In Vivo Base Editing for Precision Oncology Models

项目摘要 基因突变是癌细胞生长和治疗抗性的主要驱动力。事实上， 个体化医学是识别个体肿瘤中的特定遗传变化， 这些变化将指导更有效和更有针对性的治疗。虽然这种精确的肿瘤学方法表明 临床前景，正在进行的肿瘤测序工作继续确定潜在的新疾病驱动因素和新的 突变这些未表征的突变等位基因如何促成疾病通常并不明显，并且需要 功能检查。基因工程小鼠模型（GEMM）为研究基因工程小鼠的遗传学行为提供了理想的工具。 遗传变化对肿瘤生物学的影响，但现有的方法不够快或精确， 重现了人类癌症中的遗传变异谱。我们和其他人使用了基于CRISPR的 基因组编辑，以加速复杂的，遗传定义的动物模型的生成。然而，虽然CRISPR 系统是快速和简单的，基本的工具是不精确的，因为它们会导致插入和删除， 基因功能，但不能模仿最常见的人类癌症中的单核苷酸变异。 为了构建重现特定人类癌症相关突变的体内系统，我们的项目利用了新的 CRISPR工具将Cas9与胞苷脱氨酶偶联，并在定义的时间点实现直接DNA诱变。 基因组区域。“碱基编辑”（BE）技术提供了比现有同源性更高的效率和灵活性 定向修复（HDR）方法通过消除递送外源DNA模板的需要。我们有 系统地优化BE酶的表达和活性，以提高基因组的效率， 修改并建立了生物信息学和实验管道，以预测和验证BE工具， 重新创造已知和新的癌症突变。 在目标1中，从广泛优化的BE酶构建，我们将产生一系列敲入转基因小鼠 以最大化可以使用BE突变的可能基因组区域的数量，并验证 这些小鼠使用一种新的荧光报告系统。此外，使用一种新的传感器测定，我们将确定 所有人类和小鼠sgRNA都可以靶向复发性癌症相关突变位点。在一起，这项工作 将定义数千个独立sgRNA的BE效率，并建立第一个体内体细胞基础 编辑平台。在目标2中，我们将使用我们的体内BE工具来产生胰腺和胰腺癌的新型动物模型。 结直肠癌，并检查每种疾病中不同癌症相关突变的后果。这 这项工作不仅将提供对关键致癌突变的新理解，还将提供对基因突变的关键验证。 体内BE在多种癌症环境中的效用。 通过提供一种简单有效的途径来捕获人类疾病等位基因的多样性，我们相信这种新的 精确编辑平台有可能从根本上改变我们设计和实现鼠标的方式 转化研究的癌症模型。

PROJECT SUMMARY Genetic mutation is the predominant driver of cancer cell growth and therapy resistance. In fact, a major goal of personalized medicine is to identify specific genetic changes in individual tumors with the notion that defining these changes will guide more effective and targeted treatment. While this precision oncology approach shows clinical promise, ongoing tumor sequencing efforts continue to identify potential new disease drivers and new mutations. How these uncharacterized mutant alleles contribute to disease is often not obvious, and requires functional examination. Genetically engineered mouse models (GEMMs) provide an ideal tool to investigate the consequences of genetic changes on tumor biology, yet existing approaches are not fast or precise enough to recreate the spectrum of genetic alterations seen in human cancer. We and others have used CRISPR-based genome editing to accelerate the generation of complex, genetically defined animal models. Yet, while CRISPR systems are fast and simple, the basic tools are imprecise in that they cause insertions and deletions that ablate gene function but cannot mimic the single nucleotide variants most often seen in human cancer. To build in vivo systems that recapitulate specific human cancer-associated mutations, our project exploits new CRISPR tools that couple Cas9 to cytidine deaminase enzymes and enable direct DNA mutagenesis at defined genomic regions. ‘Base editing’ (BE) technology offers far greater efficiency and flexibility than existing homology directed repair (HDR) approaches by eliminating the need to deliver exogenous DNA templates. We have systematically optimized the expression and activity of BE enzymes to increase the efficiency of genome modification and established a bioinformatic and experimental pipeline to predict and validate BE tools that recreate known and novel cancer mutations. In Aim 1, building from extensively optimized BE enzymes, we will generate a range of knock-in transgenic mice to maximize the number of possible genomic regions that can be mutated using BE, and validate the activity of these mice using a new fluorescence-based reporter system. Further, using a novel sensor assay, we will identify all human and mouse sgRNAs that can target recurrent cancer-associated mutation sites. Together, this work will define the BE efficiency of thousands of independent sgRNAs, and establish the first in vivo somatic base editing platforms. In Aim 2 we will use our in vivo BE tools to generate novel animal models of pancreatic and colorectal cancer, and examine the consequences of distinct cancer-associated mutations in each disease. This work will not only offer a new understanding of key oncogenic mutations, it will provide critical validation of the utility of in vivo BE in multiple cancer settings. By providing an easy and efficient path to capture the diversity of human disease alleles, we believe this new precision editing platform has the potential to fundamentally change the way we design and implement mouse cancer models for translational research.