A genome‐scale yeast library with inducible expression of individual genes

Abstract 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. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single‐gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high‐throughput genetics. We characterized YETI using growth phenotyping and BAR‐seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a 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 conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.


Table of Contents
Sequence information 3

Media recipes 5
Yeast Strains 7 Plasmids 9 Appendix Figure  Sequence information p5820, Z3 promoter sequence (6 binding sites) The sequence of Z3 (6 binding sites) promoter is shown below. Modified UASGAL for Z3EV binding is highlighted in green. XbaI and NotI restriction sites are colored with gray. A Gal4 binding site outside of XbaI and NotI restriction sites was also removed.

Z3EB42 sequence
The ACT1 promoter is shown in blue; the Z3 DNA binding domain is shown in green; the human Estrogen Receptor is shown in magenta; the B42 activation domain is in red; the ENO2 terminator is in orange. The transcription factor was integrated 300 bp downstream of functional HAP1. 300 bp of downstream sequence of HAP1 was also added downstream of the Z3EB42 transcription factor. GCCTCTACCTTGCAGACCCATATAATATAATAACTAAATAAGTAAATAAGACACACGCGAGAACATATATACACAATTACAGTAAC  AATAACAAGAGGACAGATACTACCAAAATGTGTGGGGAAGCGGGTAAGCTGCCACAGCAATTAATGCACAACATTTAACCTACA  TTCTTCCTTATCGGATCCTCAAAACCCTTAAAAACATATGCCTCACCCTAACATATTTTCCAATTAACCCTCAATATTTCTCTGTCA  CCCGGCCTCTATTTTCCATTTTCTTCTTTACCCGCCACGCGTTTTTTTCTTTCAAATTTTTTTCTTCCTTCTTCTTTTTCTTCCACGT  CCTCTTGCATAAATAAATAAACCGTTTTGAAACCAAACTCGCCTCTCTCTCTCCTTTTTGAAATATTTTTGGGTTTGTTTGATCCTT  TCCTTCCCAATCTCTCTTGTTTAATATATATTCATTTATATCACGCTCTCTTTTTATCTTCCTTTTTTTCCTCTCTCTTGTATTCTTCC  TTCCCCTTTCTACTCAAACCAAGAAGAAAAAGAAAAGGTCAATCTTTGTTAAAGAATAGGATCTTCTACTACATCAGCTTTTAGAT  TTTTCACGCTTACTGCTTTTTTCTTCCCAAGATCGAAAATTTACTGAATTAACAGGGCCCCCCCTCGAGGTCGACGGTATCGATA  AGCTTGAAGCAAGCCTCCTGAAAGATGGGTACCCGCCCATATGCTTGCCCTGTCGAGTCCTGCGATCGCCGCTTTTCTCGCTC  GGATGAGCTTACCCGCCATATCCGCATCCATACCGGTCAGAAGCCCTTCCAGTGTCGAATCTGCATGCGTAACTTCAGTCGTAG  TGACCACCTTACCACCCACATCCGCACCCACACAGGCGAGAAGCCTTTTGCCTGTGACATTTGTGGGAGGAAGTTTGCCAGGA  GTGATGAACGCAAGAGGCATACCAAAATCCATACAGGTGGCGGAGGCACACCTGCAGCTGCGTCGACTCTAGAGGATCCATCT  GCTGGAGACATGAGAGCTGCCAACCTTTGGCCAAGCCCGCTCATGATCAAACGCTCTAAGAAGAACAGCCTGGCCTTGTCCCT  GACGGCCGACCAGATGGTCAGTGCCTTGTTGGATGCTGAGCCCCCCATACTCTATTCCGAGTATGATCCTACCAGACCCTTCA  GTGAAGCTTCGATGATGGGCTTACTGACCAACCTGGCAGACAGGGAGCTGGTTCACATGATCAACTGGGCGAAGAGGGTGCC  AGGCTTTGTGGATTTGACCCTCCATGATCAGGTCCACCTTCTAGAATGTGCCTGGCTAGAGATCCTGATGATTGGTCTCGTCTG  GCGCTCCATGGAGCACCCAGTGAAGCTACTGTTTGCTCCTAACTTGCTCTTGGACAGGAACCAGGGAAAATGTGTAGAGGGCA  TGGTGGAGATCTTCGACATGCTGCTGGCTACATCATCTCGGTTCCGCATGATGAATCTGCAGGGAGAGGAGTTTGTGTGCCTC  AAATCTATTATTTTGCTTAATTCTGGAGTGTACACATTTCTGTCCAGCACCCTGAAGTCTCTGGAAGAGAAGGACCATATCCACC  GAGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAGGCAGGCCTGACCCTGCAGCAGCAGCACCAGCGGCT  GGCCCAGCTCCTCCTCATCCTCTCCCACATCAGGCACATGAGTAACAAAGGCATGGAGCATCTGTACAGCATGAAGTGCAAGA  ACGTGGTGCCCCTCTATGACCTGCTGCTGGAGATGCTGGACGCCCACCGCCTACATGCGCCCACTAGCCGTGGAGGGGCATC  CGTGGAGGAGACGGACCAAAGCCACTTGGCCACTGCGGGCTCTACTTCATCGGGTATCAATAAAGAcATCGAGGAGTGCAATG  CCATCATTGAGCAGTTTATCGACTACCTGCGCACCGGACAGGAGATGCCGATGGAAATGGCGGATCAGGCGATTAACGTGGTG  CCGGGCATGACGCCGAAAACCATTCTTCACGCCGGGCCGCCGATCCAGCCTGACTGGCTGAAATCGAATGGTTTTCATGAAAT  TGAAGCGGATGTTAACGATACCAGCCTCTTGCTGAGTGGAGATGCCTCCAAGCTTTAAATCCCCGCGTGCTTGGCCGGCCGTA  GTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATC  TATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAA  AAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAACTACATGGATGATAAGAAAACATGGAGTACAGT  CACTTTGAGAACCTTCAATCAGCTGGTAACGTCTTCGTTAATTGGATACTCAAAAAAGATGGATAGCATGAATCACAAGATGGAA GGAAATGCGGGCCACGACCACAGTGATATGCATATGGGAGATGGAGATGATACCTGTTCGATGAATATGCTATTTTCGTGGTCA TACAAGAATACGTGTGTCGTCTTTGAATGGTGGCATATCAAGACCCTGCCTGGACTGAT

