Capturing the phenotypic landscape of single-nucleotide variation via systematic genome editing

项目总结 理解表型多样性、对进化中的选择建模以及 发展精确医学正在增强我们目前有限的预测疾病和表型的能力 基于基因组序列和环境暴露的结果。全面了解 遗传变异及其在条件性表型中的作用需要系统的、基于扰动的测试 在多个环境和同基因背景下跨越基因组的遗传变异。上一首 系统性的基因组干扰工作主要集中在工程功能丧失上，但这是自然而然的 发生的变异与理解医学上相关的表型最相关，比如人类的特征 和疾病。这些变异已经通过全基因组关联研究(GWAS)和定量 性状基因座(QTL)分析，但这些方法仅限于研究中出现的单倍型 只有在极少数情况下才能确定实际的致病变异体。基因组研究进展 编辑技术使工程特定的基因变异在大范围内可行。这项建议 目标是系统地设计并从功能上描述全基因组的“变异集合”，分为三个阶段 涵盖所有天然单核苷酸变种(SNV)的基因独特的菌株 酿酒酵母以及与人类疾病相关的SNV。收藏品将会 通过利用内部序列解析技术的高吞吐量CRISPR方法构建 (重组酶定向索引，或REDI)，将允许快速、廉价地分离经序列验证的 将产生的数百万个变异株中的一个。因为有些变种只在 在某些环境下，这种菌株收集将在数百种条件下进行分析，包括暴露在 各种压力和毒品。整合到每个菌株基因组中的DNA条形码将使 竞争性生长，并允许全面识别基因组中调节适应性的变异 在一次实验中，在给定的条件下。最后，为了剖析潜在途径的遗传结构 疾病和确定关键的相互作用，将分析携带SNV组合的菌株。品系 收集的样品将提供给社区进行进一步的表型调查。除 基因x环境(GxE)数据集可能是迄今为止产生的最大数据集，技术、分析、 可视化管道将被公开共享并整合到社区资源中。这项工作将 构成了对遗传变异及其依赖的后果的前所未有的调查 在为科学界提供宝贵资源的同时，也对环境产生了重大影响。它将奠定技术基础 和基于系统扰动的人类细胞遗传变异研究的概念基础 将为疾病风险的预测和基于基因组序列的治疗策略的设计提供信息。

PROJECT SUMMARY A major challenge common to understanding phenotypic diversity, modeling selection in evolution, and developing precision medicine is enhancing our currently limited ability to predict disease and phenotypic outcomes based on genome sequence and environmental exposures. A comprehensive understanding of genetic variation and its role in conditioning phenotypes requires systematic, perturbation-based testing of genetic variants across the genome in multiple environments and in an isogenic background. Previous systematic genome perturbation efforts have focused primarily on engineering loss-of-function, but naturally occurring variants have the most relevance to understanding medically relevant phenotypes like human traits and disease. Such variants have been studied via genome-wide association studies (GWAS) and quantitative trait locus (QTL) analysis, but these approaches are limited to the haplotypes that appear in the study population, and only in few cases have the actual causative variants been identified. Advances in genome editing technologies have made engineering specific genetic variants feasible at a large scale. This proposal aims to systematically engineer and functionally profile a genome-wide `variation collection' in three genetically distinct strains that cover all natural single-nucleotide variants (SNVs) in the Saccharomyces cerevisiae species as well as SNVs associated with human diseases. The collection will be constructed by a high-throughput CRISPR approach, leveraging an in-house sequence parsing technology (Recombinase Directed Indexing, or REDI) that will allow rapid, inexpensive isolation of sequence-verified variant strains among the millions that will be generated. Because some variants only exert their effects in certain environments, this strain collection will be profiled in hundreds of conditions, including exposure to various stresses and drugs. DNA barcodes integrated into the genome of each strain will enable pooled, competitive growth, and allow the comprehensive identification of variants in a genome that modulate fitness in a given condition in a single experiment. Finally, to dissect the genetic architecture of pathways underlying diseases and identify key interactions, strains carrying combinations of SNVs will be analyzed. The strain collection will be made available to the community for further phenotypic investigations. In addition to the gene x environment (GxE) dataset that will likely be the largest produced to date, the technological, analytical, and visualization pipelines will be publicly shared and integrated into community resources. This work will constitute an unprecedented investigation of the consequences of genetic variation and their dependence upon environment, while providing valuable resources for the scientific community. It will lay technological and conceptual groundwork for systematic perturbation-based studies of genetic variation in human cells that will inform the prediction of disease risk and the design of therapeutic strategies based on genome sequence.