RT Journal Article
SR Electronic
T1 Optimized CRISPR-mediated gene knock-in reveals FOXP3-independent control of human Treg identity
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.01.16.426937
DO 10.1101/2021.01.16.426937
A1 Avery J. Lam
A1 David T.S. Lin
A1 Jana K. Gillies
A1 Prakruti Uday
A1 Anne M. Pesenacker
A1 Michael S. Kobor
A1 Megan K. Levings
YR 2021
UL http://biorxiv.org/content/early/2021/01/17/2021.01.16.426937.abstract
AB 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 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 targeted the master transcription factor FOXP3 in naive and memory Tregs. Although FOXP3-knockout Tregs upregulated cytokine expression, effects on suppressive capacity manifested slowly and primarily in memory Tregs. Moreover, FOXP3-knockout Tregs retained their characteristic phenotype and had few changes in their DNA methylation landscape, with FOXP3 maintaining 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.Competing Interest StatementMKL received research funding from Sangamo Therapeutics, Bristol-Myers Squibb, Pfizer, Takeda, and CRISPR Therapeutics for work unrelated to this study. All other authors declare no competing interests.