Recombinant DNA technologies for multiplex genetic assays in human cells

项目摘要 高通量测序的进展已经揭示了数百万种蛋白质编码变体 在人类外显子组中，还有数百万可能存在但尚未发现的额外差异。 被观察到。这些变异中的许多可能在影响人类健康方面发挥重要作用，但我们缺乏足够的证据。 将每个变异基因型与其相应的表型相关联所需的临床数据。这 断开通常被称为变体解释问题。模型中的遗传实验 系统在揭示蛋白质编码变体的影响方面发挥着关键作用，但传统方法 通常逐个测试变体，并且永远不会解决这种未表征变体的过剩。复用 能够同时测试复杂变体文库的遗传测定具有所需的通量，但是 这些方法仍处于发展初期，需要改进以提高 这些技术的能力、成本、效率和可用性来成功地解决这个问题。 利用以高效Bxb 1为中心的合成生物学工具 噬菌体DNA重组酶，我开发了一个用户友好的，高度可定制的表达平台， 在单个培养的人类细胞中的复杂变体文库。我之前将这个表达系统 具有高度可推广性的测定，其鉴定由于降低的免疫原性而丧失功能的变体。 胞内稳态丰度。我应用这种检测方法全面研究了四种疾病的变异- 相关蛋白质，更多的合作项目仍在进行中。不幸的是，这些方法本身将 没有解决这个问题，需要更多的正交方法来解决数百万的问题。 存在于人体内的未表征的疾病相关变异。 该提案的目标是建立下一套所需的基本生物技术， 更高通量的蛋白质变体表征。所描述的各个方向分别是 高度概括性，可以重新应用于研究大片的蛋白质组，只有轻微的 改性直接的方向包括研究必需基因的功能互补系统， 研究细胞内信号通路扰动的荧光转录报告系统，以及 条形码化的ORFeome收集，以鉴定当它们被表达时调节感兴趣的表型的基因。 过度表达这些技术的一个主要目的是促进其他研究小组的采用， 特别是那些在其他生物领域的专家。这些发展，沿着我们 在论证其效用的过程中产生的，将直接解决变异解释问题 同时也揭示了以前隐藏的癌症相关分子机制的生物学基础， 细胞功能

PROJECT SUMMARY Advances in high throughput sequencing have already revealed millions of protein coding variants within human exomes, and there are many millions of additional differences that likely exist but have not yet been observed. Many of these variants likely play important roles influencing human health, but we lack the clinical data required to associate each variant genotype with their corresponding phenotypes. This disconnect is oftentimes referred to as the variant interpretation problem. Genetic experiments in model systems play a critical role in uncovering the effects of protein coding variants, but traditional approaches typically test variants one-by-one and will never address this glut of uncharacterized variants. Multiplex genetic assays capable of simultaneously testing complex variant libraries have the required throughput, but these approaches are still in their developmental infancy, and improvements are needed to increase the capabilities, costs, efficiency, and usability of these techniques to successfully address this problem. Harnessing a palette of synthetic biology tools centered around the highly efficient Bxb1 bacteriophage DNA recombinase, I developed a user-friendly, highly customizable platform for expression of complex variant libraries within individual cultured human cells. I previously paired this expression system with a highly generalizable assay that identifies variants that are loss-of-function due to an reduced intracellular steady-state abundance. I applied this assay to comprehensively study variants in four disease- related proteins, and more collaborative projects are still in progress. Unfortunately, these methods alone will not address the problem, and more orthogonal approaches are needed to tackle the millions of uncharacterized disease-relevant variants that exist within people. The goal of this proposal is to build the next set of fundamental biotechnologies needed to enable more high-throughput characterizations of protein variants. The individual directions described are each highly generalizable and can be reapplied to study large swaths of the proteome with only slight modification. Immediate directions include a functional complementation system to study essential genes, a fluorescent transcriptional reporter system to study perturbations to intracellular signaling pathways, and a barcoded ORFeome collection to identify genes that modulate phenotypes of interest when they are overexpressed. A major purpose of these technologies is to facilitate adoption by other research groups, especially those that are experts in other biological fields. These developments, along with the data we generate in the process of demonstrating their utility, will directly address the variant interpretation problem while also uncovering previously hidden biology underlying cancer-related molecular mechanisms critical to cell function.