CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells

Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naive T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids toward expression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients' primary T cells might be feasible.

Automated summary

Demethylation of FOXP3-TSDR is crucial for the stable differentiation and suppressive function of regulatory T cells. This study demonstrates that transfection of specific plasmids can decrease FOXP3-TSDR methylation and increase FOXP3 mRNA expression in Jurkat cells, potentially enabling the programming of primary T cells into regulatory T cells.