RNA-seq of expanded human CRISPR/HDR-mediated FOXP3-KO naive Tregs, Cas9 naive Tregs, and conventional T cells

Treg cell therapy is a promising curative approach for a variety of immune-mediated conditions. CRISPR-based genome editing allows precise insertion of transgenes through homology-directed repair, but its use in human Tregs has been limited. We report an optimized protocol for CRISPR-mediated gene knock-in in human Tregs with high-yield expansion. To establish a benchmark of human Treg dysfunction, we target the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-ablated Tregs upregulate cytokine expression, effects on suppressive capacity in vitro manifest slowly and primarily in memory Tregs. Moreover, FOXP3-ablated Tregs retain their characteristic protein, transcriptional, and DNA methylation profile. Instead, FOXP3 maintains DNA methylation at regions enriched for AP-1 binding sites. Thus, while FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knock-in with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies. Flow-sorted human naive Tregs (CD4+CD25hiCD127loCD45RA+) were edited by CRISPR-mediated gene knock-in to achieve uniformly FOXP3-ablated cells and expanded for total 20 days. Flow-sorted conventional T cells (CD4+CD25loCD127hi) were expanded in parallel, without CRISPR editing. Briefly, cells were preactivated for 5 days, edited with CRISPR and homology-directed repair to ablate FOXP3 and knock-in sfGFP, and expanded for 7 days. Edited cells were sort-purified by sfGFP+, expanded for an additional 7 days, then rested overnight. RNA-seq was performed on samples across 4 independent donors.