Viable semi-cloned mice derived from androgenetic haploid stem cells lacking DNA methylation [RNA-seq]

Among all mammalian cell types, sperm exhibit the highest level of DNA methylation, with approximately 70–80% of CpG sites being methylated. The role of this paternal hypermethylation in embryonic developmental competence, aside from the well-characterized H19-DMR and Dlk1-Dio3 intergenic germline DMR (IG-DMR) loci, remains largely unexplored. In mouse male germ cells, the lack of DNA methyltransferase (Dnmt) causes a loss of methylation, leading to meiotic catastrophe and infertility. To circumvent this limitation, we used sperm-like androgenetic haploid embryonic stem cells (AG-haESCs) for oocyte injection to produce offspring. Using CRISPR/Cas9, we effectively inactivated Dnmt1, Dnmt3a and Dnmt3b in AG-haESCs and later reactivated them in the resulting embryos, overcoming Dnmt haploinsufficiency and enabling the generation of viable offspring from methylation-deficient haploid cells. In the offspring embryos, the paternal genome derived from Dnmt-inactivated AG-haESCs rapidly reacquired methylation and restored a methylome in post-implantation embryos comparable to that of wild-type AG-haESC counterparts. These semi-cloned embryos could develop to term and survived to adulthood, exhibiting near-normal morphological and physiological parameters. These findings indicate that the functional significance of paternal genome methylation is mainly restricted to the imprinted loci Igf2-H19 and Dlk1-Dio3, while methylation of the paternal genome elsewhere appears dispensable for normal development.