采用CRISPR knockin技术实现VEGF165定点敲入HEK293T细胞系的构建

CRISPR-Cas9-based site-directed knock-in of VEGF165 gene in a HEK293T cell

目的构建定点敲入血管内皮生长因子165(VEGF165)基因的人肾上皮细胞系HEK293T,避免由慢病毒感染等方法实现过表达时所带来的脱靶效应。方法根据EZH2基因的DNA序列设计、构建两端带有同源臂的VEGF165表达载体(pUCm-T-VEGF165质粒)和能定点切割基因组DNA的小向导RNA(sgRNA)表达载体[pSpCas9(BB)-2A-Puro-sgRNA质粒],再将这两个质粒共转染HEK293T细胞,通过实时荧光定量PCR检测VEGF165和EZH2的mRNA表达情况,蛋白质印迹法(Western Blot)检测VEGF165和EZH2的蛋白表达情况。结果实时荧光定量PCR和Western Blot结果显示,与对照组、单独转染pUCm-T-VEGF165质粒和pSpCas9(BB)-2A-Puro-sgRNA质粒组比较,共转染pUCm-T-VEGF165和pSpCas9(BB)-2A-Puro-sgRNA质粒组VEGF165的mRNA和蛋白相对表达量均升高(VEGF165 mRNA:3.42±0.30比1.02±0.21、1.13±0.16、0.98±0.18,均P<0.01;VEGF165蛋白:3.12±0.10比1.02±0.06、0.88±0.03、0.80±0.05,均P<0.01),EZH2的mRNA和蛋白相对表达量均降低(EZH2 mRNA:0.14±0.06比1.08±0.11、1.02±0.12、1.13±0.16,均P<0.01;EZH2蛋白:0.23±0.03比1.05±0.13、0.91±0.04、0.81±0.06,均P<0.01)。该结果表明VEGF165基因被定点插入EZH2基因的基因组DNA序列中,并干扰了EZH2的表达。结论采用CRISPR/Cas9系统成功构建了定点敲入VEGF165基因的HEK293T细胞系。

Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system.