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.
