Recombinant DNA technologies for multiplex genetic assays in human cells

项目总结 高通量测序的进展已经揭示了数百万种蛋白质编码变体 在人类外显体内，还有数以百万计的其他差异，这些差异可能存在但尚未存在 被观察到了。这些变异中的许多可能对人类健康起着重要作用，但我们缺乏 将每个变异基因与其对应的表型相关联所需的临床数据。这 脱节通常被称为变式解释问题。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，但传统的方法 通常情况下，一个接一个地测试变体，永远不会解决这些未表征的变体的过剩问题。多路传输 能够同时测试复杂变异库的基因分析具有所需的吞吐量，但 这些方法仍处于发展阶段，需要改进以增加 这些技术的能力、成本、效率和可用性，以成功解决这一问题。 利用一系列以高效的Bxb1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、高度可定制的表达平台 单个培养的人类细胞内的复杂变异库。我之前把这个表达系统配对了 具有高度普适性的分析方法，可以识别由于减少的 细胞内稳态丰度。我应用这种分析方法全面研究了四种疾病的变异- 相关的蛋白质，以及更多的合作项目仍在进行中。不幸的是，仅靠这些方法就能 没有解决这个问题，需要更多的正交方法来解决数百万 人类体内存在的与疾病相关的未知变种。 这项提议的目标是建立下一套基础生物技术，以实现 更多高通量的蛋白质变体表征。所描述的各个方向分别为 高度泛化，可重复应用于大范围的蛋白质组研究，只需少量的 修改。近期的方向包括研究必需基因的功能互补系统， 荧光转录报告系统，研究细胞内信号通路的扰动，以及 条形码ORFeome收集，以识别在感兴趣的表型发生变化时调节它们的基因 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， 尤其是那些在其他生物领域的专家。这些发展，连同我们的数据 在展示其实用性的过程中，将直接解决异体释义问题 同时也揭示了以前隐藏的隐藏在癌症相关分子机制背后的关键 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.