Thermostable Cas9 nucleases with reduced off-target activity

Summary

ThermoCas9 is identified and characterized from the thermophilic bacterium Geobacillus thermodenitrificans T12. Experiments show how in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. Described are ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 5 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida