Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

Theodore L. Roth*, P. Jonathan Li, Franziska Blaeschke, Jasper F. Nies, Ryan Apathy, Cody Mowery, Ruby Yu, Michelle L.T. Nguyen, Youjin Lee, Anna Truong, Joseph Hiatt, David Wu, David N. Nguyen, Daniel Goodman, Jeffrey A. Bluestone, Chun Jimmie Ye, Kole Roybal, Eric Shifrut, Alexander Marson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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 β (TGF-β) 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.

Original languageEnglish
Pages (from-to)728-744.e21
Issue number3
StatePublished - 30 Apr 2020
Externally publishedYes


  • cell therapy
  • human T cell
  • knockins
  • pooled screen
  • single-cell RNA-seq


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