构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。

Construction of Plasmids for Knocking out ALOX5 Gene Using CRISPR/Cas9 Technology

This work aims to construct the plasmids targeting for ALOX5 gene knockout by CRISPR/Cas9 technology.Three pairs of oligonucleotides sgRNAs targeting exon 6 of ALOX5 gene were designed,chemically synthesized,and inserted into linearized plasmids pX458,respectively.The recombinant plasmids pX458-sgRNAs-ALOX5 was then transformed into competent Escherichia coli DH5α.Whether or not recombinant plasmids were constructed successfully was evaluated by Sanger sequencing.The constructed recombinant plasmids were transfected into 293T cells,and the transfection efficiency was observed under fluorescence microscope.Then their genomic DNA derived from the successfully-transfected cells were extracted by the reagent kit.The DNA fragment with target gene knockout was amplified by PCR,the nucleotides were obtained by sequencing,and the knockout of ALOX5 gene in the transfected cells was analyzed by DNAStar software.The sequencing results revealed that 2 pairs of double stranded sgRNA oligodeoxynucleotides were successfully inserted into the plasmids with correct sequences,and the construction of recombinant plasmid pX458-sgRNAs-ALOX5 targeting ALOX5 gene was successful.The transfection efficiency of pX458-sgRNAs-ALOX5 in 293T was about 50%;however,cleaving effect was not detected by Sanger sequencing.In summary,the CRISPR/Cas9-based plasmids pX458-sgRNAs targeting the exon 6 of ALOX5 gene is successfully constructed.