Journal
CELL
Volume 181, Issue 3, Pages 728-+Publisher
CELL PRESS
DOI: 10.1016/j.cell.2020.03.039
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Funding
- UCSF Medical Scientist Training Program [T32GM007618]
- UCSF Endocrinology Training Grant [T32 DK007418]
- NIDDK [F30DK120213]
- Care-for-Rare Foundation
- German Research Foundation (DFG)
- Burroughs Wellcome Fund at the Chan Zuckerberg Biohub
- Innovative Genomics Institute (IGI)
- American Endowment Foundation
- Cancer Research Institute (CRI)
- Lloyd J. Old STAR award
- NIH/NIGMS [P50AI150476]
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Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor beta (TGF-beta) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
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