mSphereAug 19, 2026

Higher temperature reduces the effectiveness of CRISPR gene blocking using Streptococcus pyogenes dCas9

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Aug 20, 2026

Abstract

CRISPRi knockdown efficacy is very low at high growth temperatures (45°C).

The effector protein used in CRISPRi is a modified version of Cas9, which loses effectiveness at elevated temperatures.
Temperature significantly impacts the performance of SpCas9-based CRISPRi systems in microbiological applications.
Genome-wide screens conducted in the model bacterium Bacillus subtilis reveal minimal gene repression at high temperatures.
Single-cell measurements of GFP fluorescence confirm the low repression observed in CRISPRi strains at elevated temperatures.
SpCas9-based CRISPRi systems may not be suitable for use in thermophilic organisms or under heat shock conditions.

Simplified

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.