Part:BBa_K2200006
pU6+sgRNA+pHEf1A+hCas9
This part is consisted of Human U6 promoter, sgRNA that targets the specific BRAF sequence. With its high flexibility of being devised to target different tumor gene sequences, it has a great market upon innovating cancer treatments since it will revolutionize this market by personalizing the remedy.
Usage and Biology
This composite part is specially designed to target BRAF V600E mutation. The principle behind this “device” is that when viruses invade bacteria, the self-protection system of bacteria will detect and remove the viruses DNA by a CRISPR/Cas9 system. Recently, scientists created an artificial single-guide RNA (sgRNA), which is able to combine with the Cas9 protein and recognize the target DNA. According to this principle, we designed a sgRNA that matched with the mutated BRAF gene, which brings Cas9 to its target, specifically cleaves the mutated BRAF in malenoma cells, and results in the death of the cancer cells. Through this method, we are theoretically capable of targeting any tumor cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 5117
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 863
Illegal XhoI site found at 1262 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 997
Illegal NgoMIV site found at 4214
Illegal NgoMIV site found at 5123
Illegal AgeI site found at 375 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 5242
Illegal SapI.rc site found at 2633
Illegal SapI.rc site found at 2875
Uncomment this to enable Functional Parameter display
Functional Parameters
In order to confirm the function of our CRISPR/Cas9 system, we performed our functional experiments in two different human melanoma cell lines (A375 and G361) with BRAF V600E mutation. Plasmid pHS-ACR-ZQ170 which encoded Cas9 and sgRNA was constructed for this substantiation.
We transfected A375 and G361 cells with Plasmid pHS-ACR-ZQ170 and utilized the Cell Counting Kit (CCK-8) assay to analyze the quantity of cancer cells by adding CCK solution to each distinguished two-line group every 24 hours. Growth of both cell lines has proved to be inhibited, while the result of A375 was not as obvious as that of G361.
Fig.1 Cell proliferation was significantly inhibited by the CRISPR/Cas9 system both in A375 (A) and G361 (B) cells. The error bars for each time point show the mean ± SD (*P＜0.05, **P＜0.01).
We then performed the transwell assay to detect the effects of our CRISPR/Cas9 system on cell migration. It was obvious that the cell migration rate was lower in the Treatment group both in A375 and G361 cells then in the Negative Control group. These results demonstrated that the mutant allele-specific Cas9 could selectively restrain cell migration in melanoma cells.
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Fig.2 Cell migration was restrained by the CRISPR/Cas9 system. Migration of the infected cells A375 and G361 were measured by the Transwell assay. Cell migration was significantly suppressed in the treatment group in A375 (A) and G361 (C) cells. Each experiment was performed on at least three independent occasions. Error bars show mean ± SD (*P< 0.05).
Finally, we performed flow cytometry to detect effects on cell apoptosis and it was proved that cell apoptosis was inhibited significantly by the synthetic system in melanoma cells.
Fig.3 Cell apoptosis was induced after infection with plasmid and detected by flow cytometry analysis. Apoptosis of infected cells A375 (A) and G361 (C) was measured by flow cytometry. The cell apoptotic rate was significantly increased in the Treatment group in A375 and G361 cells (A-D). Each experiment was performed on at least three independent occasions.
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