Enhancing Bacterial Crispri Specificity Using� Active ThermoCas9 and an Anticrispr Protein

CRISPR-Cas9 technologies have enabled unprecedented genome editing and transcriptional regulation efficiency in prokaryotes, accelerating the exploration and exploitation of their metabolism. However the target-DNA binding specificity of the extensively employed Streptococcus pyogenes (Sp)Cas9, as well as of currently reported engineered variants, is low. This fact compromises the accuracy of CRISPRi-based metabolic engineering studies in bacteria. Here we report the in vitro (at 37°C) and in vivo (in Escherichia coli) characterization of the thermo-tolerant Cas9 orthologue from Geobacillus thermodenitrificans T12 (ThermoCas9) that shows enhanced DNA binding and targeting specificity in comparison to previously reported data for other Cas9 orthologs as well as improved DNA binding specificity compared to its catalytically inactive variant, Thermo-dCas9. To further exploit the ThermoCas9 binding specificity for CRISPRi purposes, we harness the function of a small anti-CRISPR protein from Neisseria meningitidis (AcrIIC1_Nme). We show that AcrIIC1_Nme traps ThermoCas9 in vivo in a DNA-bound, catalytically inactive state, robustly inhibiting targeting and resulting in transcriptional silencing with comparable on-target efficiency to Thermo-dCas9.