美国加州大学旧金山分校Alexander Marson等研究人员合作发现，碱基编辑诱变绘制出调整人类T细胞功能的等位基因图谱。相关论文于2023年12月13日在线发表于国际学术期刊《自然》。

研究人员开发了一个大规模碱基编辑诱变平台，目的是精确定位编码可调节原代人类T细胞活化反应的氨基酸残基的核苷酸。研究人员生成了一个包含约117000个单向导RNA分子的文库，将碱基编辑靶向385个与T细胞功能有关的基因的蛋白质编码位点，并系统地鉴定了调节T细胞活化和细胞因子产生的蛋白质结构域和特定氨基酸残基。研究人员发现了广泛的等位基因，包括编码PIK3CD、VAV1、LCP2、PLCG1和DGKZ等蛋白中关键残基的变体，其中既有功能增益突变，也有功能丧失突变。研究人员验证了许多等位基因的功能效应，并进一步证明碱基编辑突变可以正反两方面调节T细胞的细胞毒性功能。

最后，通过使用对原间隔基序要求宽松的碱基编辑器（NG对NGG）进行更高分辨率的筛选，研究人员发现了可用于调整T细胞功能的特定结构域和蛋白质-蛋白质相互作用位点。因此，原代免疫细胞中的碱基编辑筛选提供了生化见解，并有可能加速免疫疗法的设计。

研究人员表示，CRISPR筛选技术是发现控制T细胞功能的基因的有力工具，并为免疫疗法提名了候选靶标。然而，还需要新的方法来探测关键基因中的特定核苷酸序列。在原代人类T细胞中进行系统突变可以发现调整特定表型的等位基因。DNA碱基编辑器是高效引入靶向突变的强大工具。

附：英文原文

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

Author: 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

Issue&Volume: 2023-12-13

Abstract: CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1,2,3,4,5,6. 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.

DOI: 10.1038/s41586-023-06835-6

Source: https://www.nature.com/articles/s41586-023-06835-6