Elevated temperature undermines the efficacy of Streptococcus pyogenes dCas9-based CRISPRi systems

Targeted gene repression using CRISPRi has become a foundational technique for microbiology. The effector protein in most CRISPRi systems is a catalytically inactivated version of Cas9, an RNA-guided DNA endonuclease from Streptococcus pyogenes (SpCas9). The application of SpCas9-based CRISPRi systems in increasingly ambitious experiments in diverse bacteria raises important questions about the effects of growth temperature on CRISPRi efficacy, particularly because SpCas9 loses cutting efficacy at elevated temperatures. Here, we test the effect of temperature on SpCas9 - based CRISPRi knockdown efficacy by performing a series of genome-wide screens using a well-characterized CRISPRi system in the model firmicute Bacillus subtilis . We observe very little repression at high (45°C) growth temperatures and confirm this observation using single cell measurements of GFP fluorescence in CRISPRi strains grown at different temperatures. Our data suggest that Streptococcus pyogenes- based CRISPRi systems are ill-suited for thermophiles and should not be used for elevated-temperature or heat shock experiments in mesophilic bacteria.

IMPORTANCE

CRISPRi is a common technique for inducible gene depletion in bacteria and has been used for many different types of experiments in increasingly diverse bacteria. We identify increased temperatures as an important limitation of the most common CRISPRi effector ( Streptococcus pyogenes dCas9). This finding both affects experimental design and informs the kinds of bacterial species amenable to manipulation by S. pyogenes -based CRISPRi systems.