Cas9‐Cleavage Sequences in Size‐Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells

Abstract T cell genome editing holds great promise to advance a range of immunotherapies but is encumbered by the dependence on difficult‐to‐produce and expensive viral vectors. Here, small double‐stranded plasmid DNA modified to mediate high‐efficiency homologous recombination is designed. The resulting chimeric antigen receptor (CAR)‐T cells display a similar phenotype, transcriptional profile, and in vivo potency to CAR‐T cells generated using adeno‐associated viral vector. This method should simplify and accelerate the use of precision engineering to produce edited T cells for research and clinical purposes.