26 Jan 2023
by Richa

Summary- paper 13: Gene dosage screens in yeast reveal core signalling pathways controlling heat adaptation

Cosimo Jann, Andreas Johansson, Justin D. Smith, Leopold Parts, Lars M. Steinmetz

Biorxiv, 2020

Questions/gaps addressed:

  • Heat shock induces significant transcriptional and transational changes in yeast and higher organisms but bulk of heat-induced changes seem dispensable. What mechanisms are essential for cell survival?

Key methods:

  • Inducible CRISPRi/a systems using a catalytically inactive Cas9 nuclease fused to transcriptional repression or activation domains to modulate levels of 129 protein kinases and 161 transcription factors in yeast (6 sgRNAs/gene), to screen for effects on cellular fitness at temperatures 23°C, 30°C and 38°C.

  • Employed the Streptococcus pyogenes Cas9 fused to the human Mxi1 repressor which both evolved to operate around 37°C. Tet-inducible dCas9-MxiI and dCas9-nGal4-VP64 plasmids (AddGene #73796 and #71128).

  • Chemically synthesized gRNA oligonucleotide libraries (CustomArray, Inc. (GenScript)) amplified by PCR and integrated into pRS416 dCas9-Mxi1 plasmid via Gibson Assembly with 30bp homology regions, or ligation with T4 DNA Ligase. - R code for screen analysis on Github

  • Validate repression/ activation from anhydrotetracycline (ATc)-inducible gRNA agaisnt Hsf1 transcription factor and monitor effect on growth rate. Quantify Hsf1 function with a truncated promoter of the SSA1 HSP70 gene driving GFP reporter, and FACS to monitor inheritibility.

Major takeaways:

  • CRISPRi efficiency depends on the GC content and secondary structure of gRNAs. The optimal range forgRNA target locus is between TSS-150 to TSS+25 nucleotides, with minor variation between target strands. Effective repression observed in CRISPRi strains over time and all temperatures.

  • Multiple pathways (HSR, HOG, UPR, cell cycle) controlling HSR activity that affect chaperone stability and expression levels needed for thermotolerance.