Center for the Multiplexed Assessment of Phenotype

摘要 到目前为止，已经发现了数百万个人类遗传变异，其中许多是在编码或调控序列中发现的 基因。然而，对于这些变体中的一小部分，我们理解了 编码的产品会受到影响。因此，对人类基因组进行测序以了解 人类的表型--尤其是许多带有基因成分的疾病的风险--已经在很大程度上 没有实现。需要一种简便、高通量的方法来生成人类细胞文库，这些文库含有 突变序列元件和评估这些文库以确定每个变异体对分子和 细胞表型。因此，表型多元化评估中心主要基于 华盛顿大学基因组科学系，建议开发高度概括性的、 可复制和可扩展的技术，用于生成和评估人类变种的功能影响 基因。在第一个具体目标中，该中心将建立两种主要的诱变方法来生产 变体：饱和编辑人类基因组中内源基因的位置，并在体外生成 重组成避风港的变异库。在第二个具体目标中，该中心将发展 探索非编码区突变对已有基因版本的影响的方法 最小化-削减以部分去除内含子序列，但仍能提供基本活性。 此外，它还将开发质谱学方法来分析编码序列的变异，以了解其对 蛋白质丰度、稳定性、相互作用、周转和聚集。在第三个具体目标中，中心将 通过使用一种新的显微镜来评估对细胞形态、行为和内部组织的不同影响 基于表型技术，以及通过开发大规模平行的单细胞mRNA进行全局转录 剖面法。中心开发的技术将在一组与疾病相关的人类基因上进行试点， 能够比较每个变异体的功能影响和已知致病或良性疾病的影响 变种。这一努力将使该中心的大规模功能数据在临床上得到使用 将会产生。此外，将在不同的条件下对变异进行评估，例如在多个细胞系中， 与另一种突变结合，或在药物存在的情况下。该中心还将对职业生涯早期进行培训 实验学家、临床遗传学家和数据科学家获取和使用大规模功能数据。这 培训将包括在中心实验室实习一至三个月，以及一至三个月的学徒培训 两年了。这些密切的相互作用将产生与医学和生物学相关的结果，并揭示最好的结果 用于临床使用的大量中心生成的功能数据的翻译路径。通过这些新的 技术及其在更广泛的临床社区的传播，该中心将推进 人类基因组计划通过解释人类基因变异的广阔图景。

SUMMARY To date, millions of human genetic variants have been found, many in the coding or regulatory sequence of genes. However, for only a tiny fraction of these variants do we understand how the expression or function of the encoded product is affected. As a consequence, the promise of sequencing human genomes to understand human phenotypes – especially the risk for many diseases with genetic components – has gone largely unfulfilled. What is needed are facile, high-throughput methods for generating libraries of human cells bearing mutant sequence elements and for assessing these libraries to determine each variant's effect on molecular and cellular phenotypes. Thus, the Center for the Multiplexed Assessment of Phenotype, based largely in the University of Washington's Department of Genome Sciences, proposes to develop highly generalizable, reproducible and scalable technologies to generate, and assess the functional impact of, variants in human genes. In the first specific aim, the Center will establish two workhorse methods of mutagenesis to produce variants: saturation editing of genes at their endogenous loci in the human genome, and in vitro generation of variant libraries that are recombined into safe harbor sites. In the second specific aim, the Center will develop approaches to explore the impact of mutations in noncoding regions on versions of genes that have been minimized – pared down to partially remove intronic sequence but still capable of providing essential activity. Further, it will develop mass spectrometry methods to analyze variation in coding sequences for its effect on protein abundance, stability, interactions, turnover and aggregation. In the third specific aim, the Center will assess variant effects on cell morphology, behavior and internal organization by using a novel, microscopy- based phenotyping technology, and on global transcription by developing a massively parallel single-cell mRNA profiling method. Center-developed technologies will be piloted on a set of human genes with disease relevance, enabling comparisons between each variant's functional effects and the effects of known pathogenic or benign variants. This effort will inform the use in the clinic of the large-scale functional data the Center's technologies will generate. Additionally, variants will be assessed under different conditions, such as in multiple cell lines, in combination with another mutation, or in the presence of a drug. The Center will also train early career experimentalists, clinical geneticists and data scientists to obtain and use large-scale functional data. This training will include internships in Center laboratories for one to three months, and apprenticeships for one to two years. These close interactions will generate medically- and biologically-relevant results and reveal the best paths for translating the vast amounts of Center-generated functional data for clinical use. Through these new technologies and their dissemination to the broader clinical community, the Center will advance the promise of the Human Genome Project by interpreting the vast landscape of human genetic variation.