1. Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity.
- Author
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Lam AJ, Lin DTS, Gillies JK, Uday P, Pesenacker AM, Kobor MS, and Levings MK
- Subjects
- Base Sequence, DNA Methylation genetics, DNA Repair, Dependovirus metabolism, Green Fluorescent Proteins metabolism, HEK293 Cells, Humans, Immunosuppression Therapy, Interleukin-2 metabolism, Lymphocyte Subsets immunology, Phenotype, Plasmids metabolism, Time Factors, Transcription, Genetic, Transgenes, CRISPR-Cas Systems genetics, Forkhead Transcription Factors metabolism, Gene Knock-In Techniques, T-Lymphocytes, Regulatory immunology
- Abstract
Regulatory T cell (Treg) 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 knockin 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, although FOXP3 is important for human Treg development, it has a limited role in maintaining mature Treg identity. Optimized gene knockin with human Tregs will enable mechanistic studies and the development of tailored, next-generation Treg cell therapies., Competing Interests: Declaration of interests M.K.L. 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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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