慢病毒介导的CRISPR/Cas9靶向ACTB基因sgRNA的设计及活性验证  

Design and Validation of the Effectiveness on Lentivirus⁃mediated CRISPR/Cas9 Targeting sgRNA for the ACTB Gene

作　　者：朱新宇 何燕华 李晓娇 刘子敬 张晓爱 罗成龙 ZHU XinYu;HE YanHua;LI XiaoJiao;LIU ZiJing;ZHANG XiaoAi;LUO Chenglong(College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou Guangdong 510225;Institute of Animal Science,Guangdong Academy of Agricultural Sciences/State Key Laboratory of Swine and Poultry Breeding Industry/Guangdong Key Laboratory of Livestock Breeding and Nutrition Research,Guangzhou Guangdong 510640;Agro-Biological Gene Research Center,Guangdong Academy of Agricultural Sciences,Guangzhou Guangdong 510640)

机构地区：[1]仲恺农业工程学院动物科技学院,广东广州510225 [2]广东省农业科学院动物科学研究所/猪禽种业全国重点实验室/广东省畜禽育种与营养研究重点实验室,广东广州510640 [3]广东省农业科学院农业生物基因研究中心,广东广州510640

出　　处：《广东畜牧兽医科技》2024年第2期23-29,36,共8页Guangdong Journal of Animal and Veterinary Science

基　　金：国家自然科学基金青年科学基金(32102539);广东省重点领域研发计划项目(2022B0202110002);广东省农业科学院协同创新中心项目(XT202217);广州市科技计划项目(202201011640);广东省科技创新战略专项(高水平农科院建设)“金颖之光”(R2020PY-JG008);国家肉鸡产业技术体系岗位科学家项目(CARS-41)。

摘　　要：该实验旨在利用CRISPR/Cas9基因编辑系统对鸡β-肌动蛋白(β-actin,ACTB)基因外显子2上设计的4条sgRNA进行切割活性验证,为在ACTB基因位点定点敲入外源基因提供工作基础。本实验通过网站设计四条特异性识别ACTB基因的sgRNA序列,构建到带有表达绿色荧光蛋白的慢病毒载体上,在293T细胞上包装慢病毒,然后将慢病毒液感染稳定表达Cas9蛋白的鸡成纤维细胞系(DF-1)验证sgRNA的切割活性。通过细胞荧光表达分析、T7E1内切酶和体外活性检测,鉴定出有切割活性的3条sgRNA,为后续在DF-1细胞上进行功能验证、基因定点敲入奠定了实验基础,同时为后续在鸡这一物种中进行基因编辑提供了研究材料。This study aimed to investigate the cleavage activity of four sgRNAs designed for exon 2 of the chickenβ-actin(ACTB)gene using the CRISPR/Cas9 gene editing system.The goal was to establish a framework for targeted knock-in of exogenous genes at the ACTB gene locus.Four sgRNA sequences,specific to the ACTB gene,were designed using a website and inserted into lentiviral vectors expressing green fluorescent proteins.Lentiviral fluids were then used to infect a chicken fibroblast cell line(DF-1)that stably expresses Cas9 proteins.The knockdown efficiency of the sgRNAs was validated through cell fluorescence expression analysis,T7E1 endonuclease assay,and in vitro activity assay.Three sgRNAs with cleavage activity were identified,providing a solid experimental foundation for subsequent functional validation,gene targeting knock-in on DF-1 cells,and research materials for gene editing in chickens.

关 键 词：鸡 切割活性 sgRNA ACTB基因 

分 类 号：S813[农业科学—畜牧学]