关键词: CRISPR/Cas9, A549细胞系, NRF2基因, 敲除

Abstract: Background and purpose: CNC-bZIP is a leucine zipper structure near the carboxyl terminal of homo sapiens nuclear factor (erythroid-derived 2)-like 2 (NRF2), which is necessary for DNA binding, and associated with NRF2 dimerization partners and the small masculoaponeurotic fibrosarcoma (sMaf) proteins. Then this structure binds to the antioxidant response element (ARE) for activating the transcriptional expression of a downstream target gene of NRF2, thereby increasing the cell’s ability to resist oxidative stress. This study aimed to establish the CRISPR/Cas9 lentiviral system, and produce stable NRF2 gene knockout A549 cell line. Methods: A pair of sgRNAs were designed for upstream and downstream structures, and ligated with the pLenti CRISPR v2 vector to construct the stable knockout A549 cell line. The effect was verified by sequencing maps and Western blot. The functions of NRF2 knockout cell line and the A549 cell line without gene knockout were compared by real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR), Western blot, colony formation experiments, cell counting kit-8 (CCK-8), wound-healing assay and transwell assay. Results: As expected, the results showed that the stable A549 cell line with deficient NRF2 expression was successfully obtained. The mRNA expression and protein level of the downstream target genes of NRF2 knockout strains were significantly reduced, and the proliferative, migration and invasion abilities were greatly decreased. Conclusion: CRISPR/Cas9 lentiviral system was used to obtain a highly efficient permanent NRF2 knockout lung cancer cell line, and the proliferation, migration and invasion abilities of A549 lung cancer cells were greatly reduced after NRF2 gene knockout.

Key words: CRISPR/Cas9, A549 cell lines, NRF2 gene, Knockout