Team:SCUT-China B/Results
Transfection
We co-transfected the dCas-VP64-GFP expression under Bax-1 minimal promoter, and under survivin promoter, and Bax-sgRNA-1 into the A549 cells. We analyzed the fluorescence by image pro plus 6.0. The mass ratio of Bax-1-dCas9-VP64-GFP to survivin-dCas9-VP64-GFP is 1:1.
Figure 1.Left: dCas9-VP64 expression under survivin promoter and Bax-1 promoter (B+S).Right: dCas9-VP64 expression under Bax-1 promoter(B).
Figure2. Left: dCas9-VP64 expression under survivin promoter(S). Right: dCas9-VP64 expression under EF1α promoter(E).
Figure 3. The expression of dCas9-VP64-GFP is analyzed by image pro plus 6.0 after 48h.
The dCas9-VP64-GFP expression under survivin promoter(S) was about 5% compared to EF1α promoter(E), and the expression under Bax-1 promoter(B) was about 50% compared to EF1α promoter(E). It shows that our amplifier works as expect that it indeed amplifies the activity of surviving promoter. And the leakage expression of Bax-1 promoter (B) is relative high compared to the expression under EF1α (B+S).
Transcription level of bcl-2 and bax
CRISPRi: we did real-time PCR to validate the transcription level of bcl-2.
Figure 5.dCas9-KRAB co-transfected with Bcl-2-sgRNA-1 to Bcl-2-sgRNA4 (BBa_K2080004, BBa_K2080005, BBa_K2080006, BBa_K2080007) respectively, and we detected the mRNA level after 48 hour.
After analysis of data, we got to validate that the transcription level of bcl-2 under the regulation of CRISPRi is nearly 2 to 3 fold repressed. It confirmed that CRISPRi worked and the sgRNA we designed was efficient.
CRISPRa:
Figure 6.dCas9-VP64 co-transfected with Bax-sgRNA-4(BBa_K2080002) and Bax-sgRNA-10 (BBa_K2080003)respectively, and we detected the mRNA level after 48 hour.
The transcription level of bax is confirmed by real-time PCR, bax is nearly 2 fold to 4 fold activation.
CRISPRi Apoptosis Analysis:
Apoptosis was mostly observed after 48 h. Early apoptosis (lower right quadrant) andlate apoptosis/necrosis (upper right quadrant) were clearly evident in dot plots of Figures below.
Four sgRNAs that we design is validated for dCas9-KRAB to downregulate the bcl-2 mRNA level, and we chose bcl-2-sgRNA1 and bcl-2-sgRNA2, which both show strong repression of bcl-2.
About 10^5 A549 cells were harvested after 48h for apoptosis analysis. Resuspended cells were incubated with Annexin V-FITC for 15 min in the dark. Propidium iodide was used as a counterstain to discriminate necrotic/ dead cells from apoptotic cells.
Figure 7. Apoptosis analysis after Bcl-2-sgRNA1 and dCas9-KRAB were co-trasfected into A549 cells after 48h
Figure 8. Apoptosis analysis after Bcl-2-sgRNA2 and dCas9-KRAB were co-trasfected into A549 cells after 48h.
Figure 9. Apoptosis analysis after negative sgRNA and dCas9-KRAB were co-transfected into A549 cells after 48h.
Figure 10. Apoptosis analysis for no treatment group after 48h.
Apoptosis detection after applying CRISPRi:
Figure 11. Apoptosis index of Bcl2-sgRNA1 and Bcl2-sgRNA2.
These apoptosis results verified the rate of apoptosis ,regulating by the sgRNA1-dcas9, sgRNA2-dcas9, are 14.35%, 11.71%. Both them are nearly increased 2 fold comparing the wild type(7.57%). And the rate of sgRNA1-dcas9 increased 1.83% more than the negative control(12.52%).
Then, we further upregulated the bcl-2 expression by co-transfecting all the bcl2-sgRNAs into the A549 cells. The apoptosis analysis and the apoptotic index results are listed below.
CRISPRa Apoptosis Analysis:
Three sgRNAs that we design is validated for dCas9-VP64 to upregulate the bax mRNA level, and we chose bax-sgRNA-4 and bax-sgRNA-10, which both show 4 fold of activation of bax.
10^5 A549 cells were used for analysis. Resuspended cells were incubated with Annexin V-FITC for 15 min in the dark. Propidium iodide was used as a counterstain to discriminate necrotic/ dead cells from apoptotic cells.
Figure 12. Apoptosis analysis after Bax-sgRNA4 and dCas9-VP64 were co-trasfected into A549 cells after 48h.
Figure 13. Apoptosis analysis after Bax-sgRNA10 and dCas9-VP64 were co-trasfected into A549 cells after 48h.
Figure 14.Apoptosis analysis after negative sgRNA and dCas9-VP64 were co-transfected into A549 cells after 48h
Apoptosis detection after applying CRISPRa:
Figure 15. Apoptosis analysis of Bax-sgRNA-4 and Bax-sgRNA-10.
As expected, the apoptosis rate of sgRNA10(16.03%) increased two more than WT cells(7.57%). And the rate of sgRNA4(14.45%) increased nearly two than wt cells. Besides, both of these rates are larger than negative control(13.10%).
We further upregulated the bax expression by co-transfecting all the bax-sgRNAs into the A549 cells. The apoptosis analysis and the apoptotic index results are listed below.
Taken together, it strongly demonstrated all of the sgRNAs are working in CRISPRi/a in A549. Our project can truly reach the results to promote cancer cells apoptosis.
