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.