Induction Of Foxp3 Gene Over-expression And Regulatory T Cells Differentiation In Primary Culture Of T Cells By Using CRISPR-dCas9 Technology

Autoimmune diseases are characterized by aberrant immune responses against healthy cells and normal tissues.Treg cells(CD4+CD25+ regulatory T cells)are a subset of mature T cells which play an important role in maintaining immune homeostasis and preventing autoimmune diseases.In addition,large amounts of studies suggested that Treg cells unnormal may be a potential cause of human autoimmune diseases.Such as systemic lupus erythematosus(SLE),immune dysregulation,polyendocrinopathy and enteropathy,X-linked(IPEX syndrome),rheumatoid arthritis(RA),and many other autoimmune diseases are all related to the Treg cells.As the member of the fork head transcription factor family,Forkhead box p3(Foxp3)is recognized as a marker gene of Treg cells.The present study suggests that Foxp3 gene is expressed high level in Treg cells of thymus and peripheral blood lymphocytes.Knockout Foxp3 gene will lead to the developmental disorders of Treg cells and eventually caused severe autoimmune diseases.Foxp3 gene is necessary for the development and maintain the function of Treg cells.Deactivated Cas9 system(dCas9),which is generated by inhibiting the enzymatic activities of both the RuvC and HNH domains in the Cas9 nuclease.dCas9 only has the ability to target genomic sequence without the ability to edit the DNA sequence.dCas9 system provides two opposite functions when dCas9 fused to a repressor(CRISPRi)and a activator(CRISPRa),respectively.Clustered regularly interspaced short palindromic repeats(CRISPR)are segments of prokaryotic DNA containing short,repetitive base sequences.The CRISPR/Cas systems is RNA-based immune systems thatconfers resistance to foreign genetic elements of viruses and plasmids in prokaryotic and provides a form of acquired immunity.CRISPR/Cas9 is a simple version of the CRISPR/Cas system,is use to modify and edit genomes.Under the guide of single or multiple synthetic single guide RNA(sgRNA),the Cas9 nuclease complex passed into a cell and cut the genome at a desired location,then remove the existing genes or add some new sequence.So far,CRISPR system has been modified and edited to be a biotechnological tool for making programmable transcription factors that allow scientists to target and activate or repress any genes which they want.In this study,we activated the expression of Foxp3 gene by CRISPR /Cas9 gene editing technique and Foxp3 gene overexpression and other molecular biology methods.The activation of Foxp3 gene in human peripheral blood by endogenous activation and exogenous activation was carried out by using Casilio system and overexpression in order to promote the cell fate change,and to obtain the expression of Foxp3 gene regulatory T cells.The main conclusions are as follows:1)The traditional CRISPR / Cas9 and the Casilio gene editing system are verified feasible and stable by liposome transfection and activated Oct4 gene in 293 T cell lines(human renal epithelial cell lines)and the system is optimized.The Real-time PCR analysis displays that the activation effect of the Casilio gene editing system on Oct4 gene was much higher than that of the traditional CRISPR / Cas9 gene editing system.While the activation effect of the hybrid system was more superior than that of Casilio Gene editing system.Above all,the orders the activation effect as follows : hybrid system> Casilio Gene editing system>traditional CRISPR / Cas9 system.This provided favorable technical support for later experiments.2)The activating experiment of Foxp3 gene in the HEK293 T cell line by the above activation system and the Foxp3 expression level is analyzed by Real-time PCR.The result shows that the activation effect of Foxp3 gene and Oct4 gene is similar.This indicates that the activation efficiency of these system in the HEK293 T cell line is stable.It provides an efficient and stable system for the activation of other genes in the future.3)We used the results of the previous studies to construct the Foxp3 gene overexpression vector in the suspension cell line--Jurkat cell line(human peripheral blood leukemia T cells,which is cultured in vitro with the primary T cells closest to a class of cell lines),Foxp3 gene was expressed in a very significant activation effect.4)Finally,we worked with the hospital to establish a small amount of humanperipheral blood and isolated mononuclear cells(PBMC).The primary T cells were further isolated and the transient expression of Foxp3 gene was used to promote the change of primary T cell fate,and got a part of T cells with high expression of Foxp3 gene;In addition,they were found have the function of inhibiting the proliferation of CD4+ CD25-cells,indicating that the cells had regulatory T cell function(proliferation inhibition)In summary,we obtained the CRISPR /dCas9 gene editing system with a large number of experiments over a longer period of time and obtained high expression of Foxp3 gene and a certain function of the regulatory T cells from the human primary T cells by overexpressing the Foxp3 gene.In the future,the focus of our experiment is to combine several efficient gene editing tools in the activation experiments of primary T cells to optimize the effect of maximizing it.Through the experiment it is not difficult to find that scientific research is not at one stroke,and it would be through a large number of experiments to explore and optimize,but the end result is bright.We are confident that CRISPR / dCas9 gene editing techniques and overexpression will induce the production of T cells with high expression of Foxp3 gene from primary T cells,laying the foundation for future research on the treatment of autoimmune diseases,and reducing the incidence of autoimmune diseases.