| At present,the CRISPR/Cas9 system has become a new technology of targeted gene editing by virtue of the cleavage activity of Cas9 nuclease and the targeting ability of guide RNA.Later,the CRISPR/dCas9 system with the loss of nuclease activity has gradually entered people's vision due to its strong gene regulation ability.The CRISPR/dCas9 system targets THE dCas9 protein at the target site through a short segment of single-stranded guide RNA(sgRNA)with a specific base sequence.sgRNA makes base complementary pairing with the target site,and dCas9 protein also binds to the target DNA strand.By combining the CRISPR/dCas9 system with a variety of effectors,including transcriptional regulators and epigenetic modifications,people have become more flexible in manipulating genome editing.The editable and binding capacity of the CRISPR/dCas9 system,which can recruit effector factors to specific genomic sites,plays a crucial role in this process.Moreover,the system has a wider range of genomic target selection.Combining transcriptional regulators with the CRISPR/dCas9 system can regulate the expression of specific genes in the genome.In this project,a system of protein interaction regulation of gene expression has been established in the laboratory,in which dCas9 protein is connected with a pair of interacting protein pairs and a transcription factor protein is connected at the end of the protein pairs to realize transcriptional regulation of the target genes.And protein in this subject to the above system to replace of p53 and Mdm2 protein,using the flexible connecting peptide dCas9 protein and Mdm2 protein p53 protein and transcriptional activation function of VPR connection into a fusion protein,through and p53 protein Mdm2 protein interaction,combined with the function of target sgRNA,implementation by protein interaction to complete the activation of red fluorescent protein gene of the report..In order to verify the principle of the system is through the protein interaction activation report gene expression,joined the destruction of protein interactions in the system of the specificity of small molecule inhibitors nutlin-3a,the results of the system of transcription activation efficiency drops,proved that the system is achieved by the principle of protein interaction of reporter gene activation.The main results of this paper are as follows:(1)Two fusion protein expression vectors,dCas9-Mdm2 and p53-VPR,were successfully constructed.(2)The plasmids of dCas9-Mdm2,p53-VPR,sgRNA and reporter-dtomato were transferred into 293T cells,which demonstrated the transcriptional activation of this system in cells.(3)Fluorescence microscopy and flow cytometry were used to detect the expression of red fluorescent protein without nutlin-3a and with increasing concentrations of nutlin-3a,and compared with another independent small molecule inhibitor.Results There was a negative correlation between the concentration of nutlin-3a and the expression of red fluorescent protein.The results indicated that nutlin-3a could reduce the transcriptional activation efficiency of the system.(4)Fluorescent quantitative PCR was used to evaluate the mRNA content of red fluorescent protein in the condition of increasing concentration of nutlin-3a and nutlin-3a without nutlin-3a,and compared with another independent small molecule inhibitor.The results indicated that nutlin-3 a concentration was negatively correlated with the expression of red fluorescent protein mRNA,but not the control group.It is further proved that nutlin-3a can reduce the transcriptional activation efficiency of the system.In summary,the present study successfully established a CRISPR/dCas9 transcriptional activation system through p53-Mdm2 protein interaction,and found that the transcriptional activation efficiency of this system was regulated by the small molecule inhibitor nutlin-3 a. |
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