The effects of the CRISPR/Cas9 system on the proliferation, migration and apoptosis in melanoma cells.
Cell proliferation was inhibited by the CRISPR/Cas9 system in melanoma cells. The CCK-8 assay was used to investigate the effects of the CRISPR/Cas9 system on cell growth in melanoma cells. Compared with the negative control, cell growth was significantly inhibited in A375 cells (Fig.1A) and G361 cells (Fig.1B) after treatment of the CRISPR/Cas9 system.
Cell migration was suppressed by CRISPR/Cas9 system in melanoma cells. The transwell assay was performed to detect the effects of these devices on cell migration. Compared with the NC group, the cell migration rate was obviously lower in the Treatment group both in A375 and G361 cells (Fig 2. A-D).
Cell apoptosis was significantly promoted by the CRISPR/Cas9 system in melanoma cells. Flow cytometry was performed to detect the effects of the CRISPR/Cas9 system on cell apoptosis. As shown in Fig. 3 A-D, compared with the negative control group, cell apoptosis was promoted significantly in A375 and G361 cells in the Treatment group. The results show that cell apoptosis was inhibited significantly by the synthetic system in melanoma cells.
DNA sequencing demonstrated that the CRISPR/Cas9 system specifically cleaved the mutant, but not wide-type BRAF gene in melanoma cells
With TA cloning technology, twenty-one single clones from the transfected cells were selected for DNA sequencing, and the results showed that the mutated BRAF gene was cleaved in 3 of 21 clones (Fig. 4), but no any wild-type BRAF gene was cleaved in the 21 clones (Fig. 4).
The future plan
In the future, we would like to verify the system if it can work as expected. Then we would put the regulatory system and CRISPR/Cas9 system together and measure the growth of normal cells and melanoma cells to see whether Cas9 protein only expresses in melanoma cells.
Next, we will pack the vectors containing the amiRNA, amiRNA binding-site, Cas9 and sgRNA into lentivirus in order to increase the transfection efficiency and use them to deliver the constructs into the target cells in the functional experiments.
Finally, we also want to sequence the whole genome of the transfected cell and see whether we can reduce the off-target effects on the melanoma cells by the system.