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