Methods for engineering S. cerevisiae strains carrying multiple precise deletions

描述（由申请人提供）：酿酒酵母单基因缺失研究的一个惊人结论是，约80%的基因在富培养基中是非必需的。在所有21种不同生长条件下，其中约84%为非必需物质。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所缓冲。因此，通常需要多个基因的突变来揭示基因的功能。当存在一个功能重叠的大基因家族时，必须删除许多基因才能消除其功能。例如，至少有20个基因必须被删除才能消除葡萄糖运输。在另一个例子中，在酵母菌中29个ABC转运蛋白同源物（药物外排泵）之间存在大量重叠功能。功能缺陷的多突变株允许通过功能互补克隆同源基因，或者允许分离研究单个基因的“加回”实验。在此，我们提出了“绿色怪物”方法，一种高风险/高回报的技术，用于快速工程菌株携带许多精确缺失：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，用GFP精确替换一个目标基因；2) ProMonster菌株聚集在一起，彼此重复交配，形成孢子；3)通过荧光活化细胞分选（FACS）选择增加GFP基因剂量，从而选择缺失较多的菌株。突变等位基因的数量预计在每一轮早期增加一倍，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、产孢和FACS选择。由此产生的绿怪菌株可以使用位点特异性PCR引物或“条形码”微阵列进行分型。这种策略也适用于其他类型的等位基因，例如：可诱导的启动子等位基因，使一个蛋白质复合体的所有成员的表达同步改变，或插入许多外源基因（允许在一个更易于处理的遗传系统中研究复杂的哺乳动物途径）。潜在的未来扩展是一种“遗传下拉”技术，用于发现在给定查询基因中表现出掩蔽上位或抑制突变的基因集。许多与公众健康高度相关的基因——例如：这些基因编码的蛋白质通过从细胞输出药物来提供对病原体和肿瘤的耐药性，是相关基因大家族的成员，而其他疾病相关基因则与其他基因协同作用，编码蛋白质复合物。我们提出了一种有效的酵母菌工程方法，其中许多选定的基因已经被精确地从另一个生物体中删除或插入。多重缺失菌株允许对基因家族中的基因进行功能研究，而多重插入菌株允许在可处理的模式生物中研究人类蛋白质复合物。

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.