Fig. 1

Bioinformatics analysis for identification of regulatory sites in the FOXP3 gene for targeting by a CRISPR. The genomic segment encompassing the human FOXP3 locus on chromosome X (49,114,400–49,123,900 GRCh37/hg19 assembly) is shown. Regions named CNS1–3, Core promoter, Cage1 and Cage2 were identified (vertical broken gray lines) and targeted by sgRNA–dCas9VPR or sgRNA-dCas9-KRAB fusions. UCSC browser (https://genome.ucsc.edu) default graphics have been modified and customized tracks pertinent to our study were included. Targeted regions were selected based on the literature review of FOXP3 regulatory sites and if differential regulatory activity of FOXP3 upstream and downstream sequences in Treg vs. Tconv cells existed [11]. Cell types are: CD4+CD25highCD45RA+naïve (eRA+ Treg) and in vitro expanded CD4+ CD25-conventional T cells (eTconv). Additionally, FOXP3, ETS1, and STAT5 cell type-specific enhancer architecture and gene regulation by chromatin immunoprecipitation sequencing (ChIP-seq), differential epigenetic modifications such as histone H3K27 acetylation (H3K27ac) and H3K4 methylation (H3K4me1) signals, DNaseI hypersensitive sites, conserved transcription binding sites and 100-vertebrates basewise conservation segments are shown to be clustered in these targeted regions (more details on http://www.ag-rehli.de/NGSdata.htm, by following the link “HeliscopeCAGE, H3K27ac, and H3K4me1 as well as FOXP3, STAT5, ETS1 and RUNX1 ChIPs for T cell subpopulations