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Figure 3 Correction of the Pathogenic FBN1T7498C Mutation by Base Editing in Heterozygous Human Embryos Show full caption (A) Schematic illustration of the experimental procedure for the correction of the pathogenic mutation by base editor in human embryos. (B) The development stage of the corrected embryos and the control embryos before and after treatment. (C) The representative chromatogram of the sequencing of PCR products from the corrected human embryos. The human embryos treated with base editor were collected, and the genomic DNA was extracted and amplified by PCR. The PCR products were analyzed by DNA sequencing. The red stars indicate the target base; the arrow indicates another base substitution in the target region. (D) The representative chromatogram of the sequencing of PCR products from the control human embryos. The control human embryos were collected, and the genomic DNA was extracted and amplified by PCR. The PCR products were analyzed by DNA sequencing. The red stars indicate the target base; the mixed peaks at the target site in Control-1 indicate the different kinds of nucleotides. (E) The genotyping analysis by deep sequencing. All of the samples, including three control and seven corrected human embryos, were collected, and the genomic DNA was extracted and amplified by PCR. The PCR products were analyzed by deep sequencing. The percentage of different genotypes of all samples was calculated.

The efficient and specific correction of the MFS pathogenic FBN1mutation encouraged us to test this process in human embryos. The immature oocytes were collected for research with the informed consent from individual donors. The collected oocytes were first subjected to in vitro maturation. Then the matured oocytes were used for in vitro fertilization (IVF) by ICSI (intracytoplasmic sperm injection) of single sperm donated by the MFS patient with the heterozygous FBN1mutation ( Figure 3 A). 16–18 hr later, all of the zygotes were collected, and some of them were microinjected with the mRNA of BE3 and the correctional sgRNA at concentrations of 100 and 50 ng/μL, respectively. As the control, the other zygotes were microinjected similarly with the mRNA of BE3 and the scrambled sgRNA. The embryos were further cultured for 2 days and then collected for analysis. A total of seven testing and seven control embryos that showed apparently normal development were obtained ( Figure 3 B). All samples were used for whole-genome amplification and then genotyped by Sanger sequencing. As shown in the sequence chromatograms, all of the testing embryos showed A at the 7498 site (Gsite), whereas three of seven control embryos harbored G (about 50%, as expected), indicating that allele correction in testing embryos occurred at a rate of near 100% ( Figures 3 C and 3D; Embryo-1∼7 in Figures S3 A and S3B). Nevertheless, an unwanted C-to-T conversion in addition to the desirable correction was detected in Embryo-7 ( Figure 3 C). To further characterize the BE3-mediated base editing, another 11 embryos were used to repeat the test by microinjection of BE3 and the correctional sgRNA as above. Sequencing the PCR products of the target site showed that 10 of the edited embryos yielded completed conversion at the 7498 site, besides Embryo-9, which harbored about 60% G-to-A at the 7498 site. These results showed BE3 mediated efficient correction of the pathogenic mutation in embryos.