Pcmv be4max

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.

All plasmids used were either purchased from Addgene (Watertown, MA, United States), specifically pCMV_BE4max (Addgene ID: 112093), or previously published (Patsali et al., 2019b (link)), and retargeting of BEs was achieved by co-transfection with alternative synthetic gRNAs. Purchased plasmids were received as bacterial stabs, which were purified and verified. Briefly, bacteria were spread in an agar plate following overnight (O/N) incubation at 37°C. A single colony was isolated and added in Luria Broth (LB) (Invitrogen, Carlsbad, CA, United States), Supplemented with antibiotic (ampicillin or kanamycin), and was incubated O/N at 37°C. The next day, the plasmids were purified with the Nucleobond Xtra Midi Endotoxin-free plasmid DNA purification kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following the manufacturer’s instructions. Approximately 1 μg of each plasmid was used to verify identities by informative restriction digests and comparison with the corresponding plasmid map.