Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies

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. [Display omitted] •Method for pooled knockin screens of large DNA sequences at targeted genomic loci•Rapid discovery of novel synthetic constructs to enhance primary human T cell fitness•PoKI-seq combines pooled knockins with single-cell RNA sequencing in vitro and in vivo•Novel chimeric TGF-βR2-41BB receptor hit promotes solid tumor clearance Development of a platform to assess the functional effects of pools of knockin constructs targeting one locus allows discovery of constructs promoting anti-tumor activity in T cells.