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

CRISPR（成簇规律间隔短回文重复序列，Clustered Regularly Interspaced Short Palindromic Repeats）介导的基因筛选是挖掘调控T细胞功能基因的有力工具，已为免疫治疗筛选出候选靶基因[1-6]。然而，当前仍需开发新方法以探究关键基因内的特定核苷酸序列。对原代人T细胞进行系统性诱变，有望发掘调控特定表型的等位基因。
DNA碱基编辑器（DNA base editor）是高效引入靶向突变的有力工具[7,8]。本研究开发了一款大规模碱基编辑诱变平台，旨在精准定位调控原代人T细胞活化应答的氨基酸残基对应的编码核苷酸。我们构建了包含约117,000条sgRNA（单向导RNA，single guide RNA）的文库，将碱基编辑器靶向至385个与T细胞功能相关基因的蛋白质编码位点，并系统性鉴定了调控T细胞活化与细胞因子产生的蛋白质结构域及特定氨基酸残基。
我们发掘了涵盖大量关键残基变异的等位基因（涉及PIK3CD、VAV1、LCP2、PLCG1、DGKZ等基因），其中既包含功能获得性突变，也包含功能丧失性突变。我们验证了多种等位基因的功能效应，并进一步证实碱基编辑筛选得到的靶点可正向或负向调控T细胞的细胞毒性功能。
最后，使用具备宽松PAM（原间隔序列临近基序，Protospacer Adjacent Motif）识别需求的碱基编辑器（NG vs NGG）开展的高分辨率筛选，揭示了可用于靶向调控T细胞功能的特定结构域与蛋白质相互作用位点[9]。
对原代免疫细胞开展的碱基编辑筛选，可为免疫治疗的研发提供生化层面的见解，具备加速免疫治疗设计的潜力。
实验整体设计：原代人T细胞的CRISPR碱基编辑筛选数据分为两组：① 采用NGG型Cas9（3名人类供体；使用CBE（胞嘧啶碱基编辑器，Cytosine Base Editor）与ABE（腺嘌呤碱基编辑器，Adenine Base Editor）；检测指标为TNFα（肿瘤坏死因子α，Tumor Necrosis Factor α）、IFNγ（干扰素γ，Interferon γ）、PD-1、CD25；检测细胞类型为CD4 T细胞）；② 采用NG型Cas9（2名人类供体；使用ABE；检测指标为TNFα；检测细胞类型为CD4及CD8 T细胞）