Abstract: This study aimed to investigate the cleavage activity of four sgRNAs designed for exon 2 of the chicken β?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 awebsite and inserted into lentiviral vectors expressing green fluorescent proteins. Lentiviral fluids were then used to infectachicken 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.

Key words: Chicken；Knockout efficiency；sgRNA；ACTB gene

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。

关键词: 鸡；切割活性；sgRNA；ACTB 基因