干扰素-γ（IFN-γ）信号传导介导宿主对感染、炎症和抗肿瘤免疫的反应。IFN-γ信号通路中的突变引起免疫系统疾病、血液恶性肿瘤和癌症免疫检查点阻滞（ICB）的耐药性。然而，大多数临床观察到的变异的功能仍未知。因此，我们采用CRISPR-Cas9筛选和碱基编辑突变原理，系统地研究了IFN-γ响应在结直肠癌细胞中的遗传决定因素。我们使用胞嘧啶和腺嘌呤碱基编辑酶对JAK1进行深度突变，并结合通路广泛筛查，揭示了功能丧失和增强变异，包括造成血液恶性肿瘤的因果变异和检测到的ICB耐药患者的变异。我们在原代肿瘤器官样品中功能验证了不确定意义的变异，通过在JAK1中进行错义突变工程，增强或降低了对自主抗肿瘤T细胞的敏感性。我们鉴定了300多个预测的换义突变，改变了IFN-γ通路活性，生成了一个有价值的资源，用于解释基因变异功能。Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Interferon-γ (IFN-γ) signaling mediates host responses to infection, inflammation and anti-tumor immunity. Mutations in the IFN-γ signaling pathway cause immunological disorders, hematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer; however, the function of most clinically observed variants remains unknown. Here, we systematically investigate the genetic determinants of IFN-γ response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations, including causal variants in hematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumor organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumor-reactive T cells. We identify more than 300 predicted missense mutations altering IFN-γ pathway activity, generating a valuable resource for interpreting gene variant function.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.