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Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity

Lam, AJ; Lin, DTS; Gillies, JK; Uday, P; Pesenacker, AM; Kobor, MS; Levings, MK; (2021) Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity. Cell Reports , 36 (5) , Article 109494. 10.1016/j.celrep.2021.109494. Green open access

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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.

Type: Article
Title: Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.celrep.2021.109494
Publisher version: https://doi.org/10.1016/j.celrep.2021.109494
Language: English
Additional information: Copyright 2021 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Infection and Immunity
URI: https://discovery.ucl.ac.uk/id/eprint/10132987
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