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

Camilla Wilk; Laura Effenberg; Hanna Abberger; Laura Steenpass; Wiebke Hansen; Michael Zeschnigk; Carsten Kirschning; Jan Buer; Jan Kehrmann

doi:10.1016/j.cellimm.2021.104471

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

Cell Immunol. 2022 Jan:371:104471. doi: 10.1016/j.cellimm.2021.104471. Epub 2021 Dec 20.

Authors

Camilla Wilk¹, Laura Effenberg¹, Hanna Abberger¹, Laura Steenpass², Wiebke Hansen¹, Michael Zeschnigk³, Carsten Kirschning¹, Jan Buer¹, Jan Kehrmann⁴

Affiliations

¹ Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany.
² Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, Germany; Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.
³ Institute of Human Genetics, University Hospital Essen, University of Duisburg-Essen, Germany.
⁴ Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen, Germany. Electronic address: [email protected].

PMID: 34954490
DOI: 10.1016/j.cellimm.2021.104471

Abstract

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 naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression 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.

Keywords: CRISPR-Cas9; Epigenome editing; FoxP3; T cell differentiation; TET1.

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Cas Systems / genetics*
Cell Differentiation / immunology
Cell Line, Tumor
DNA Methylation / genetics*
Demethylation
Forkhead Transcription Factors / genetics
Forkhead Transcription Factors / metabolism*
GATA3 Transcription Factor / genetics
Gene Editing / methods*
Humans
Jurkat Cells
Mixed Function Oxygenases / genetics*
Nuclear Receptor Subfamily 1, Group F, Member 3 / genetics
Proto-Oncogene Proteins / genetics*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Messenger / genetics
T-Lymphocytes, Regulatory / cytology*
T-Lymphocytes, Regulatory / immunology

Substances

FOXP3 protein, human
Forkhead Transcription Factors
GATA3 Transcription Factor
GATA3 protein, human
Nuclear Receptor Subfamily 1, Group F, Member 3
Proto-Oncogene Proteins
RNA, Guide, CRISPR-Cas Systems
RNA, Messenger
RORC protein, human
Mixed Function Oxygenases
TET1 protein, human
CRISPR-Associated Protein 9