Abstract
AbstractThe 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.
Publisher
Cold Spring Harbor Laboratory
Cited by
4 articles.
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