(A) Comparison of total (x axis) versus FOXP3-proximal (y axis) levels of epigenetic regulators in induced Tregs with or without TCR stimulation (anti-CD3/CD28) (28). (B) Schematic of in vivo CRISPR screen. A retroviral sgRNA library targeting 21 FOXP3-proximal epigenetic regulators whose proteins were differentially represented in proximity to FOXP3 (in A), plus Zap70 and Il2ra, was transduced into Tregs from Foxp3^gfp Rosa^Cas9 mice. After 3–4 days of in vitro expansion, these CD45.2⁺ cells were sorted, and GFP⁺ Tregs were cotransferred with total CD45.1⁺ CD4⁺ T cells, including Tregs, into Rag1^–/– mice. Then, 2 weeks later, cells were recovered from spleens, lymph nodes, liver, lungs, and small and large intestines. CD45.2⁺GFP⁺ Tregs were sorted to assess sgRNA representation by high-throughput sequencing. (C) Fold changes (FCs) of sgRNAs targeting genes identified by FOXP3 PSI in Tregs recovered from lymphoid (spleen and lymph nodes) and nonlymphoid organs (liver, lung, small and large intestines) after CRISPR deletion and adaptation in Rag1^–/– mice, as described in B. Data are summarized from 4 biological replicates. Read counts of 4 sgRNAs targeting the same gene were combined for a paired Student’s t test by comparing with NCs. Log2 FCs were determined by comparing recovered cells and inputs. (D) Kdm6b expression in induced Tregs was quantified by RNA-Seq under conditions with or without TCR stimulation. Data are reported as the mean ± SD (n = 2); DESeq2 with Benjamini-Hochberg correction (***q < 0.001). (E) Log₂(TPM) Kdm6b expression (RNA-Seq) in control and Foxp3 CRISPR-deleted rTregs with or without TCR restimulation. DESeq2 with Benjamini-Hochberg correction (****q < 0.0001). TPM, transcripts per million RNA molecules.