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

Ralf Schmidt (), Carl C. Ward (), Rama Dajani, Zev Armour-Garb, Mineto Ota, Vincent Allain, Rosmely Hernandez, Madeline Layeghi, Galen Xing, Laine Goudy, Dmytro Dorovskyi, Charlotte Wang, Yan Yi Chen, Chun Jimmie Ye, Brian R. Shy, Luke A. Gilbert, Justin Eyquem, Jonathan K. Pritchard, Stacie E. Dodgson and Alexander Marson ()
Additional contact information
Ralf Schmidt: Gladstone–UCSF Institute of Genomic Immunology
Carl C. Ward: Gladstone–UCSF Institute of Genomic Immunology
Rama Dajani: Gladstone–UCSF Institute of Genomic Immunology
Zev Armour-Garb: Gladstone–UCSF Institute of Genomic Immunology
Mineto Ota: Gladstone–UCSF Institute of Genomic Immunology
Vincent Allain: Gladstone–UCSF Institute of Genomic Immunology
Rosmely Hernandez: Gladstone–UCSF Institute of Genomic Immunology
Madeline Layeghi: Gladstone–UCSF Institute of Genomic Immunology
Galen Xing: Gladstone–UCSF Institute of Genomic Immunology
Laine Goudy: Gladstone–UCSF Institute of Genomic Immunology
Dmytro Dorovskyi: Gladstone–UCSF Institute of Genomic Immunology
Charlotte Wang: Gladstone–UCSF Institute of Genomic Immunology
Yan Yi Chen: Gladstone–UCSF Institute of Genomic Immunology
Chun Jimmie Ye: Gladstone–UCSF Institute of Genomic Immunology
Brian R. Shy: Gladstone–UCSF Institute of Genomic Immunology
Luke A. Gilbert: University of California, San Francisco
Justin Eyquem: Gladstone–UCSF Institute of Genomic Immunology
Jonathan K. Pritchard: Stanford University
Stacie E. Dodgson: Gladstone–UCSF Institute of Genomic Immunology
Alexander Marson: Gladstone–UCSF Institute of Genomic Immunology

Nature, 2024, vol. 625, issue 7996, 805-812

Abstract: Abstract CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1–6. 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 efficiency7,8. 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 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 thus provide biochemical insights with the potential to accelerate immunotherapy design.

Date: 2024
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DOI: 10.1038/s41586-023-06835-6

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