Differentiation of T cells by CRISPR/Cas9-mediated epigenetic DNA modification

Regulatory T cells (Tregs) play an essential role in maintaining immune tolerance and homeostasis by suppressing excessive immune reactions. The imbalance between Treg cells and pro-inflammatory T helper cells can even lead to autoimmunity. A demethylated FOXP3-TSDR is supposed to be fundamental for the differentiation, stability and suppressive function of Tregs, but a specific proof thereof has been unfeasible until now. Therefore, I aimed to actively demethylate FOXP3-TSDR of Jurkat T cells by using a deactivated Cas9 fused to the catalytic domain of the TET1 enzyme as a model for subsequent differentiation of stable Tregs. For this purpose, eight sgRNAs were cloned into a sgRNA plasmid expressing Cas9 nuclease to verify their functionality in HEK293T and Jurkat T cells by T7 endonuclease assay and TIDE analysis. Functional sgRNAs and a P2A-BFP fluorochrome were cloned into pdCas9-TET1-CD plasmid encoding for a deactivated Cas9 fused to the catalytic domain of the TET1 enzyme for active demethylation. Jurkat T cells were transfected with an efficiency of 20% using pdCas9-TET1-CD-P2A-BFP-sgRNA plasmid constructs. Western Blot and fluorescence microscopy confirmed protein expression in the nuclear fraction. The overexpression of dCas9-TET1 significantly decreased the DNA methylation of FOXP3-TSDR in BFP-sorted cells transfected with eight different plasmid constructs compared to negative controls. Furthermore, a minimum of three sgRNA constructs was sufficient to induce a similar extent of demethylation as induced by eight sgRNA. The additional transfection of one sgRNA exhibited a strong reduction in the methylation level in CpG 9-11 of FOXP3-TSDR, indicating a different potential of individual sgRNAs to demethylated CpGs within FOXP3-TSDR of Jurkat T cells. Interestingly, I observed an increase in the mRNA expression of the Treg markers Foxp3 and Garp, whereas expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. Moreover, I tested various conditions to improve the electroporation efficiency of human primary CD4+ T cells and confirmed that sgRNAs are functional in primary human CD4+ T cells for guiding the Cas9 enzyme to FOXP3-TSDR. However, active demethylation of FOXP3-TSDR in human CD4+ T cells transfected with eight pdCas9-TET1-CD-P2A-BFP plasmid constructs was not observed at 24 hours post transfection. The combination of Treg cell inducing culture conditions with targeted epigenetic modifications of the FOXP3-TSDR might improve functional Treg cell induction protocols.


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