ABSTRACT Genetically-engineered immune cell therapies have been in development for decades 1–3 and recently have proven effective to treat some types of cancer 4 . CRISPR-based genome editing methods, enabling more flexible and targeted sequence integrations than viral transduction, have the potential to extend the clinical utility of cell therapies 5,6 . Realization of this potential depends on improved knowledge of how coding and non-coding sites throughout the genome can be modified efficiently and on improved methods to discover novel synthetic DNA sequences that can be introduced at targeted sites to enhance critical immune cell functions. Here, we developed improved guidelines for non-viral genome targeting in human T cells and a pooled discovery platform to identify synthetic genome modifications that enhance therapeutically-relevant cell functions. We demonstrated the breadth of targetable genomic loci by performing large knock-ins at 91 different genomic sites in primary human T cells, and established the power of flexible genome targeting by generating cells with Genetically Engineered Endogenous Proteins (GEEPs) that seamlessly integrate synthetic and endogenous genetic elements to alter signaling input, output, or regulatory control of genes encoding key immune receptors. Motivated by success in introducing synthetic circuits into endogenous sites, we then developed a platform to facilitate discovery of novel multi-gene sequences that reprogram both T cell specificity and function. We knocked in barcoded pools of large DNA sequences encoding polycistronic gene programs. High-throughput pooled screening of targeted knock-ins to the endogenous T cell receptor (TCR) locus revealed a transcriptional regulator and novel protein chimeras that combined with a new TCR specificity to enhance T cell responses in the presence of suppressive conditions in vitro and in vivo . Overall, these pre-clinical studies provide flexible tools to discover complex synthetic gene programs that can be written into targeted genome sites to generate more effective therapeutic cells.

Rapid discovery of synthetic DNA sequences to rewrite endogenous T cell circuits