Next, we co-transfected the gRNA and the BE3 expression vectors into clone #3. Cells transfected with GFP were included as a control. After 48 h, the cells were harvested. Target sites were amplified with FP1 and RP1 primers. Sanger sequencing of the PCR amplicons revealed obvious G>A conversion using the three gRNAs (Fig. S2). TA cloning and sequencing further confirmed active conversion in these cells (Fig. 1C). The conversion efficiency was 46.7% (14/30) for gRNA-1 (Fig. 1C). And consistent with previous findings in human cells and mouse embryos, we found proximal-site deamination using gRNA-2 (Fig. 1C) (Liang et al., 2017). Off-target deamination could be a concern in base editing, so we further investigated off-target deamination in this HBB −28 (A>G) mutant cell line. We again co-transfected BE3 together with either gRNA-1 or gRNA-2 into clone #3. GFP transfected cells were used as a control. The cells were harvested for genomic DNA extraction 48 h after transfection. The exogenously integrated HBB DNA fragment and 10 potential off-target sites were PCR amplified for deep sequencing. We found 16.3% and 26.0% G>A conversions at the target sites for gRNA-1 and gRNA-2 respectively, significantly higher than the rate of 1.2% in GFP control cells (Fig. 1D). And in line with data in Fig. 1C, we found that both the G−28 and G−25 at the target region could be deaminated by BE3 (Fig. 1D). We found higher G>A conversion efficiency at G−28 and G−25 using gRNA-2 (Fig. 1D). Moreover, we did not found any off-target deamination at the 10 potential off-target sites examined for both gRNAs, indicating high specificity (Fig. 1D). Taken together, these results clearly indicate the feasibility of repairing HBB −28 (A>G) in human cells in situ by base editing.