In mammals, a new life starts with the fusion of an oocyte and asperm cell. Parthenogenesis, a way of generating offspring solelyfrom female gametes, is limited because of problems arising fromgenomic imprinting. Here, we report live mammalian offspringderived from single unfertilized oocytes, which was achieved by tar-geted DNA methylation rewriting of seven imprinting control regions.Oocyte coinjection of catalytically inactive Cas9 (dCas9)-Dnmt3a ordCpf1-Tet1 messenger RNA (mRNA) with single-guide RNAs (sgRNAs)targeting specific regions induced de novo methylation or demethyla-tion, respectively, of the targeted region. Following parthenogeneticactivation, these edited regions showed maintenance of methylationas naturally established regions during early preimplantation develop-ment. The transfer of modified parthenogenetic embryos into fostermothers resulted in significantly extended development andfinally inthe generation of viable full-term offspring. These data demonstratethat parthenogenesis can be achieved by targeted epigenetic rewrit-ing of multiple critical imprinting control regions. The success of parthenogenesis in mammals opens many opportunities in agriculture, research, and medicine. In mammals, a new life starts with the fusion of an oocyte and a sperm cell. Parthenogenesis, a way of generating offspring solely from female gametes, is limited because of problems arising from genomic imprinting. Here, we report live mammalian offspring derived from single unfertilized oocytes, which was achieved by targeted DNA methylation rewriting of seven imprinting control regions. Oocyte coinjection of catalytically inactive Cas9 (dCas9)-Dnmt3a or dCpf1-Tet1 messenger RNA (mRNA) with single-guide RNAs (sgRNAs) targeting specific regions induced de novo methylation or demethylation, respectively, of the targeted region. Following parthenogenetic activation, these edited regions showed maintenance of methylation as naturally established regions during early preimplantation development. The transfer of modified parthenogenetic embryos into foster mothers resulted in significantly extended development and finally in the generation of viable full-term offspring. These data demonstrate that parthenogenesis can be achieved by targeted epigenetic rewriting of multiple critical imprinting control regions. oocyte mammal offspring early embryo genomic imprinting pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement yes pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes pmc-license-ref CC BY-NC-ND In mammals, a new life begins with the sperm successfully meeting the oocyte. Fine coordination between the paternal and the maternal genomes is essential for mammalian development ( 5 – 8 ). However, this coordination is disrupted in parthenogenetic embryos because of the 2-fold establishment of the maternal-specific imprinting of the diploid genome. Several paternally methylated imprinting control regions (ICRs), including the H19 ( 9 ) and Gtl2 ( 10 ) ICRs, function in the regulation of genes that are essential for embryonic development. Several maternally methylated ICRs, such as Igf2r ( 11 ), Snrpn ( 12 ), Kcnq1ot1 ( 13 ), Nespas ( 14 ), and Peg10 ( 15 ) ICRs, have been shown to play pivotal roles in the regulation of fetal and/or postnatal growth and development. In addition, some studies have identified several imprinting regions that are critical for supporting the full-term development of bimaternal and bipaternal embryos ( 16 , 17 ). In this study, we examined whether the targeted epigenetic rewriting of these regions could improve parthenogenetic development. By the targeted methylation editing of seven ICRs, we were able to generate viable full-term offspring directly from single unfertilized mouse oocytes. The dotted line (set as 1) represents the average of expression levels of each gene from controls. ( F ) SNuPE assays for allele-specific expression of the indicated genes. Lanes 1 and 2 contained DNA derived from the pooled RNA of reconstructed parthenogenetic and fertilized control 2 F , lane 1) in these modified embryos, which is similar to the monoallelic expression pattern seen in the fertilized control embryos ( Fig. 2 F , lane 2). After transfer of 158 E 3.5 blastocysts into 12 pseudopregnant foster female mice, we were able to obtain 13 viable fetuses from three females at E 13.5, as evidenced by clear heartbeats. These data suggest that further methylation editing significantly improved parthenogenetic development. Of these 13 embryos, we selected 6 for methylation analysis of all seven ICRs edited in each single embryo. Together, these data demonstrate that parthenogenesis can be achieved in mammals by appropriate epigenetic regulation of multiple ICRs. This is consistent with the famous parental conflict hypothesis (also known as the Haig hypothesis) ( 6 , 7 ), which proposes that the imprinting-mediated balance between paternal and maternal genomes is critical for mammalian development. We considered several possibilities to explain the low efficiency of parthenogenetic mouse generation. First, only a small portion of embryos with all seven imprinting regions corrected can support full-term development. Consistent with this idea, two of six E 13.5 embryos ( SI Appendix , Fig. were able to generate both bimaternal and bipaternal mice by genetic modification of multiple imprinting regions, among which Grb10 is involved in the full-term development of bipaternal embryos ( 16 ). The success of parthenogenesis in mammals opens many opportunities in agriculture, research, and medicine. Further identification and editing of additional ICRs might improve the efficiency of parthenogenetic development.