利用CRISPR/Cas9基因编辑技术敲除团头鲂socs1重复基因  

Knock-out analysis of duplicated socs1 using CRISPR/Cas9 in Megalobrama amblycephala

作　　者：赵心愉 刘娟[1] 邹曙明[1] ZHAO Xinyu;LIU Juan;ZOU Shuming(Genetics and Breeding Center for Blunt Snout Bream,Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rurall Affairs,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学,农业农村部团头鲂遗传育种中心,农业农村部淡水水产种质资源重点实验室水产科学国家级实验教学示范中心,上海201306

出　　处：《水产学报》2020年第12期1937-1947,共11页Journal of Fisheries of China

基　　金：国家自然科学基金(31572220,2012BAD26B00);上海高校知识服务平台(ZF1206)。

摘　　要：以团头鲂细胞因子信号传导抑制蛋白1(SOCS1)基因socs1a和socs1b为编辑对象,在线分析并筛选出合适的靶点合成向导RNA (guide RNA, gRNA),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。通过荧光定量PCR技术在转录水平检测socs1a和socs1b的表达,并通过测序平台在DNA水平鉴定基因序列的突变,成功建立了多种socs1的突变品系。与同期野生型(WT)相比,socs1a突变杂合体(SOCS1a^+/-)和socs1b突变杂合体(SOCS1b^+/-)团头鲂生长性能增强、体质量显著增加,同时炎症因子基因中肿瘤坏死因子α基因(TNF-α)和白细胞介素6基因(IL-6)表达量显著增加,而白细胞介素1β基因(IL-1β)表达无明显变化。注射嗜水气单胞菌后,与野生型不同的是,IL-6和TNF-α在SOCS1a^+/-和SOCS1b^+/-团头鲂肝脏中的表达均有持续显著上升。本实验通过CRISPR/Cas9系统成功敲除了团头鲂socs1a和socs1b,为进一步探讨socs1的作用提供了一定的研究基础,同时为CRISPR/Cas9基因编辑技术在养殖鱼类中的合理应用提供了依据和借鉴。In this study, the suppressor of cytokine signaling 1(SOCS1) genes socs1 a and socs1 b of Megalobrama amblycephala were used as genetic editing objects, and we screened out appropriate target synthetic guide RNA(gRNA) by online analysis. According to the mixture injection(gRNA and Cas9 protein) in the 1-2 cell stage embryos, the results of identifications of socs1 expression level by qRT-PCR and gene mutants by sequencing showed that we successfully established socs1 knock-out mutant. Compared with wild type, the growth performance and body mass of SOCS1 a^+/-and SOCS1 b^+/-were significantly increased, Meanwhile, the expression levels of inflammatory cytokines TNF-α and IL-6 were significantly increased, while the expression levels of IL-1β were not changed. After Aeromonas hydrophila injection, in contrast to the wild type, significant increases in levels of IL-6 and TNF-α mRNA were observed in both socs1 a and socs1 b heterozygous mutants. The duplicated socs1 knockout blunt snout bream has 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 will provide a basis and reference for the CRISPR/Cas9 gene editing techniques in other aquaculture species.

关 键 词：团头鲂 CRISPR/Cas9 SOCS1 基因编辑 

分 类 号：Q789[生物学—分子生物学] S965.1[农业科学—水产养殖]