1. dCas9-mediated repression of transcription.
dCas9-mediated repression of transcription. (A) Genetic constructs used to test repression. Design of the CRISPR interference systems requires the expression of dCas9 or of a dCas9 fused to the repressor Nrg1 and a sgRNA that targets the nuclease death protein to the CAT1 promoter in a tester strain which harbors the promoter fused to GFP. (B) Flow cytometry profiles of the indicated strains after 24 h of growth in SD medium at 37°C. (C) Percentage of transcriptional repression of the indicated strains normalized to the MFI level of the corresponding control strain which expresses only the sgRNA4 or sgRNA4F in the pCAT1-GFP background. Values represent means ± standard deviations of the results from at least three measurements from three independent transformants. (D) Oxidant susceptibility of cells expressing dCAS9 or dCAS9-NRG1 and sgRNA4 in both guide systems. Cells of the indicated strains from the stationary phase were serially diluted 10-fold and plated on SD media containing hydrogen peroxide (H2O2) at the indicated concentration or subjected to 75 mM H2O2 in liquid SD medium and spotted at different times (indicated in minutes) onto YPD plates. Images were taken after 24 h of growth at 37°C.
Elvira Román et al. mSphere 2019; doi: /mSphere