Design and Validation of the Effectiveness on Lentivirus-mediated CRISPR/Cas9 Targeting sgRNA for the ACTB Gene
ZHU XinYu
HE YanHua
LI XiaoJiao
LIU ZiJing
Zhang XiaoAi
LUO Chenglong
ZOU Xian
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 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.
Keywords：ChickenKnockout efficiencysgRNAACTB gene
Publication Date：2024-04-18
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：8( 23-29,36 )