ABSTRACT Engineered T cell-based cancer therapies are a major advancement in cancer treatment; however the majority of cancers still do not respond to adoptive cellular therapy. We need to “design” new T cell therapies with increased potency, and we need to overcome cell dysfunction that occurs as T cells face chronic tumor antigen stimulation. We and others have screened for genes that can be “knocked out” in antigen-specific T cells to enhance their functions, but enormous opportunities still remain to “knock-in” new synthetic DNA sequences at targeted genome sites. This proposal is focused on detailed evaluation of genes and inducible gene programs that will enable next-generation cellular therapies for cancer. We have developed several complementary technologies to discover synthetic gene programs that can be “inserted” into T cell genomes to enhance therapeutic functions. We developed a CRISPR technology for high throughput pooled knock-ins to accelerate discovery of synthetic knock-in programs (Roth et al., Cell, 2020), and have now have conducted two screens with ~100-member libraries that include transcription factors and synthetic chimeric receptors (“switch receptors”) to discover programs that make chronically stimulated T cells resistant to dysfunction. In addition, we have optimized a complementary robust platform for genome-wide CRISPR activation (CRISPRa) gain-of-function forward genetic screens in human T cells, and have already completed systematic discovery of factors that regulate stimulation-dependent cytokine production (Schmidt and Steinhart et al., Science, 2022). We propose to translate insights from these high-throughput discovery efforts into preclinical testing of novel knock-in designs with screen hits in vivo using xenotransplanted mouse models. In this proposal, we will test validated candidates from gain-of-function CRISPR PoKI (Aim 1) and CRISPRa (Aim 2) screens to discover new components of knock-in constructs that improve cell-based T cell therapies. We also recognize that these genetic components may be more beneficial if they are not expressed constitutively. In Aim 3, we draw on the power of synthetic biology to engineer synthetic circuits that can induce or repress genetic programs in response to antigen stimulation. This precise and dynamic regulation of genetic elements has great potential to further enhance efficacy and safety of next-generation immune cell therapies. Taken together, we present a proposal that leverages recent discoveries from CRISPR discovery platforms and deep expertise in synthetic biology to engineer powerful “knock-in” circuits that we will validate and study in preclinical cancer models. We leverage functional genomics, CRISPR engineering and synthetic cell program design expertise to address insufficient T cell potency and T cell dysfunction, which remain significant barriers to adoptive cell therapy for cancer.