Base editing mutagenesis maps functional alleles to tune human T cell activity

CRISPR-enabled genetic screening is a powerful tool to discover genes that control T cell function and has nominated candidate target genes for immunotherapies1–6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could discover alleles that tune specific phenotypes. DNA base editors are powerful tools to introduce targeted mutations with high efficiency7,8. Here, we develop a large-scale base editing mutagenesis platform with the goal of pinpointing nucleotides encoding amino acid residues that tune primary human T cell activation responses. We generated a library of ~117,000 sgRNAs targeting base editors to protein coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We discovered a broad spectrum of alleles with variants encoding critical residues (in PIK3CD, VAV1, LCP2, PLCG1 and DGKZ and others), comprising both gain-of-function and loss-of-function mutations. We validated the functional effects of diverse alleles and further demonstrated that base edit hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed PAM requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base editing screens in primary immune cells provide biochemical insights with potential to accelerate immunotherapy design. Overall design: CRISPR base editing screen data in primary human T cells with NGG Cas9 (3 human donors; CBE and ABE; assessing TNFa,IFNg,PD-1,CD25; CD4 T cells) or NG Cas9 (2 human donors; ABE; assessing TNFa; CD4 and CD8 T cells)