A Method For Constructing CRISPR-based Vector To Target DNA Demethylation

Biologists have dreamed of having a technology to regulate gene expression at will.The ransom of the CRISPR/Cas9 system meets this demand.Cas9 is like magic scissors,it specifically recognizes the cleavage target sequence to form a double strand break gap of DNA under the guidance of sgRNA.In recent years,some scientists have studied the structure of Cas9 and then inactivate the activity of Cas9 endonuclease by site-directed mutagenesis and obtained dead Cas9(abbr.dCas9).Under the guidance of sgRNA,dCas9 is able to specifically bind to the target DNA without cutting.If we fuse some activation elements with dCas9,we can specifically over-express the target gene to realize the dream of regulating gene expression.This method has a very significant advantage over traditional over-expression because it is not limited to the size of the gene.It is very convenient to simultaneously up-regulate the expression of multiple genes and the over-expression can be more “nature”.It provides a way to study the complicated human diseases caused by polyprotein and polygenic factors.Current vector-mediated gene activation based on dCas9 has been widely used in vitro studies,but the use of dCas9 to control specific target gene expression in vivo has not been achieved.DNA methylation is an epigenetic process which mainly adds a methyl group to the fifth carbon of the cytosine base in the CpG dinucleotide of DNA.Methylation can change DNA fragment activity without changing its sequence.In mammalian cells,DNA methylation can precisely regulate the expression of genes and thus play a key role in many physiological and pathological processes including cell development and differentiation,gene expression silencing at specific times,genome imprinting and tumorigenesis and so on.Therefore,the development of technologies regulating the DNA methylation status of target genes in vivo will not only help us understand how DNA methylation regulates the expression of genes in specific time and space,but also can actively manipulate gene expressions.And there is a bright future in relevant clinical treatment.DNA demethylation with the Tet-catalytic domain(Tet-CD)can be promoted by Ten-eleven translocation(Tet)dioxygenase-catalyzed oxidation of 5-methylcytosine.In this project,we aimed at the use of CRISPR-dCas9 system fused with Tet-CD protein targeting to DNA demethylation and investigated as following.First,we constructed the All-in-one vector and optimized the dCas9-based CRISPR system.In addition to the fusion of the Tet-CD protein behind the dCas9 protein,MS2-NLS-Tet-CD was also connected via the ribosome binding site IRES.The MS2-NLS-Tet-CD protein allows more expression of the Tet-CD protein in order to increase the efficiency of target DNA demethylation.The promoter region of Makorin ring finger 3(Mkrn3)gene was selected as a target,and the dCas9-sgRNA-Tet1CD-IRES-MS2-NLS-Tet-CD plasmid was transfected into Neuro-2a and fetal mouse primary neurons.The QPCR technique was used to detect expression of the Mkrn3 gene in Neuro-2a cells and fetal rat primary neurons by Crispr-dCas9 activation system.The results showed that the expression of Mkrn3 gene was significantly up-regulated,which was approximately two-fold higher than the control group.This provides good technical support for future experiments.Second,we further constructed the CRISPR-dCas9 activation system targeting Mkrn3 Promoter to demethylate on the basis of lentiviral vectors,and used QPCR technology to detect the expression of activated Mkrn3 gene.We found that the expression of Mkrn3 gene in the experimental group was significantly improved.The expression increase was about 4-fold higher than that of the control group.We hope that the lentiviral system will be more widely used.Finally,the Crispr-dCas9 activation system based on the dCas9-VP64 vector targeted the Mkrn3 Promoter to demethylation.Using the QPCR technique to detect the expression of the activated Mkrn3 gene,we can conclude that the system has a very high efficiency,about 3 Times.While our previous Crispr-dCas9 activation system based on a lentiviral Crispr-dCas9 vector was found to be a higher efficiency which was approximate 4-fold.The proble reason may be due to the fact that the All-in-one vector vector omposition wass as large as15 kb which led to low efficiency of plasmid transfection,thus affecting the detection of Mkrn3 gene expression.In the future,AAV will be used to further verify the efficiency of demethylation in this system.We will also use this system to fuse the methylases DNMT3 a with DNMT3 L to test the efficiency of methylation of certain genes in this system.Due to the close relationship between DNA methylation and some human diseases such as cancers,tumor suppressor genes are prevalently over-methylated and their expression issuppressed or silenced.If we use the dCas9 demethylation system as we have constructed,simultaneous regulation of multiple genes will accelerate the clarification of disease mechanisms and the development of drugs so as to provide potential therapeutic approaches for future gene therapy.In summary,the sgRNA-mediated dCas9 demethylation system developed by us will have a bright future in epigenetics editing and clinical research.