Efficient conversion of AD risk variant ε4 to neutral variant ε3 in ε4 carrying 2PN embryos. (A) Experimental diagram showing FNLS-YE1-mediated gene correction of the APOE4 locus in human 2PN embryos. Sperm from a heterozygous male (APOE4/APOE3) was used to fertilize the oocytes to obtain 2PN embryos by ICSI. FNLS-YE1 mRNA, APOE4-targeting sgRNA, and GFP mRNA were co-injected into six blastomeres of 8-cell embryos whereas the other blastomeres were left un-injected. When embryos developed to the early morula stage, GFP-positive and negative blastomeres were separated and analyzed by targeted deep sequencing. (B and C) Blastomere genotyping results of embryos injected with FNLS-YE1 targeting the APOE4 or APOE3 loci (158R) in 2PN human embryos. (D) Genotype change induced by FNLS-YE1 in single blastomere from human 2PN embryos carrying APOE3/E3, APOE3/E4, and APOE4/E4. (E) Percentage of alleles with targeted C to T conversion for FNLS-YE1 in human 2PN embryos carrying APOE3/E3, APOE3/E4, and APOE4/E4. Each dot represents an embryo. (F–H) Allelic frequency changes induced by FNLS-YE1 in human embryos carrying APOE3/E3 (F), APOE3/E4 (G), and APOE4/E4 (H) respectively. (I) Summarized allelic frequency change before and after FNLS-YE1 injection. (J) Developmental rate from MI-stage oocyte to blastocyst for un-injected and injected embryos with FNLS-YE1 targeting APOE. Data are presented as the mean ± SEM.