Base Editing Constructs Preparation

Plasmids for the following four base editing constructs: pCMV_BE4max (Addgene plasmid # 112093), pCMV_BE4max_P2A_GFP (Addgene plasmid # 112099), pCMV_AncBE4max (Addgene plasmid # 112094), and pCMV_AncBE4max_P2A_GFP (Addgene plasmid # 112100) [40 (link)] were kind gifts from Dr. David Liu. Plasmid DNA was purified from an overnight culture of TOP10 cells (Thermo Fisher Scientific, Waltham, MA, USA) using a miniprep kit (Qiagen, Germantown, MD, USA). All four plasmid DNAs were linearized with SapI (New England BioLabs, Ipswich, MA, USA) and mRNA was synthesized with the T7 mMessage mMachine kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions with the following modifications: addition of 1 µL GTP, an incubation time of 3 h and LiCl precipitation.