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OBJECTIVE To study the potential resistance gene and molecular mechanism of osimertinib in non-small cell lung cancer cells. METHODS Using genome-wide CRISPR/Cas9-based screening, we identified the potential resistance gene ferredoxin 1 (FDX1) from NCI-H1975 cells. Then, the stable cell lines sg-FDX1 and sg-adeno-associated virus integration sitel (sg-AAVS1) were established after knockout of FDX1 and AAVS1 as control. Genomic DNA was extracted from sg-FDX1 cells and the DNA sequences on both sides of the sgRNA were amplified and connected to the T carrier for sequencing. Western blotting was used to detect the protein expression levels of FDX1 at the same time. sg-FDX1 or sg-AAVS1 cells were treated with osimertinib at 0, 30, 50 and 100 nmol-L'1 for 30 h and the expression levels of cleaved poly (ADP-ribose) polymerase (c-PARP), phosphorylation of epidermal growth factor receptor (p-EGFR) and phosphorylation of extracellular regulated protein kinases (ERK) were detected by Western blotting. The clones were grown to about 10 cells that were treated with osimertinib at 12 nmol-L'1 for 10 d, and the number of clones with ≥10 cells was counted under the microscope using the plate cloning assay. CCK8 assay was used to detect the survival rate of tumor cells after they were treated with osimertinib from 0 to 30 nmol • L-1 for 72 h, or 30 nmol • L-1 for 3, 5 and 7 d. RESULTS Sequencing results showed that FDX1 gene was deleted and FDX1 expression was knocked out effectively. The level of c-PARP expression was significantly reduced in sg-FDX1 cells, compared with sg-AAVS1 cells after osimertinib 50 nmol-L-1 treatment (P<0.01). The inhibition of osimertinib on phosphorylation level of EGFR and ERK was reduced in sg-FDX1 cells, compared with sg-AAVS1 cells (P<0.01). Cloning formation and CCK8 experiments showed that osimertinib inhibition on cloning formation rate and proliferation rate were decreased significantly in sg-FDX1 cells compared with sg-AAVS1 cells (P<0.05). CONCLUSION NCI-H1975 cells with FDX1 knockout can weaken osimertinib-induced apoptosis, which accounts for the mechanism by which FDX1 is involved in the resistance of NCI-H1975 cells to osimertinib.
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Objective CRISPR/Cas9 genome-editing technique provides an novel method for whole genome editing in eukaryotic cells.Recently,we found that gene subtype library with smaller size and focused pur-pose is more economical and practical. In this study,we aimed to target kinases,a group of pivotal cell signal transducers,to construct a kinase knock-out library using CRISPR/Cas9 technique.The construction strategy wll al-so be discussed. Methods 10 sgRNA was designed for each kinase target.After oligo pool synthesis by semicon-ductor chip,the oligos were eluted from the chip. The oligo templates were amplified and cloned into Cas9 vector and transformed into Stble3 competent cells.Monoclonal colonies were selected for DNA sequencing. Results(1) GO analysis of 507 cell kinases showed that the cell kinases took part in a wide range of cell signaling.(2)The sgRNA pool with about 140 bp in length was successfully amplified by using oligo pool as the template and univer-sal PCR primers.(3)In 40 identified library clones,34 clones were sequenced successfully. Among them,the DNA sequencing results of 25 samples were completely consistent with the designed target sequences.But there are some mutations in the primers of 9 samples.Failure in bacteria shaking,DNA sequencing and other factors were ex-isted in the other clones. Conclusion The CRISPR/Cas9 kinase knock-out library can be widely used for screen-ing the important kinases which may mediate cell proliferation,metastasis,drug resistance and autophagy.This li-brary will play an important role in clarifying the development of disease associated with kinases.