Methods for engineering S. cerevisiae strains carrying multiple precise deletions

描述(申请人提供)：来自酿酒酵母单基因缺失研究的一个引人注目的结论是，在富媒体中，约80%的基因是非必需的。其中，约84%在所有21种不同的生长条件下都是非必需的。双突变研究证实，基因丢失通常被其他具有补偿功能的基因的存在所缓冲。因此，经常需要多个基因的突变来揭示基因的功能。如果存在一个功能重叠的大基因家族，就必须删除许多基因来消除该功能。例如，必须至少删除20个基因才能消除葡萄糖运输。在另一个例子中，在酵母中的29个ABC转运体同系物(药物外排泵)之间存在大量重叠的功能。功能缺陷的多突变菌株允许通过功能互补来克隆同源基因，或允许分离研究单基因的“加回”实验。在这里，我们提出了一种高风险/高回报的方法，用于快速设计携带许多精确缺失的菌株：1)对于每个目标基因，构建一个“ProMonster”菌株，在可诱导的Tet启动子下将其中一个目标基因精确替换为GFP；2)将ProMonster菌株汇集在一起，相互重复交配，形成孢子；3)通过荧光激活细胞分类(FACS)选择增加GFP基因剂量的菌株，从而选择更多缺失的菌株。预计突变等位基因的数量在每一轮早期都会翻一番，并继续快速增长。模拟表明，携带24个缺失的菌株只需要8轮交配、产孢子和FAC选择。由此产生的绿色怪物菌株可以使用特定于基因座的聚合酶链式反应或条形码微阵列进行分型。这一策略也可以应用于其他类型的等位基因--例如，允许蛋白质复合体所有成员表达同步变化的可诱导启动子等位基因，或许多外源基因的插入(允许在更容易处理的遗传系统中研究复杂的哺乳动物途径)。一个潜在的未来扩展是一种“基因下拉”技术，用于发现一组基因，这些基因表现出掩蔽上位或抑制给定查询基因中的突变。许多与公共健康高度相关的基因--例如，那些编码通过从细胞输出药物对病原体和肿瘤产生抗药性的蛋白质--是相关基因大家族的成员，而其他与疾病相关的基因与其他基因协同作用，编码蛋白质复合体。我们提出了有效地设计酵母菌株的方法，在这些酵母菌株中，许多选定的基因从另一个生物体中被精确地删除或插入。多重缺失的菌株可以对基因家族中的基因进行功能研究，而多重插入的菌株可以在易于处理的模式生物中研究人类蛋白质复合体。

DESCRIPTION (provided by applicant): A striking conclusion from single-gene deletion studies in S. cerevisiae is that ~80% of genes are non- essential in rich media. Of these, ~84% are non-essential in all of 21 diverse growth conditions. Double mutant studies confirm that gene loss is often buffered by the presence of other genes with compensatory function. Thus, mutations in multiple genes are often required to reveal gene function. Where a large gene family with overlapping function exists, one must delete many genes to eliminate the function. For example, at least 20 genes must be deleted to eliminate glucose transport. In another example, there is substantial overlapping function among the 29 ABC transporter homologs (drug efflux pumps) in yeast. Multi-mutant strains deficient in a function allow cloning of orthologous genes by functional complementation, or 'add-back' experiments allowing study of single genes in isolation. Here we propose the "Green Monster" method, a high risk / high reward technology for rapidly engineering strains carrying many precise deletions: 1) for each target gene, a "ProMonster" strain is constructed, carrying a precise replacement of one of the target genes with GFP under an inducible Tet promoter; 2) ProMonster strains are pooled and repeatedly mated en masse with one another, sporulated; 3) increasing GFP gene dosage is selected by fluorescence-activated cell sorting (FACS), thereby selecting strains with more deletions. The number of mutant alleles is expected to double in each early round, and continue to grow quickly. Simulations show that a strain carrying 24 deletions would require as few as 8 rounds of mating, sporulation, and FACS selection. Resulting Green Monster strains can be typed using locus- specific PCR primers or 'bar-code' microarrays. This strategy can also be applied to alleles of other types-e.g., inducible promoter alleles allowing synchronous change in expression of all members of a protein complex, or insertion of many exogenous genes (allowing study of complex mammalian pathways in a more tractable genetic system). A potential future extension is a 'genetic pull-down' technique for discovering sets of genes that exhibit masking epistasis or suppression of a mutation in a given query gene. Many genes of high relevance to public health--e.g., those encoding proteins that provide drug resistance to pathogens and tumors by exporting drugs from the cell--are members of large families of related genes, while other disease-relevant genes act in concert with other genes to encode protein complexes. We propose methods for efficiently engineering yeast strains in which many selected genes have been precisely deleted or inserted from another organism. Multiply-deleted strains allow functional study of genes in gene families, while multiply-inserted strains allow the study of human protein complexes in a tractable model organism.