Optimized CRISPR-mediated gene knockin reveals FOXP3-independent maintenance of human Treg identity.
Cell Rep
; 36(5): 109494, 2021 08 03.
Article
em En
| MEDLINE
| ID: mdl-34348163
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Linfócitos T Reguladores
/
Fatores de Transcrição Forkhead
/
Técnicas de Introdução de Genes
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Sistemas CRISPR-Cas
Tipo de estudo:
Guideline
Limite:
Humans
Idioma:
En
Revista:
Cell Rep
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Canadá