CRISPR/Cas13d介导猪胎儿成纤维细胞SUV39H1/SUV39H2基因敲降

CRISPR/Cas13d-mediated SUV39H1/SUV39H2 Gene Knockdown in Porcine Embryonic Fibroblasts

研究旨在利用CRISPR/Cas13d系统对猪胎儿成纤维细胞(PEF)中的胚胎发育相关基因SUV39H1/SUV39H2进行RNA水平的敲降,从而在猪上建立CRISPR/Cas13d介导的基因敲降系统。根据SUV39H1、SUV39H2基因的编码序列各设计3个靶向编码链的sgRNAs,并以单链寡核苷酸的形式合成,退火后与BspQⅠ线性化的sgRNA表达载体进行连接,构建SUV39H1-sgRNA和SUV39H2-sgRNA表达载体,用Sanger软件进行测序;将Cas13d表达载体和靶向SUV39H1/SUV39H2基因的sgRNA载体按照1∶1、1∶2、1∶4、2∶1和4∶1比例转染猪胎儿成纤维细胞,48 h后收集细胞,用流式细胞术分选检测细胞转染效率,用半定量PCR和实时荧光定量PCR检测敲降效率;用半定量PCR和免疫荧光检测敲降SUV39H1/SUV39H2基因后,靶基因转录水平及组蛋白H3K9me3水平的变化。测序结果表明,针对2个基因设计的各3条sgRNAs均成功连入载体中;流式细胞术分选结果显示,转染效率约70%;半定量PCR结果表明,与对照组相比,3个sgRNAs均极显著降低了SUV39H1和SUV39H2基因的表达(P<0.01),其中SUV39H1-sgRNA-2和SUV39H2-sgRNA-1均可使SUV39H1和SUV39H2的表达降低50%;Cas13d∶sgRNA为1∶1、1∶2和1∶4组细胞的存活率高于2∶1和4∶1组;实时荧光定量PCR结果表明,Cas13d∶sgRNA为1∶2、1∶4、2∶1和4∶1组敲降效率均显著高于1∶1组(P<0.05),且Cas13d∶sgRNA为1∶2组敲降效率最高(70%)。半定量PCR结果显示,转染SUV39H1-sgRNA-2和SUV39H2-sgRNA-1极显著降低SUV39H1和SUV39H2的表达,表达量为对照组的25%~30%(P<0.01)。免疫荧光检测结果表明,敲降SUV39H1和SUV39H2基因后,组蛋白H3K9me3水平显著降低(P<0.05)。因此,本研究利用CRISPR/Cas13d系统在猪胎儿成纤维细胞中成功敲降SUV39H1和SUV39H2,并下调其催化的H3K9me3水平。

This study was aimed to knockdown the embryonic development related genes SUV39H1/SUV39H2 in porcine embryonic fibroblasts(PEFs)by CRISPR/Cas13d system,so as to establish a CRISPR/Cas13d mediated gene knockdown system in pigs.According to the coding sequences of SUV39H1/SUV39H2 genes,3 sgRNAs were designed and synthesized in the form of single stranded oligonucleotides.After annealing,they were connected with the sgRNA expression vector linearized by BSPQⅠ.The sgRNA-expressing vectors of SUV39H1 and SUV39H2 genes sgRNA were constructed and sequenced by Sanger software.The expression vector of Cas13d and sgRNA vector targeting SUV39H1/SUV39H2 genes were transfected into PEFs at the ratio of 1∶1,1∶2,1∶4,2∶1 and 4∶1.After 48 h,the cells were collected,and the transfection efficiency was detected by flow cytometry.The knockdown efficiency was detected by semi-quantitative PCR and Real-time quantitative PCR.Semi-quantitative PCR and immunofluorescence were used to detect the level of target gene transcripts and histone H3K9me3 after SUV39H1/SUV39H2 genes knockdown.Sequencing results showed that 3 sgRNAs for each of two genes were successfully inserted into the vector.Flow cytometry results showed that the transfection efficiency was about 70%.Semi-quantitative PCR results showed that 3 sgRNAs extremely significantly reduced the expression of SUV39H1/SUV39H2 genes compared with control group(P<0.01).SUV39H1-sgRNA-2 and SUV39H2-sgRNA-1 could reduce the expression of SUV39H1 and SUV39H2 by 50%.The survival rate of Cas13d∶sgRNA in 1∶1,1∶2 and 1∶4 groups were higher than that of 2∶1 and 4∶1 groups.The knockdown efficiency of Cas13d∶sgRNA in 1∶2,1∶4,2∶1 and 4∶1 groups were significantly higher than that of 1∶1 group(P<0.05),and the knockdown efficiency of Cas13d∶sgRNA in 1∶2 group was the highest(70%).Semi-quantitative PCR results showed that transfection of SUV39H1-sgRNA-2 and SUV39H2-sgRNA-1 extremely significantly reduced the expression of SUV39H1 and SUV39H2,and the expression level was 25%-30%of control group(P<0.01).The results of immunofluorescence showed that the level of histone H3K9me3 decreased significantly after SUV39H1 and SUV39H2 genes knockdown(P<0.05).Therefore,in this study,CRISPR/Cas13d system was used to knockdown SUV39H1 and SUV39H2 successfully,and downregulate H3K9me3 level catalyzed by them.