶点合成gRNA(guide RNA),然后对1-2细胞期的团头鲂胚胎进行gRNA和Cas9蛋白的混合注射。通过检测发现SOCS1a和SOCS1b基因的碱基都出现缺失和错位,表明CRISPR/Cas9系统对SOCS1基因的敲除是有效的。随后经过对3月龄已敲除团头鲂个体检测,成功筛选出存在靶点序列突变的F0代个体。与野生型(WT)相比,SOCS1a和SOCS1b杂合突变品系团头鲂生长性能增强、体重显著增加,同时炎症因子TNF-α和IL-6显著增加,而IL-1β表达无明显变化。随即通过嗜水气单胞菌对敲除SOCS1团头鲂突变品系进行攻毒试验,与野生型不同的是,SOCS1a和SOCS1b杂合突变体中都出现IL-6和TNF-α mRNA水平持续显著增加的现象。本实验通过CRISPR/Cas9系统成功敲除了团头鲂SOCS1a和SOCS1b基因,为进一步探讨SOCS1基因的作用提供了一定的研究基础。同时,也为以养殖鱼类为对象的研究,合理选用CRISPR/Cas9基因编辑技术提供了依据和借鉴。
英文摘要:
      The suppressor of cytokine signaling 1 (SOCS1) is an essential feedback regulator extensive involved in many different cytokine signaling pathways, regulating immune and growth of organism. In this study, the obtained genes of blunt snout bream, SOCS1a and SOCS1b, were used as genetic editing objects. Then we screened for appropriate target synthetic gRNA (guide RNA) by online analysis. According to the mixture injection (gRNA and Cas9 protein) in the 1-2 cell stage embryos, By detecting the effectiveness of the target, it was found that the bases of the SOCS1a and SOCS1b genes were deleted and misplaced, indicating that the CRISPR/Cas9 system is effective for the knockout of the SOCS1 gene. After the detection of 3-month-old F0 generation blunt snout bream, the F0 generation individuals with the mutation of the target sequence were successfully screened.Compared with the wild type (WT), the overall growth of heterozygosis mount was largely retared abnormal inflammatory. There are significant variations in the expression levels of the typical inflammatory cytokines, such as IL-6 and TNF-α, between the wild-type control and heterozygous duplicated socsl-deficient mounts, rather than IL-1β. After knock-out SOCS1a and SOCS1b blunt snout bream were challenged by intraperitoneal injection with A. hydrophila, In contrast to the wild type, significant increases in levels of IL-6 and TNF-α mRNA were observed in both SOCS1a and SOCS1b heterozygous mutants. The dupliacated socs1 knockout blunt snout bream have been successfully obtained by the CRISPR/Cas9 gene editing system, which provides a basis for further study of the socs1 gene. Meanwhile, our experimental results provide a basis and reference for the study of CRISPR/Cas9 gene editing techniques in other aquaculture species.
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