Journal Article

Base-editing mutagenesis maps alleles to tune human T cell functions

Chen, Yan Yi, Hernandez, Rosmely, Ward, Carl C., Layeghi, Madeline, Goudy, Laine, Dorovskyi, Dmytro, Marson, Alexander, Armour-Garb, Zev, Allain, Vincent, Eyquem, Justin, Schmidt, Ralf, Dodgson, Stacie E., Pritchard, Jonathan K., Shy, Brian R., Wang, Charlotte, Gilbert, Luke A., Dajani, Rama, Xing, Galen, Ota, Mineto, Ye, Chun Jimmie

2024

Overview

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies 1 – 6 . However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency 7 , 8 . Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules 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 found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements 9 (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 thus provide biochemical insights with the potential to accelerate immunotherapy design. Massive-scale mutational screening across 385 genes reveals a wide spectrum of alleles that govern tunable T cell functions, including cytokine production and cytotoxicity.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 42/47 / 45 / 45/23 / 631/208/212 / 631/250/248 / 631/61/191 / Adaptor Proteins, Signal Transducing / Alleles / Amino acids / Amino Acids - genetics / Cancer / Cell activation / Class I Phosphatidylinositol 3-Kinases / CRISPR / CRISPR-Cas Systems - genetics / Cytokines / Cytokines - biosynthesis / Cytokines - metabolism / Cytotoxicity / Discovery tools / Editing / Editors / Gain of Function Mutation / Gene Editing / Gene mapping / Genes / Genetic engineering / Humanities and Social Sciences / Humans / Immune system / Immunotherapy / Loss of Function Mutation / Lymphocyte Activation / Lymphocytes / Lymphocytes T / multidisciplinary / Mutagenesis / Mutagenesis - genetics / Mutation / Nominations / Nucleotides / Phenotypes / Phospholipase C gamma / Phosphoproteins / Protein expression / Protein interaction / Proteins / Proto-Oncogene Proteins c-vav / Residues / RNA, Guide, CRISPR-Cas Systems - genetics / Science / Science (multidisciplinary) / Screening / T cell receptors / T-Lymphocytes - immunology / T-Lymphocytes - metabolism