Fig. 2.

Correction of a pathogenic heterozygous mutation in human embryos with base editors. a Diagram of MUT c.299 A>G mutation locus for a male patient. Exon is labeled with a gray box; c.299A>G mutation site is indicated with a red line. In addition, mutant and wild-type alleles of this patient can be distinguished by two adjacent neutral SNPs. b Experimental diagram showing BE3-medicated gene correction in human embryos. Sperm from a heterozygous patient was used to fertilize the oocytes. BE3 mRNA and MUT sgRNA were co-injected into each blastomere of the two-cell embryos 36 h after fertilization. Embryos were cultured to eight-cell embryos and used for targeted deep sequencing or single-cell sequencing. c Blastomere genotyping results in injected embryos. WT, wild-type; Mut, mutant c.299A>G. d, e Allele frequency and blastomere genotypes in BE3-treated heterozygous embryos. f Schematic of off-targeting analysis using whole genome sequencing of BE3-treated embryos. BE3 mRNA, OCT4 sgRNAs, and GFP mRNA were co-injected into one blastomere of two-cell embryos whereas another blastomere left uninjected. When embryos developed to the eight-cell stage, GFP-positive and negative blastomeres were separated and analyzed by WGS. g Alignments and percentage of mutant and corrected sequences from embryos injected with BE3 mRNA and MUT sgRNA. The target sequence is underlined. PAM site and substitutions are shown in blue and red, respectively. The column on the right indicates frequencies of mutant alleles. WT, wild-type. h Variant calling results revealing no off-target event detected by WGS. Indels, insertion or deletion; SNV, single nucleotide variants. i Targeted deep sequencing analysis of on-target and 11 potential off-target loci in MUT c.299 A>G mutant embryos with or without base editing