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

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.