Next, we tested the feasibility of repairing HBB −28 (A>G) mutation in human embryos. To model disease embryos, we generated cloned human embryos by nuclear transfer (Fig. 3A). The 1st polar body (PB1) and spindle of the in vitro matured oocytes were removed, and then the oocytes were fused with lymphocyte cells from peripheral blood of the patient. The reconstructed oocytes were activated and cultured until the appearance of pronucleus (PN). Approximately 5–6 h later, BE3 mRNA (200 ng/μL) and gRNA-1 (100 ng/μL) were injected into the cytoplasm after the appearance of pronucleus (Fig. S4). Of the 30 embryos injected, 26 survived (Fig. 3B). 48 h later, the HBB site of each embryo was PCR amplified individually. And then the PCR products were detected by Sanger sequencing and deep sequencing. HBB site was successfully amplified in 22/26 embryos (Fig. 3B). Interestingly, in these cloned embryos, we found high point mutation repairing efficiency, which was between 7.0% and 25.9% among the repaired embryos (Figs. 3C, S5 and Table S1). Analysis of the data showed that G−28 was converted to either A or C in 45.4% (10/22) of the injected embryos (Fig. 4B). In embryo #17, G−28 was converted to C. In the other 9 embryos, G−28 was converted to A, representing precise mutation repairing (Fig. 3B and 3C). Furthermore, we did not find deamination at G−25, indicating highly efficient and specific point mutation repairing in these embryos (Fig. 3C).Figure 3 Effective HBB gene correction in human embryos by BE3. (A) Schematic of repairing HBB −28 (A>G) in cloned human embryos by BE3 and gRNA-1. Cloned HBB −28 (A>G) mutant homozygous human embryos were generated by fusing lymphocyte cell, from peripheral blood of the patient, with in vitro matured oocytes. And the BE3 mRNA and gRNA mixture was injected after the appearance of pronucleus. HBB site from each embryo was amplified by PCR and deep sequenced. PB1, the 1st polar body. PN, pronucleus. ZP, zonapellucida. (B) Summary of base editing-mediated point mutation repairing by BE3 in cloned human embryos. The repaired embryo contains G>A conversion at the HBB −28 site. *, The target G at the HBB −28 site was converted to C instead of A. (C) Deep sequencing to detect successful repairing by BE3 in human embryos. Target site PCR amplicons from these embryos were deep sequenced Figure 4 Improving the precision of gene correction in human embryos by YEE-BE3. (A) Schematic of repairing HBB −28 (A>G) in cloned human embryos by YEE-BE3 and gRNA-1. Firstly, cloned HBB −28 (A>G) mutant homozygous human embryos were generated by fusing skin fibroblast cell from the patient with in vitro matured oocytes. And YEE-BE3 mRNA and gRNA mixture was injected after removing PB1. And 1 h later, the injected oocytes were fused with skin fibroblast cells. Then the fused embryos were activated and cultured for another 48 h. Single blastomere was isolated and MDA amplified. Then HBB site was amplified and sequenced. PB1, the 1st polar body. PN, pronucleus. ZP, zonapellucida. (B) Summary of base editing-mediated point mutation repairing by YEE-BE3 in cloned human embryos. The numbers of homozygous mutant blastomere (G−28G−25/G−28G−25), heterozygous blastomeres (A−28G−25/G−28G−25), and wild-type blastomeres (A−28G−25/A−28G−25) were calculated. #, 4 embryos did not develop into 2-cell stage. *, HBB site failed to be amplified by PCR. (C) Sanger sequencing to detect successful repairing by YEE-BE3 in each blastomere. Representative sequencing chromatographs of homozygous mutant blastomeres, heterozygous blastomeres and wild-type blastomeres