Of the two base editors (BE), BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI), BE3 showed higher editing efficiency (Kim et al., 2017a). We therefore decided to repair HBB −28 (A>G) mutation using BE3. HBB −28 (A>G) mutation, in which the wild-type A at position −28 (A−28) is replaced with G in patients (G−28), locates in the ATA box upstream of the first exon of HBB (Fig. 1A) (Orkin et al., 1983). Three gRNAs targeting this mutant HBB allele were designed to convert C (on the complementary strand) to T (Figs. 1A and S1). We found that G at position −25 (G−25) might also be converted to A by these gRNAs (Fig. 1A). To test the deamination activity of these three gRNAs, we cloned the DNA fragment surrounding the HBB −28 (A>G) mutation into a lentiviral vector for stable integration in 293T cells. After selection with puromycin, three different cell clones were picked and verified by PCR (Fig. 1B). PCR primers (FP1 & RP1), that could specifically amplify this exogenous HBB −28 (A>G) mutant fragment, were designed (Fig. 1B). Sanger sequencing of this PCR amplicons indicated a clear G at HBB position −28 in these cell clones (Fig. 1B).Figure 1 Correcting HBB −28 (A>G) mutation in human cell line. (A) Schematic of HBB −28 (A>G) mutation. The exons are labeled with blue boxes. −28 (A>G) mutation was in red and indicated with red line (G−28). The −25 (G), next to G−28, was in blue and indicated with blue line (G−25). And gRNAs were labeled with black arrow. (B) Generation of HBB −28 (A>G) mutant stable cell lines. A fragment of HBB gene, containing the −28 (A>G) mutation, was cloned into a lentiviral vector. Packaged lentivirus was used to infect 293T. Virus-infected cells were selected by puromycin. 7 days after selection, single clones of cells were picked. The up panel showed the design of the recombined lentivirus vector. HBB gene fragment containing −28 (A>G) mutation was labeled with green box. LTR (long terminal repeat) region of lentiviral vector was labeled with blue arrowhead. PCR primer used to specifically amplify HBB fragment from integrated provirus were showed. The down panel showed the results of one wild-type 293T cells and three clones, amplified using FP1 and RP1. Representative sequencing chromatographs of the PCR amplicons of #3 clone were shown. The mutant base (G−28) was indicated by red arrowheads. (C) Precise repairing of HBB −28 (A>G) mutation by base editor 3 in the HBB −28 (A>G) mutant stable cell line. TA cloning sequencing showed clear G>A conversion at the target site. The frequency of each allele is shown. (D) Deep sequencing to detect on-target and off-target deamination at 10 potential off-target sites in HBB −28 (A>G) mutant stable cell line. Bars represent mean ± SEM (n = 3). Significance was calculated using a two-tailed unpaired t test (*P < 0.05, **P < 0.01)