利用CRISPR/Cas9编辑系统构建ACTA1基因敲除的PEFs细胞系

doi:10.16431/j.cnki.1671-7236.2024.06.003

摘要/Abstract

摘要： 【目的】利用CRISPR/Cas9技术获得α-肌动蛋白1(actin alpha 1,ACTA1)基因敲除的猪胎儿成纤维细胞(porcine embryo fibroblasts,PEFs),为后续在细胞水平上探究猪ACTA1基因功能及发掘友好基因座位奠定基础。【方法】通过实时荧光定量PCR检测ACTA1基因在巴马小型猪心脏、肝脏、脾脏、肺脏、肾脏、背部皮下脂肪和背最长肌7种组织中的表达情况;利用CRISPOR在线网站在猪ACTA1基因第7外显子区域设计3条向导RNA(single guide RNA,sgRNA),并将其连接至pX330载体;通过T7E1酶切检测不同sgRNA活性,选择效率较高且符合目标的载体质粒共转染至PEFs中;利用有限稀释法筛选单克隆细胞,并对获得的单克隆细胞进行基因型鉴定和脱靶分析。【结果】实时荧光定量PCR结果表明,ACTA1基因在背最长肌中的表达量极显著高于其他组织(P＜0.01)。设计的3条sgRNA的基因编辑效率分别为24.87%(sgRNA1)、39.59%(sgRNA2)、36.93%(sgRNA3),综合切割位置和基因编辑效率,选用sgRNA1和sgRNA2共转染细胞。基因型鉴定结果表明,获得的69株单克隆细胞中有20株单克隆细胞发生了编辑,其中单等位基因片段敲除细胞3株,双等位基因片段敲除细胞1株,片段敲除效率为5.8%(4/69)。脱靶分析结果显示,在预测的脱靶位点未检测到脱靶效应。【结论】本研究利用CRISPR/Cas9基因编辑系统成功构建了ACTA1基因纯合敲除的PEFs,研究结果为进一步探究ACTA1基因对猪骨骼肌发育调控提供了技术支撑和理论基础,同时为挖掘猪组织特异性表达的友好位点提供新思路。

关键词: 猪; CRISPR/Cas9; ACTA1基因; 猪胎儿成纤维细胞(PEFs)

Abstract: 【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.

Key words: pig; CRISPR/Cas9; ACTA1 gene; porcine embryo fibroblasts (PEFs)

中图分类号:

S828.9

张雪萍, 刘嘉仪, 王彦芳, 吴添文. 利用CRISPR/Cas9编辑系统构建ACTA1基因敲除的PEFs细胞系[J]. 中国畜牧兽医, 2024, 51(6): 2273-2284.

ZHANG Xueping, LIU Jiayi, WANG Yanfang, WU Tianwen. Construction of ACTA1 Gene Knockout PEFs Cell Lines by CRISPR/Cas9 Editing System[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(6): 2273-2284.

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