Fig. 1: The design and validation of parental allele-specific site targeting methods.

a A mixture of Cas9 and an individual sgRNA targeting Anapc2 was injected into MII oocytes. The injected oocytes and wild-type oocytes underwent IVF simultaneously. When the injected embryos developed into pronuleus PN3–4 stage 7–9 h post IVF, their paternal or maternal pronuclei (red ovals) were isolated and transferred into wild-type embryos with the maternal or paternal pronuclei (green ovals) depleted. The pronuclei were fused with the cytoplasm by the Sendai virus and generated reconstructed normal diploid zygotes. Some of the reconstructed embryos were transplanted into oviducts of pseudopregnant females, and others were cultured in vitro. Embryos and offspring were generated for PCR and sequencing. b Immunostaining for detecting SpCas9 signals in the nuclear region of zygotes and 2-cell embryos resulting from Past-CRISPR and traditional zygote injection. White dashed lines demarcate the nuclear membrane. The data shown are representative of at least two independent experiments. Scale bars, 5 µm. c A bar graph shows the developmental potential (counted 96 h after fertilization) of Anapc2-targeted embryos, Anapc2^{mat-edited/pat} embryos, Anapc2^{mat/pat-edited} embryos, and the wild-type groups (mat/pat-swapped wild-type embryos and wild-type embryos). d Bright field images of Anapc2-targeted embryos, Anapc2^{mat-edited/pat} embryos, Anapc2^{mat/pat-edited} embryos and wild-type embryos. One representative result of three independent experiments is shown. Scale bars: 100 µm. e TA cloning sequences of the Anapc2 gRNA target sites from one of the potential parental allele-specific edited embryos. Sequences of ten alleles present in individual embryos reveal two different types of alleles: half are wild-type alleles, and the other half are mutant alleles. f A pie chart demonstrates proportions of targeting alleles and wild-type alleles in the tested Anapc2 potential parental allele-specific edited embryos. Number, total alleles counted.