采用CRISPR/Cas9系统构建HIF-1α基因敲除质粒及功能验证

Construction of HIF-1αgene knockout plasmid using CRISPR/Cas9 system and its functional verification

目的采用簇状规则间隔短回文重复序列(CRISPR)/Cas9基因编辑技术构建质粒并敲除HEK-293细胞的HIF-1α基因。方法通过在线软件设计3号外显子上靶向HIF-1α的sgRNA,并将其构建到CRISPR/Cas9载体中。经Sanger测序验证质粒构建正确后,将重组质粒通过Lipofectamine 3000转染到HEK-293细胞中。通过药物筛选提取细胞DNA和蛋白,用T7E1酶和Western blotting检测基因变化和蛋白表达情况。结果Sanger测序结果显示,所设计的sgRNA成功插入到CRISPR/Cas9载体中,序列验证正确,重组质粒构建成功。T7E1酶切实验成功切除3条带,sgRNA的打靶效率为33.8%。Western blotting结果显示,经过氯化钴诱导、转染药筛后的HEK-293细胞蛋白表达水平较野生型细胞明显下降,差异有统计学意义(P<0.05)。结论成功构建靶向HIF-1α基因的CRISPR/Cas9载体,载体靶向敲除HIF-1α基因功能验证成功。

Objective To use Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/Cas9 gene editing technology to construct plasmids and knock out HIF-1αgene in HEK-293 cells.Methods The sgRNA targeting HIF-1αon exon 3 was designed by online software and constructed into the CRISPR/Cas9 vector.After Sanger sequencing verified that the plasmid was constructed correctly,the recombinant plasmid was transfected into HEK-293 cells by Lipofectamine 3000.Cell DNA and protein were extracted after drug screening,and gene changes and protein expression were detected by T7E1 enzyme and Western blotting.Results The Sanger sequencing results showed that the designed sgRNA was successfully inserted into the CRISPR/Cas9 vector,the sequence was verified correctly,and the recombinant plasmid was successfully constructed.The T7E1 digestion experiment successfully excised 3 bands,and the targeting efficiency of sgRNA was 33.8%.Western blotting results showed that the protein expression level of HEK-293 cells induced by cobalt chloride and transfected with drug screening was significantly lower than that of wild-type cells,and the difference was statistically significant(P<0.05).Conclusion The CRISPR/Cas9 vector targeting the HIF-1αgene is successfully constructed,and the function of the vector targeting and knocking out the HIF-1αgene is successfully verified.