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
- 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?
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.
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.