Construction of ACTA1 Gene Knockout PEFs Cell Lines by CRISPR/Cas9 Editing System
[Objective]The purpose of this study was to establish actin alpha 1(ACTA1)gene knockout porcine embryo fibroblasts(PEFs)by CRISPR/Cas9 system,laying the foundation for subsequent research on the function of ACTA1 gene and exploration of harbor locus in pigs.[Method]Expression of ACTA1 gene in seven tissues(heart,liver,spleen,lung,kidney,dorsal subcutaneous fat and longissimus dorsi muscle)of Bama Miniature pigs was detected by Real-time quantitative PCR.3 single guide RNAs(sgRNAs)were designed in the exon 7 region of the porcine ACTA1 gene by the CRISPOR online website and were ligated into the pX330 vector.The activity of different sgRNA vectors was detected by T7E1 digestion assay,and vector plasmids with higher efficiency and meeting the target were selected for co-transfection into PEFs.Monoclonal cells were screened by limited dilution method and conducting genotype identification and off-target analysis.[Result]The results of Real-time quantitative PCR showed that the expression of ACTA1 gene was extremely significantly higher in the longissimus dorsi muscle than other tissues(P＜0.01).The gene editing efficiencies of 3 sgRNAs were 24.87％(sgRNA1),39.59％(sgRNA2)and 36.93％(sgRNA3),respectively.Based on the cutting position and gene-editing efficiency chose sgRNA1 and sgRNA2 for co-transfected into cells.Genotyping results showed that gene editing occurred in 20 of the 69 monoclonal cells were obtained,of which 1 was biallelic fragment knockout cells and 3 were monoallelic fragment knockout cells,with a fragment knockdown efficiency of 5.8％(4/69).Off-target analysis showed that no off-target effects were detected at the predicted off-target sites.[Conclusion]This study successfully constructed a PEFs with a biallelic knockout of ACTA1 gene using CRISPR/Cas9 editing system,which provided technical and theoretical bases for further research on the regulation of ACTA1 gene on porcine skeletal muscle development,and also provided new idea for exploration of tissue-specific harbor locus in pigs.
万方数据
维普
