High-throughput precision genome editing to characterize natural genetic variants

项目摘要 遗传学和进化生物学的一个主要目标是了解基因型的变化如何影响 表型。影响适应性的遗传变异对于研究进化特别有用，因为自然 选择只对这些变体起作用。通过识别影响适应性的特定变体，我们可以开始 去理解驱动所有生物体令人难以置信的适应性的分子机制， 环境. 我们最近开发了一种方法，使我们能够以前所未有的效率编辑基因组 （~100%）和吞吐量。在我们最初的筛选中，我们测量了16，000个自然基因的适应性效应， 两种酿酒酵母菌株之间的不同变体。在这个试点实验中，我们测量了 在一个单一的条件下，每个变量的影响。我们发现，几乎所有的强适应性效应都是 来自启动子变体，而不是蛋白质编码区，这些在转录时特别丰富 因子结合位点。 在这里，我们建议利用这个强大的系统来调查两种类型的相互作用的基本 重要性：目标1中的基因-环境（GxE），目标2中的基因-基因（GxG，或上位性）。 理解适应性效应的上下文依赖性将揭示进化过程的关键见解 如果没有我们的高通量精确基因组编辑技术，这将是无法实现的。 !

Project summary A major goal of genetics and evolutionary biology is to understand how changes in genotype affect phenotype. Genetic variants affecting fitness are especially informative for investigating evolution, since natural selection acts exclusively on these variants. By identifying specific variants that influence fitness, we can begin to understand the molecular mechanisms driving the incredible adaptations of all organisms to their respective environments. We recently developed an approach that allows us to edit genomes with unprecedented efficiency (~100%) and throughput. In our initial screen, we measured the fitness effects of 16,000 natural genetic variants differing between two strains of Saccharomyces cerevisiae. In this pilot experiment, we measured the effects of each variant in isolation, in a single condition. We found that nearly all strong fitness effects were from promoter variants, rather than protein-coding regions, and these were especially enriched at transcription factor binding sites. Here we propose to utilize this powerful system to investigate two types of interactions of fundamental importance: gene-by-environment (GxE) in Aim 1, and gene-by-gene (GxG, or epistasis) in Aim 2. Understanding the context-dependence of fitness effects will reveal key insights into the evolutionary process that would be unapproachable without our high-throughput precision genome editing technology. !