Difference between revisions of "Part:BBa K2200006"

Revision as of 18:34, 30 October 2017

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

The 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 detect the viruses’ DNA. The bacteria produces two kinds of RNA, one of which contains a sequence that matches the DNA of invading viruses, and this sequence is regarded as guide RNA(also small guide RNA, sgRNA). These two RNA form a complex with Cas9 protein. When sgRNA finds its target, Cas9( the DNA scissor) then cuts the target DNA sequence. Jennifer Doudna and Emmanuelle Charpentierwe found a way to simplify the long pre-crRNA and tracrRNA sequence by physically linking them and creating an artificial single-guide RNA (sgRNA). The 20nt-long sgRNA has the same function as normal crRNA-tracrRNA, and is able to combine with the protein Cas9 and recognize the target DNA. In order to conduct this principle, which is called CRISPR-Cas9, we designed a sgRNA that matches mutant BRAF gene. This sgRNA then brings Cas9 to its target to cleave the mutant DNA. It then causes the death of those cancer cells. Using this method, we are theoretically capable of targeting any tumor cells. This kind of innovated and precise methodology can be adopted to track patients throughout their life without getting their normal cells into danger and within suppressing the dispersion of cancer cells. U6 promoter drives the expression of sgRNA and HEf1A promoter drives the expression of Cas9.

Sequence and Features

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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.