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| Status |
Public on Aug 03, 2021 |
| Title |
Genome-wide DNA methylation profile of expanded human CRISPR/HDR-mediated FOXP3-KO naive Tregs, Cas9 naive Tregs, and conventional T cells |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by array
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| Summary |
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.
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| Overall design |
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. Genome-wide DNA methylation was assessed by Illumina MethylationEPIC from samples across 5 independent donors.
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| Contributor(s) |
Lam AJ, Lin DT, Levings MK |
| Citation |
https://0-doi-org.brum.beds.ac.uk/10.1016/j.celrep.2021.109494
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| Submission date |
Jan 02, 2021 |
| Last update date |
Aug 04, 2021 |
| Contact name |
Megan K Levings |
| E-mail(s) |
megan.levings@ubc.ca
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| Phone |
604-875-2000
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| Organization name |
University of British Columbia
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| Street address |
950 West 28th Ave
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| City |
Vancouver |
| ZIP/Postal code |
V5Z 4H4 |
| Country |
Canada |
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| Platforms (1) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA689222 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE164149_RAW.tar |
389.8 Mb |
(http)(custom) |
TAR (of IDAT) |
| GSE164149_betas.txt.gz |
106.4 Mb |
(ftp)(http) |
TXT |
| GSE164149_signal_intensities.csv.gz |
86.8 Mb |
(ftp)(http) |
CSV |
| Processed data included within Sample table |
| Processed data are available on Series record |
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