Recombinant DNA technologies for multiplex genetic assays in human cells

用于人体细胞多重基因检测的重组 DNA 技术

基本信息

批准号：
10275903
负责人：
Kenneth A Matreyek
金额：
$ 40.25万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2026-05-31
项目状态：
未结题

项目摘要

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.

项目概要高通量测序的进步已经揭示了数百万种蛋白质编码变体在人类外显子组中，还有数百万个可能存在但尚未存在的额外差异被观察到。其中许多变异可能对影响人类健康发挥重要作用，但我们缺乏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。这断开连接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异的影响方面发挥着关键作用，但传统方法通常会逐一测试变体，并且永远不会解决大量未表征变体的问题。多路复用能够同时测试复杂变异文库的基因检测具有所需的通量，但是这些方法仍处于发展初期，需要改进以提高这些技术成功解决这一问题的能力、成本、效率和可用性。利用以高效 Bxb1 为中心的一系列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、高度可定制的平台，用于表达单个培养的人类细胞内的复杂变体库。我之前配对过这个表达系统具有高度通用性的检测方法，可识别由于基因减少而导致功能丧失的变异体细胞内稳态丰度。我应用这种测定法来全面研究四种疾病的变异—— 相关蛋白，更多合作项目仍在进行中。不幸的是，仅靠这些方法就无法不能解决问题，需要更多正交方法来解决数以百万计的问题人体内存在的与疾病相关的未表征的变异。该提案的目标是构建下一组所需的基础生物技术，以实现蛋白质变体的更多高通量表征。所描述的各个方向分别是高度通用化，只需轻微的操作即可重新应用于研究大范围的蛋白质组修改。直接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，用于研究细胞内信号通路的扰动，以及条形码 ORFeome 集合，用于识别调节感兴趣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，尤其是那些其他生物领域的专家。这些进展以及我们的数据在展示其实用性的过程中生成，将直接解决变体解释问题同时还揭示了以前隐藏的与癌症相关的分子机制的生物学基础，这对于癌症治疗至关重要细胞功能。