Base editing tools

Thermostable, type II-C Cas9 variants of Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) use 23 nt spacers to edit sites adjacent to an N4CVAA/N4CCCA or N4CRAA PAM, respectively. These Cas9 variants are linked to a cytidine deaminase enzyme from the sea lamprey Petromyzon marinus (PmCDA1) to provide: (a) dThermoTarget-AID, (b) dGeoTarget- AID, (c) AcrThermoTarget-AID, and (d) AcrGeoTarget-AID. Compared to known base editors, these systems exhibit much larger base editing windows: (a) from -5 to -27 positions relative to the PAM (23 bp); (b) from -5 to -24 positions relative to the PAM (20 bp); (c) from -5 to -27 positions relative to the PAM (23 pb); (d) from -3 to - 28 positions relative to the PAM (26 bp). The first two editors employ the catalytically inactive ThermoCas9 or GeoCas9, respectively, while the last two editors co-express a small anti-CRISPR protein from Neisseria meningitidis (AcrIIC1Nme) with active ThermoCas9 or GeoCas9, respectively. Methods of gene editing are described.