Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

Cell. 2020 Apr 30;181(3):728-744.e21. doi: 10.1016/j.cell.2020.03.039. Epub 2020 Apr 16.


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.

Keywords: CRISPR; cell therapy; human T cell; knockins; pooled screen; single-cell RNA-seq.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Cells
  • CRISPR-Cas Systems / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Gene Knock-In Techniques / methods*
  • Genetic Engineering / methods*
  • Humans
  • Immunotherapy / methods*
  • Mice
  • Mice, Inbred NOD
  • Mice, SCID
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Single-Cell Analysis / methods
  • T-Lymphocytes
  • Transcriptome / genetics


  • RNA, Guide, CRISPR-Cas Systems