Team:SDU CHINA/design
In our project, we aimed to construct two plasmids based on CRISPR technique to cleave PD-L1 gene in cancer cells and make sure they are safe for normal cells simultaneously. With the help of sgRNA and tumor specific promoters, our device become sensitivity and specificity, which means sgRNA can guide plasmids to the correct site for cleavage and the tumor specific promoter can switch on the cas 9 expression at the proper time and proper location. The specificity of our project is that after the gene of PD-1 ligands is cut off, the transferred plasmids will commit suicide. Even if the plasmids are transferred into normal cells, plasmids will also cleave and degrade themselves. The information of basic plasmid we used is as follows:
Figure1:Plasmid lentiCRISPR V2 map
The skeleton of plasmid is lentiCRISPR V2, which is shown in Figure1. Based on the lentiCRISPR V2, we constructed our first plasmid, shown in Figure 2. U6 promoter is used for expression gRNA1 targeting for PD-L1, and the hTERT (human telomerase reverse transcriptase) or survivin promoter is inserted between gRNA1 and Cas9 gene, which is followed by tetR. Here, the gRNA1 is to guide cas9 enzyme to find the cutting position; hTERT or survivin promoter are tumor-specific promoters which can only start expression in tumor cells; the protein of tetR can bind with the tetracycline operon.
Figure2:The schematic diagram of our first plasmid
The hTERT promoter only starts transcription in tumor cells and acts as a regulatory element for antitumor genes to initiate antitumor gene-specific expression in the downstream of tumor cells, which can avoid damage to normal cells. Survivin belongs to the family of apoptosis inhibitors (IAPs), which antagonizes the induction of cell death. Dysregulated expression of IAPs is frequently observed in cancers, and the high levels of survivin expression in tumor cells makes it an attractive target for pharmacological interventions.
Construction of the second plasmid is similar to the first one. tetR repressible promoter was used for starting cas9 and gRNA2 expression. Here, the tetR repressible promoter can be repressed by tetR protein which is expressed by the first plasmid, so it can not start the expression of cas9 and gRNA2 in the case of tetR present. gRNA2 is designed targeting for the original replication region of the two plasmids, and it can guide cas9 to find cutting site on the plasmid which is used for plasmids suicide.
Figure3:The schematic diagram of our second plasmid
Due to the specificity of hTERT and survivin promoter in tumor cell, when the constructed two plasmids were transfected into the cancerous cells, the first constructed plasmid would express and the second constructed plasmid would be inhibited by tetR protein. In addition, gRNA1 would guide cas9 protein to cut PD-L1, and the cancer cells would gradually differentiate to normal celsl or die. So, we will achieve our goal to cure cancer. However, if the two plasmids were transfected into normal cells, hTERT or survivin promoter wouldn’t work, tetR does not expression, but tetR repressible promoter would initiate the expression of Cas9 and gRNA2. The latter would guide cas9 protein to cleave the original replication region of the two plasmids to commit suicide which make safe for the normal cells.
Figure4:The mechanism of our two plasmids working together