10X SC solution (for preparation of 1L)
-Add 20 g of SC mix (Sunrise Cat# 1300-030) into a beaker with stir bar -Add 900 mL of water -Stir until dissolved (heat liquid up to 60°C to dissolve) -Transfer into a graduated cylinder, and add water to 1000 mL -Filter sterilize using 1000 mL stericup

10X SC-arg-his-lys-ura solution (for preparation of 1L)
-Add 16.6 g of SC-arg-his-lys-ura mix (Sunrise Cat# 6103-030) into a beaker with stir bar -Add 900 mL of water -Stir until dissolved (heat liquid up to 60°C to dissolve) -Transfer into a graduated cylinder, and add water to 1000 mL -Filter sterilize using 1000 mL stericup Appendix Figure S3: Appendix Figure S7: Non-essential toxic genes encode proteins that are bigger and more disordered than non-toxic genes A. Density plots of YETI-NE genes identified as non-toxic/toxic when overexpressed based on the percent of the protein that belongs to an intrinsically disordered region. The sample sizes are 687 for toxic genes, and 3970 for non-toxic strains. B. Density plots of YETI-E genes identified as non-toxic/toxic when overexpressed based on the percent of the protein that belongs to an intrinsically disordered region. The sample sizes are 301 for toxic genes, and 721 for non-toxic strains. C. Density plots of YETI-NE genes identified as non-toxic/toxic when overexpressed based on protein length. D. Density plots of YETI-E genes identified as nontoxic/toxic when overexpressed based on protein length.
Appendix Figure S10: Reversibility analysis for 19 strains that impair growth when overexpressed A. The Response Ratio, ResRatioi = AUGCi,0,0 / AUGCi,10,10, for each strain. For AUGCi,j,k, i is the gene, j is the concentration of β-estradiol during the first round of growth, and k is the level of β-estradiol during the second round of growth. For a given gene, the larger the ResRatio value, the greater the toxicity when the gene is induced. If log2(ResRatio) is negative, the gene improves growth when induced. Genes are ranked from least toxic to most toxic when induced in the presence of β-estradiol B. The Reversibility Ratio, RevRatioi = AUGCi,10,0 / AUGCi,0,10i, for each strain. If log2(RevRatioi) > 0, the growth phenotype is at least partially reversible. C-D.
Examples of reversible (C) and non-reversible (D) phenotypes. Barplots show total area under growth curve (AUGC) for cells grown at two β-estradiol concentrations and transferred to plates under four different conditions: 0 to 0_avg -a control transfer from no β-estradiol to no βestradiol (no β-estradiol); 10 to 10_avg -another control transfer from β-estradiol to β-estradiol (continuous β-estradiol); 0 to 10_avg -transfer from no β-estradiol to β-estradiol (addition of βestradiol) and; 10 to 0_avg -transfer from β-estradiol to no β-estradiol (removal of β-estradiol).