The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships, including instances of feedback control. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which 5,687 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains Synthetic Genetic Array (SGA) screening markers and a unique molecular barcode, enabling high-throughput yeast genetics. We characterized YETI using quantitative growth phenotyping and pooled BAR-seq screens, and we used a YETI allele to characterize the regulon of ROF1, showing that it is a transcriptional repressor. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from other eukaryotic and prokaryotic systems shows that low native expression is a critical variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3EB42, that gives lower expression than Z3EV, a feature enabling both conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.

中文翻译：

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具有可诱导表达单个基因的基因组规模的酵母文库

将基因从关闭切换到打开并监视动态变化的能力为探测基因功能和阐明因果调节关系（包括反馈控制实例）提供了强大的方法。在这里，我们开发并表征了YETI（具有Titratable诱导的酵母雌二醇菌株），该集合中的5687个酵母基因经过工程改造，可在其天然位点处以单基因精度进行转录诱导，并且无需质粒。每个菌株均包含合成遗传阵列（SGA）筛选标记和独特的分子条形码，可实现高通量酵母遗传。我们使用定量生长表型和合并的BAR-seq筛选对YETI进行了表征，并使用YETI等位基因表征了ROF1的调控子，表明它是转录阻遏物。我们观察到具有可诱导的必需基因且天然表达较低的菌株通常可以在没有诱导剂的情况下生长。对来自其他真核和原核系统的数据的分析表明，低天然表达是一个关键变量，可能会影响包括CRISPRi在内的干扰启动子的筛选。我们设计了第二个表达系统Z3EB42，该表达系统的表达低于Z3EV，该功能可以有条件地激活和抑制在YETI文库中不存在诱导子的低表达必需基因的生长。