Fig. 3: Comparison of ABEmax and ABE8e for editing and phenotypic correction in FA LCLs.

a Schematics of experimental design to edit FA-55 or FA-75 LCLs by ABEmax or ABE8e using mRNA base editor and synthetic gRNAs. Electroporated cells were collected at day 5 to measure editing efficiency and at day 9 to measure MMC resistance and activation of FANCD2 monoubiquitination. (Created with BioRender.com). b Quantification of editing levels by amplicon NGS on day 5 in edited LCL populations. Dot or rhombus indicate individual experiments, bars represent the mean of 4 independent experiments and error bars indicate SD. c Representative Sanger traces show initial editing 5 days after electroporation of FA-55 and FA-75 with ABE8e mRNA and with or without indicated synthetic sgRNAs. Arrows indicate the presence of edited alleles. Dots indicate the presence of bystander edits. d MMC survival of edited FA-A LCLs. Black lines indicate healthy donor (HD) LCL response to increasing doses of MMC. Dashed lines represent FA-55 or FA-75 LCLs electroporated only with ABEmax or ABE8e. Solid colored lines represent FA-55 or FA-75 electroporated with sgRNAs and ABEmax or ABE8e. The graphs summarize the mean of 4 (FA-55) and 3 (FA-75) biological replicates, error bars indicate SD. e Representative Western blots. Indicated cell populations were challenged with 1 μM MMC for 24 h. Protein extracts were analyzed by Western blot with indicated antibodies (anti-FANCD2, anti-HSP60). FANCD2 or FANCD2-Ub bands are indicated by the arrows. (n = 2, biologically independent experiments). Source data are provided as a Source Data file.