利用CRISPR/Cas9技术构建猪的LDLR基因敲除细胞

Generation of LDLR-knockout pig cells using by CRISPR/Cas9 technology

利用CRISPR/Cas9系统构建猪的低密度脂蛋白受体基因LDLR敲除细胞:根据NCBI数据库猪LDLR基因(Gene ID为396801)序列设计sg RNA靶位点,构建敲除打靶载体,通过电转染猪胎儿成纤维细胞,经G418筛选获得细胞克隆,进行PCR扩增和T载体克隆测序,经序列比对计算基因敲除效率。结果表明,依照CRISPR/Cas9系统GN20GG法则,在LDLR基因第一外显子上设计1条sg RNA,测序结果显示靶序列已正确连接,转染、筛选后共获得30个单细胞克隆,23个测序成功,其中有7个细胞克隆为LDLR基因敲除细胞克隆,敲除效率为30.4%。

The objective of this study was to disrupt the low density lipoprotein receptor gene(LDLR) of pig cells using the CRISPR/Cas9 system.Specific guide RNA(sgRNA) sequences were designed based on the LDLR sequence from the NCBI database(Gene ID 396801).A CRISPR/Cas9 target vector was constructed and porcine embryonic fibroblasts colonies were generated through electrotransfection and G418 screening.Genotype identification was performed on the cell colonies.PCR products were cloned into a T vector and subjected to sequence alignment.The efficiency of gene knockout was analyzed statistically.The sg RNA was designed to target the first exon of the LDLR following the GN20GG rule.DNA sequence assays confirmed the correct construction of the target vector,which was subsequently introduced into porcine fetal fibroblasts using electroporation.In total,30 monoclonal cells were isolated,with successful sequencing of 23 colonies.Among these,seven cell colonies exhibited gene modifications,resulting in a fragment knockout efficiency of 30.4%.