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

Cell Rep. 2021-08; 
Avery J Lam, David T S Lin, Jana K Gillies, Prakruti Uday, Anne M Pesenacker, Michael S Kobor, Megan K Levings
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GenParts™ DNA Fragments … The resulting HDR-edited cells could be enriched with a reporter gene and exhibited … method for gene knockin in human Tregs to generate a uniform population of gene-edited cells. As … DNA, plasmids, or synthesized (IDT gBlocks or GenScript GenParts), then cloned by Gibson … Get A Quote

摘要

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, transcripti... More

关键词

CRISPR, DNA methylation, FOXP3, cell therapy, genome editing, homology-directed repair, regulatory T cells