Fig. 1

a Schematic of expression construct for regulatable CRISPRi in yeast. Key features include ORFs expressing dCas9-Mxi1 and the tetracycline repressor (TetR), as well as a tetracycline inducible gRNA locus containing the RPR1 promoter with a TetO site, a NotI site for cloning new gRNA sequences encoding target complementarity, and the constant part of the gRNA. b In the absence of anhydrotetracycline (ATc) TetR binds the gRNA promoter and prevents PolIII from binding and transcribing the gRNA. This in turn prevents dCas9-Mxi1 from binding the target site. In the presence of ATc, TetR dissociates and gRNA is expressed, allowing dCas9-Mxi1 to bind its target locus, and repress gene expression. c CRISPRi-induced drug-sensitivity. Transformants expressing gRNAs directed against CRG1, ERG11, ERG25, and SEC14 (indicated above each panel), were grown in the presence of a specific small molecule (that is, cantharidin, fluconazole, 1181-0519, and 4130-1276, respectively). Growth of the gRNA-expressing strain and the empty vector control, was measured in the presence and absence of ATc (see legend). Growth relative to the ‘no-drug’ control is indicated on the y-axis (see Methods), in increasing concentrations of each small molecule (x-axis)