Although we did not find off-target deamination at G−25, we could not rule out the possibility of off-target deamination at G−25 in human embryos according to the data in human cells (Figs. 1D and 2E). We therefore turned to YEE-BE3, a BE3 variant with a smaller deamination window (Kim et al., 2017d). We injected gRNA-1 and YEE-BE3 mRNA before fusing the skin fibroblast cell with oocytes in which spindle and PB1 had been removed. Injecting YEE-BE3 mRNA before fusion will leave more time for protein translation and deamination before cell division. At about one hour after fusion, the reconstructed embryos were activated and cultured for another 48 h, when embryos were at 4–8 cell stage (Fig. 4A). The zona pellucidas of these embryos were removed, and 73 blastomeres were isolated from 20 embryos (Fig. 4B). Then the blastomeres were MDA-amplified individually (Fig. 4A). The HBB site was PCR amplified from these MDA products and sequenced by Sanger sequencing (Fig. 4C). We successfully amplified the HBB loci in 48 blastomeres (48/73, 65.8%) (Fig. 4B), and found that 37 blastomeres were still homozygous mutants (G−28G−25/G−28G−25), while the other 11 blastomeres (11/48, 22.9%) had been repaired (Fig. 4B and Table 1). A total of 3 out of 11 (6.3%) repaired blastomeres were heterozygous, and the other 8 (16.7%) were WT with both mutant alleles repaired perfectly (Table 1). More importantly, no off-target deamination at G−25 was observed, suggesting highly precise deamination at G−28.Table 1Summary of base editing-mediated point mutation repairing by YEE-BE3 in human embryosEmbryo IDBlastomere No.PCR-amplified blastomere No.* <Homozygous>G−28G−25/G−28G−25 blastomere No. (%)<Heterozygous>A−28G−25/G−28G−25 blastomere No. (%)<Wild-type>A−28G−25/A−28G−25 balstomere No. (%)#121100#252200#37431(25)0#431100#563300#663201(33.3)#722200#811100#944400#1063300#1111100#124431(25)0#1333201(33.3)#1421001(100)#156541(20)0#1621100#1721100#1854103(75)#1953102(66.7)#2011100A total of 20 embryos were harvested for single blastomere genotyping. In some blastomeres, both alleles were repaired. In one blastomere, only one mutant allele was repaired* Some blastomeres failed to be amplified by PCR