摘要
[目的]本文旨在利用Cas9/sgRNA体系,提高猪胎儿成纤维细胞的基因组编辑效率。[方法]针对猪ROSA26位点的启动子区(包含第一外显子区)设计2条sgRNA,将其构建在PX459-cas9-puro质粒上,以获得PX459-cas9-sgRNA质粒。将sgRNA靶序列连同原间隔序列(protospacer-adjacent motif,PAM)构建在RGS-CR质粒上,以获得RGS-sgRNA质粒。使用PX459-cas9-sgRNA质粒与RGS-sgRNA质粒系统共转染,筛选打靶效率较高的一条sgRNA,并使用该sgRNA对猪胎儿成纤维细胞(PEF)进行后续基因打靶试验。分别使用PX459-cas9-sgRNA质粒和Cas9/sgRNA 2种方法对PEF的靶位点进行基因敲除,将转染后的细胞提取基因组DNA,以桑格尔测序检测打靶效率。[结果]成功构建了打靶猪ROSA26靶序列的PX459-cas9-sgRNA质粒和含PAM序列的RGS-sgRNA,质粒测序结果显示序列插入正确;RGS-sgRNA筛选结果表明,sgRNA2的打靶效率极显著高于sgRNA1(P<0.01),且sgRNA2无脱靶效应,因此后续使用PX459-cas9-sgRNA2和Cas9/sgRNA2在PEF中进行基因打靶试验,并比较两者的基因打靶效率。在猪胎儿成纤维中,PX459-cas9-sgRNA2质粒的打靶效率为15.67%,Cas9/sgRNA2体系的打靶效率为33.33%。[结论]Cas9/sgRNA体系较PX459-cas9-sgRNA质粒打靶效率提高了约1倍,这对提高猪体细胞的基因编辑效率和促进基因编辑在猪上的应用有重要意义。
[Objectives]The aim of our study was to improve the efficiency of genome-editing in porcine embryonic fibroblasts(PEF)by Cas9/sgRNA system.[Methods]Two sgRNA were designed for the promoter region(first exon included)of porcine ROSA26 locus,and then they were constructed into PX459-cas9-puro plasmid to form PX459-cas9-sgRNA plasmids.The target sequence of sgRNA and their protospacer-adjacent motif(PAM)was constructed into RGS-CR to form RGS-sgRNA plasmid.The PX459-cas9-sgRNA and RGS-sgRNA plasmid system were combined to screen the sgRNA which had higher genome-editing efficiency,and this sgRNA was used to conduct subsequent transfection experiments on PEF.PEF cells were transfected with PX459-cas9-sgRNA2 plasmid and Cas9/sgRNA2,respectively.Genomic DNA was extracted from the transfected cells,and the targeting efficiency was measured by Sanger sequencing.[Results]PX459-cas9-sgRNA targeting ROSA26 sequence and RGS-sgRNA2 plasmids containing PAM were successfully constructed,and Sanger sequencing showed that the sequence of sgRNA1 and sgRNA2 was inserted correctly.RGS-sgRNA system showed that the editing efficiency of sgRNA2 was significantly higher than that of sgRNA1(P<0.01),and sgRNA2 showed no off-target in porcine genome,therefore,PX459-cas9-sgRNA2 and Cas9/sgRNA2 were used to compare the editing efficiency in PEF cells.The editing efficiency of PX459-cas9-sgRNA2 plasmid in PEF was 15.67%,and the editing efficiency of Cas9/sgRNA2 system was 33.33%.[Conclusions]Compared with PX459-cas9-sgRNA2 plasmid,Cas9/sgRNA2 can improve the targeting efficiency by about 1 fold,which improves genome-editing efficiency of pig somatic cell and is of great significance for promoting application of gene-editing in pigs.
作者
姜爱文
郭鸿运
吴望军
刘红林
JIANG Aiwen;GUO Hongyun;WU Wangjun;LIU Honglin(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2023年第3期545-554,共10页
Journal of Nanjing Agricultural University
基金
江苏省种业振兴揭榜挂帅项目(JBGS〔2021〕026)。
