Editing LCLs with base editor plasmids
For base editing experiments, LCLs were run through Ficoll gradient and the death cells and debris were cleared. 5 × 10^5 LCLs were electroporated with ABEmax (750 ng) and sgRNA (250 ng) using 4D Nucleofector™ X unit from Lonza (SF solution, DN-100 and CM-137). Cells were cultured in a 24 well dish after nucleofection and transferred into a T25 flask after recovery for the long term culturing.
Editing LCLs with in vitro transcribed mRNA
For base editing with ABEmax mRNA, 2 × 10^5 FA-55 or FA-75 LCLs were electroporated with 3 µg or 6 µg BE mRNA and 100 or 200 pmol of synthetic sgRNAs (Synthego), respectively. For both experiments the Lonza nucleofector was used with SF solution and the EW-113 nucleofection program.
Electroporation was performed using Lonza 4D Nucleofector (V4XP- 3032 for 20-µl Nucleocuvette Strips or V4XP-3024 for 100-µl Nucleocuvette Strips) according to the manufacturer’s instructions. The modified synthetic sgRNA (2'-O-methyl 3' phosphorothioate modifications in the first and last three nucleotides) were purchased from Synthego and BE mRNA was obtained through in vitro transcription using mMESSAGE mMACHINETM T3 Transcription kit (Invitrogen). A total of 2 × 10^5 HSPCs from healthy donor were resuspended in 20 µL P3 solution and electroporated in 20-µL Nucleocuvette wells using program EO-100 with increasing concentration of BE mRNA and sgRNA (3 µg of BEmRNA and 3.2µg sgRNA for HD CB cells and 6 µg of BE mRNA and 6.4 µg sgRNA for HD mPB cells). For 100-µL cuvette electroporation, 1 × 10^6 HSPCs were resuspended in 100 µL P3 solution and electroporated using 30 µg of BE mRNA and 32 µg of sgRNA with program EO-100. FA Lineage negative cells were electroporated using similar conditions. Electroporated cells were resuspended in StemSpan medium (StemCell Technologies) with corresponding cytokines.