Construction Of Fluorescent Labeled IFNGR2 Knock-in Cell Line Based On CRISPR/Cas9 Technology

Interferon?(IFN?)signaling pathway plays an important role in innate immunity,inflammatory response,adaptive immunity,anti-tumor immunity,and its functional status is the key to determine the success or failure of tumor immunotherapy.IFN-? plays a role mainly by binding to the membrane surface receptor IFN-?R and activating JAK1 and JAK2 downstream,and further phosphorylating the transcription factor STAT1.The phosphorylated STAT1 translocates into the nucleus and activates the expression of downstream related genes.IFN-?R consists of two subunits: IFN?R1(IFNGR1)and IFN?R2(IFNGR2).IFNGR1 is relatively overexpressed in cells and plays a role in binding IFN?,while the expression level of IFNGR2 is strictly regulated.The reactivity level of IFN? signaling pathway is determined by the expression level of IFNGR2.Therefore,Regulating the expression of IFNGR2 is a potential breakthrough for improving the effect of immunotherapy.And elucidating the regulatory mechanism of IFN?R2(IFNGR2)is helpful for understanding the immune response and disease occurrence process related to IFN? signaling pathway,and has potential clinical significance.In order to discover genes and small molecular compounds that regulate the expression of IFNGR2 and the IFN? signaling pathway,and to find new ideas and strategies to study their regulation methods and molecular mechanisms,based on CRISPR / Cas9 gene editing technology,this study knocked the green fluorescent protein(EGFP)into the end of the Ifngr2 gene,and established the Ifngr2 reporter cell line expressing the fusion-expression protein.The cell lines characterize the expression of endogenous IFNGR2 in living cells by the intensity of green fluorescenceto achieve rapid and quantitative monitoring of the degree of regulation of IFNGR2.We have adopted two strategies to achieve EGFP knock-in,namely CRISPRmediated microhomologous end joining(MMEJ)and CRISPR-mediated homologous recombination(HR).Both strategies cause the DNA double-strand break at the end of the last exon of the IFNGR2 gene through the CRISPR-Cas9 system,and introduce the donor vector containing the EGFP coding sequence and the homologous sequence to the DNA break position,using the intracellular various DNA repair mechanisms to complete knock-in.The main difference between the two methods is that MMEJ uses the intracellular micro-homologous recombination repair mechanism with a homologous sequence of about 20 bp and the HR uses the homologous recombination repair mechanism with a long homologous sequence of about 200 bp.The MMEJ is not limited by the cell cycle,and theefficiency is higher;while the HR has a longer homologous sequence which is convinent for selecting the CRISPR-Cas9 target position.Botn of the two strategies have their stengths and has a certain complementarity.When constructing the knock-in cell line,we selected two mouse cell lines(prostate cancer cell line My C-Ca P and mouse melanoma cell line B16),and a human cell line(colon cancer cell line HCT116).We transferred the Cas9 vector(carrying the IFNGR2 gene-targeted sg RNA)and the donor vector(carrying the EGFP coding sequence and homologous sequence of knock-in site)into the cell line at the same time,using antibiotic resistance gene screening to obtain candidate cell clones.By extracting the genome and performing PCR identification,it is clear that the EGFP coding sequence is correctly inserted.And candidate clones are screened and selected according to EGFP expressional level.Finally,these clones are verified to have normal IFN? reactivity,and the construction was successful.on this basis,we further examined the regulatory effect of IFNGR2 regulatory factors on the expression levels of IFNGR2 and EGFP in the knock-in cells.We found that tumor necrosis factor alpha(TNF-?)and Oxaliplatin,a chemotherapy agent,can significantly up-regulate the expression levels of Ifngr2 and EGFP in the knock-in cell lines.TNF-? is one of the most vital cytokinesin our body,mainly secreted by activated macrophages,regulates various immune processes and induce tumor cell apoptosis.Studies have shown that TNF-? can regulate IFNGR2 expression through the NF-?B signaling pathway.Oxaliplatin,as an anti-tumor drug,is widely used in a variety of tumor chemotherapy regimens.Recent studies have shown that it can significantly promote anti-tumor immunity.Our results indicate that the effect of Oxaliplatin on anti-tumor immunity is likely to be achieved by up-regulating the expression of IFNGR2 in tumor cells and thereby increasing the sensitivity of tumor cells to IFN?.Based on the CRISPR gene editing knock-in technology,EGFP gene was knocked into My C-Ca P prostate cancer cell line,mouse B16 melanoma cell line and human colon cancer cell line HCT116.Genomic PCR confirmed the successful knocking of EGFP gene into the end of Ifngr2 in B16 cell line and My C-Ca P cell line,where B16 cells can detect the expression of green fluorescence.Furthermore,we conducted a preliminary study on the regulation of IFNGR2 expression by cytokines and antitumor drugs in the constructed B16 Ifngr2-EGFP cell line.The results showed that in B16 Ifngr2-EGFP cell line,TNF? and Oxaliplatin can up-regulate the expression of IFNGR2,and the expression of green fluorescent protein can reflect the expression of IFNGR2.The cell line established in this paper provides a strong technical support for the in-depth study of IFNGR2 expression,regulation of IFN? signaling pathway and screening of immunomodulatory active drugs.