Difference between revisions of "Part:BBa K2200006"

Revision as of 14:04, 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 5117
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 863
Illegal XhoI site found at 1262
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE 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
1000
INCOMPATIBLE 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.

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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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.

@@ Line 9: / Line 9: @@
 ===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 [1] 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.
+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.