(PQ4) Novel tools for in vivo study of genetic interactions in cancer progression

项目总结： 人类癌症的进化是一个由多个分子和细胞事件驱动的复杂过程。我们 努力利用单效应子RNA引导的内切酶(RGNs)进行基因组编辑，并行 人类癌症的筛查和体内建模。最近，我们创建了一个平台，系统地 直接在活体内审问数百个基因座。为了克服目前多基因编辑和 为了更准确地控制多等位基因肿瘤建模的同时性和顺序性，我们利用 Cpf1，一种RGN，它可以简单地使用独立于trrRNA的crRNA编辑其目标，从而允许同时 用单个crRNA阵列编辑多个基因。我们开发了一个初步的基于Cpf1的crRNA阵列 在哺乳动物细胞中筛选(CCAS)系统，并将其应用于小鼠的进展和转移模型。 在我们的第一个目标中，我们将对CCAS进行体内双敲除表型的验证和优化 癌症的副驾驶。我们将建立其同步编辑的技术严谨性、效率和专用性，如 以及开发一套计算管道，用于准确调用统计上有意义的基因对。我们 将应用这种方法来研究肺癌患者中发现的肿瘤抑制基因的相互作用 耶鲁大学癌症中心和医院，并确定转移到重要器官的潜在共同驱动因素。在第二个 目的：我们将对Cpf1-Flip系统进行验证和优化，以用于肿瘤的顺序突变 目标。我们将通过测试与临床相关的基因集来证明其更广泛的适用性 对转移的基因组学以及收集的大型多样本转移数据集的公开研究 耶鲁大学癌症患者。然后，我们将应用这一方法作为无偏见的耗尽筛查，以确定目标 在特定致癌背景下生存所必需的基因。我们将开发新的多功能转基因 用于直接模拟小鼠多基因肿瘤发生的小鼠品系和伴生病毒载体。我们会 将这些工具结合在一起，可以直接在健康细胞中进行高通量基因交互筛选 识别驱动肿瘤发生的致病突变对的天然器官。我们预计正在发展中的 建立这些工具将改变人类癌症的多基因肿瘤建模和临床前研究， 直接回答NCI的挑衅性问题4。这些强大的工具包将使科学家能够针对任何 基因对或组合同时或顺序地，评估它们在体内的表型结果 肿瘤进展、转移、合成致命性、药物敏感性或其他癌症过程中的相互作用 进化论。本补编的目的是(1)扩大和加强目标3所述的现有研究；以及 (2)通过让代表不足的少数族裔受训人员参与 项目。这将主要涉及父母奖的目标3由于这个项目的阶段。

PROJECT SUMMARY: The evolution of human cancer is a complex process driven by multiple molecular and cellular events. We endeavored to harness single-effector RNA-guided endonucleases (RGNs) for genome editing, parallel screening and in vivo modeling of human cancer. Recently, we generated a platform to systematically interrogate several hundred loci directly in vivo. To overcome current limitations in multigene editing and achieve more accurate control of simultaneity and sequentiality of multi-allelic tumor modeling, we utilized Cpf1, an RGN that can edit its target simply with crRNAs independent of tracrRNA thus allowing simultaneous editing of multiple genes with a single crRNA array. We developed a preliminary Cpf1-based crRNA array screening (CCAS) system in mammalian cells, and applied it in mouse models of progression and metastasis. In our first aim, we will perform validation and optimization of CCAS for in vivo double-knockout phenotyping of cancer co-drivers. We will establish its technical rigor, efficiency and specificity for simultaneous editing, as well as developing a set of computational pipelines for accurate calling of statistically significant gene pairs. We will apply this approach to study the genetic interactions of tumor suppressors found in lung cancer patients at Yale Cancer Center and Hospital, and identify potential co-drivers of metastasis to vital organs. In the second aim, we will carry out validation and optimization of a Cpf1-Flip system for sequential mutagenesis of cancer targets. We will demonstrate its broader applicability by testing clinically relevant gene sets identified from public studies of the genomics of metastasis as well as a large multi-sample metastasis dataset gathered on Yale cancer patients. We will then apply this methodology as an unbiased depletion screen to identify targets that are essential for survival in specific oncogenic backgrounds. We will develop novel versatile transgenic mouse strains and companion viral vectors for direct modeling of multigenic tumorigenesis in mice. We will combine these tools to enable high-throughput genetic interaction screening in healthy cells directly in the native organ to identify causative mutation pairs that drive tumorigenesis. We anticipate that developing and establishing these tools will transform multigenic tumor modeling and pre-clinical studies of human cancer, directly addressing NCI Provocative Question 4. These powerful toolkits will enable scientists to target any gene pairs or combinations simultaneously or sequentially, assessing the phenotypic outcome of their in vivo interactions in tumor progression, metastasis, synthetic lethality, drug sensitivity or other processes in cancer evolution. This supplement’s goal is to (1) extend and strengthen the existing research described in Aim 3; and to (2) promote diversity in health-related research by involving an under-represented minority trainee in the project. This will mostly involve Aim 3 of the parent award due to the stage of this project.