Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins.