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

• 成果类型: Article
• 署名作者: Schmidt, Ralf; Ward, Carl C.; Dajani, Rama; Armour-Garb, Zev; Ota, Mineto; Allain, Vincent; Hernandez, Rosmely; Layeghi, Madeline; Xing, Galen; Goudy, Laine; Dorovskyi, Dmytro; Wang, Charlotte; Chen, Yan Yi; Ye, Chun Jimmie; Shy, Brian R.; Gilbert, Luke A.; Eyquem, Justin; Pritchard, Jonathan K.; Dodgson, Stacie E.; Marson, Alexander
• 署名单位: Medical University of Vienna; Stanford University; University of California System; University of California San Francisco; Institut National de la Sante et de la Recherche Medicale (Inserm); Assistance Publique Hopitaux Paris (APHP); Universite Paris Cite; Hopital Universitaire Saint-Louis - APHP; University of California System; University of California Berkeley; University of California System; University of California San Francisco; UCSF Medical Center; UCSF Helen Diller Family Comprehensive Cancer Center; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; Stanford University; University of California System; University of California San Francisco; University of California System; University of California Berkeley
• 刊物名称: Nature
• ISSN/ISSBN: 0028-6200
• DOI: 10.1038/s41586-023-06835-6
• 发表日期: 2024-01-25
• 关键词: crispr screen; genomic dna

摘要

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. Massive-scale mutational screening across 385 genes reveals a wide spectrum of alleles that govern tunable T cell functions, including cytokine production and cytotoxicity.