围脂滴蛋白基因CRISPR/Cas9载体的活性分析

Construction and Activity Analysis of the PLIN1 Gene CRISPR/Cas9 Vector

设计并验证能够靶向切割PLIN1基因的sgRNA,为建立高效的PLIN1基因敲除动物模型奠定基础。利用预测软件设计3条评分较好的sgRNA。添加T7启动子后体外转录相应的sgRNA,构建酶切体系并验证其体外切割活性。构建相应的基因敲除质粒载体,通过电转染将质粒转到3T3-L1前脂肪细胞内并进行诱导分化。RT-PCR法检测细胞中PLIN1 mRNA的表达,Western blot法检测细胞中PLIN1蛋白的表达。成功在体外转录3条PLIN1的sgRNA并构建酶切体系,测得sgRNA-PLIN1-1的切割效率为61.8%±9.0%,sgRNA-PLIN1-2的切割效率为64.1%±9.6%,sgRNA-PLIN1-3的切割效率为34.1%±7.2%,sgRNA-PLIN1-3组的切割效率明显低于sgRNA-PLIN1-1组和sgRNA-PLIN1-2组(P<0.05)。成功构建3条sgRNA的质粒载体并电转染进入3T3-L1前脂肪细胞内,转染效率约为30%。诱导分化的第4天,各阳性干扰组PLIN1 mRNA的表达量显著降低(P<0.01);与sgR NA-PLIN1-3组相比,sgRNA-PLIN1-1组和sgRNA-PLIN1-2组PLIN1 mRNA的表达量有显著性差异;各阳性干扰组PLIN1蛋白的表达量显著降低(P<0.01),阳性干扰组相互之间PLIN1蛋白的表达量无显著性差异。3条sgRNA皆可以靶向切割PLIN1基因的2号外显子,其中gRNA-PLIN1-1组和sgRNA-PLIN1-2组的切割活性略高于sgRNA-PLIN1-3组。体外活性切割实验可以很好的预测细胞内切割效果,是筛选高活性sgRNA的有效手段。

The objectives of this work are to design and validate the sgRNA being capable of targeted cleaving the PLIN1 gene and to lay a foundation for the establishment of a highly efficient PLIN1 gene-cleaved animal model.Three sgRNAs with favorable evaluation results were designed using predictive software.After adding T7 promoter,the corresponding sgRNA was transcribed in vitro to construct the digestive system and to verify the cleavage activity in vitro.The corresponding gene knockout plasmid vector was constructed,and the plasmid was transferred into 3 T3-L1 preadipocytes by electroporation and induced to differentiate.The expression of PLIN1 mRNA in cells was detected by RT-PCR.The expression of PLIN1 protein in cells was detected by Western blot.Three sgRNAs of PLIN1 were successfully transcribed in vitro and the restriction enzyme system was constructed.The cleavage efficiency of sgRNA-PLIN1-1 was 61.8%±9.0%,and that of sgRNA-PLIN1-2 was 64.1%±9.6%.The cleavage efficiency of sgRNA-PLIN1-3 was 34.1%±7.2%,and that of sgRNA-PLIN1-3 group was significantly lower than that of sgRNA-PLIN1-1 group and sgRNA-PLIN1-2 group(P<0.05).The plasmid vector of three sgRNAs was successfully constructed and electroporated into 3 T3-L1 preadipocytes,and the transfection efficiency was about 30%.The expression of PLIN1 mRNA in each positive interference group(P<0.01)significantly decreased at the 4 th day after differentiation.Compared with sgRNA-PLIN1-3 group,there was a significant difference in the expression level of PLIN1 mRNA in sgRNA-PLIN1-1 group and sgRNA-PLIN1-2 group.The expression of PLIN1 protein in each positive interference group significantly decreased(P<0.01),and there was no significant difference in the expression of PLIN1 protein between the positive interference groups.In conclusion,all three sgRNAs may cleave the exon 2 of the PLIN1 gene in a target manner,and the cleavage activity of the gRNA-PLIN1-1 group and the sgRNA-PLIN1-2 group is slightly higher than that of the sgRNA-PLIN1-3 group.In vitro active cleavage experiments may well predict intracellular cleavage and are an effective means of screening highly active sgRNA.