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.