Methods for engineering S. cerevisiae strains carrying multiple precise deletions

描述（由申请人提供）：从S.酿酒酵母的一个重要原因是，在丰富的培养基中，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是非必需的。双突变体研究证实，基因丢失往往是缓冲的存在，其他基因的补偿功能。因此，通常需要多个基因的突变来揭示基因功能。如果存在一个具有重叠功能的大基因家族，则必须删除许多基因以消除功能。例如，必须删除至少20个基因以消除葡萄糖转运。在另一个实例中，在酵母中的29种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直向同源基因，或允许单独研究单个基因的“加回”实验。在这里，我们提出了“绿色怪物”方法，一种用于快速工程化携带许多精确缺失的菌株的高风险/高回报技术：1）对于每个靶基因，构建“ProMonster”菌株，其携带在诱导型泰特启动子下的GFP对其中一个靶基因的精确替换; 2）将ProMonster菌株汇集并重复地彼此交配，形成孢子; 3）通过荧光激活细胞分选（FACS）选择增加GFP基因剂量，从而选择具有更多缺失的菌株。预计在每一轮早期，突变等位基因的数量将翻一番，并继续快速增长。模拟显示，携带24个缺失的菌株将需要少至8轮的交配、孢子形成和FACS选择。所得的绿色Monster菌株可以使用基因座特异性PCR引物或“条形码”微阵列进行分型。这种策略也可以应用于其他类型的等位基因-例如，诱导型启动子等位基因，允许蛋白质复合物的所有成员的表达同步变化，或插入许多外源基因（允许在更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个“遗传下拉”技术，用于发现一组表现出掩蔽上位性或抑制给定查询基因中的突变的基因。许多与公共健康高度相关的基因-例如，那些编码蛋白质的基因通过从细胞中输出药物来提供对病原体和肿瘤的抗药性，这些基因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一起编码蛋白质复合物。我们提出了有效的工程酵母菌株，其中许多选定的基因已被精确删除或插入从另一个有机体的方法。多位点缺失的菌株允许基因家族中基因的功能研究，而多位点插入的菌株允许在易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.