在胚胎发育过程中,DNA甲基化通过DNMT3A/3B建立并随后通过DNMT1维持。虽然在这个领域已经做了很多研究,DNA甲基化在胚胎发育中的功能意义尚不清楚。这里,我们通过筛选能有效引入终止密码子的碱基编辑,建立了一个同时失活受精卵中多个内源基因的系统。在Dnmts和/或Tets中具有突变的胚胎可以用IMGZ在一个步骤中产生。Dnmt-null胚胎在E7.5时显示出原肠胚形成失败。有趣的是,虽然DNA甲基化是不存在的,在Dnmt无效胚胎中,与胃泌素相关的途径下调。此外,DNMT1,DNMT3A,而DNMT3B对胃泌素至关重要,它们的功能与TET蛋白无关。过度甲基化可以通过DNMT1或DNMT3A/3B在一些启动子处维持,它们与miRNA的抑制有关。六个miRNA和父系IG-DMR的单个突变等位基因的引入部分恢复了Dnmt空胚胎中的原始条纹伸长。因此,我们的研究结果揭示了启动子甲基化与胃泌膜miRNA表达抑制之间的表观遗传相关性,并证明IMGZ可以加速体内多个基因的功能破译.
During embryo development, DNA methylation is established by DNMT3A/3B and subsequently maintained by DNMT1. While much research has been done in this field, the functional significance of DNA methylation in embryogenesis remains unknown. Here, we establish a system of simultaneous inactivation of multiple endogenous genes in zygotes through screening for base editors that can efficiently introduce a stop codon. Embryos with mutations in Dnmts and/or Tets can be generated in one step with IMGZ. Dnmt-null embryos display
gastrulation failure at E7.5. Interestingly, although DNA methylation is absent,
gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1, DNMT3A, and DNMT3B are critical for
gastrulation, and their functions are independent of TET proteins. Hypermethylation can be sustained by either DNMT1 or DNMT3A/3B at some promoters, which are related to the suppression of miRNAs. The introduction of a single mutant allele of six miRNAs and paternal IG-DMR partially restores primitive streak elongation in Dnmt-null embryos. Thus, our results unveil an epigenetic correlation between promoter methylation and suppression of miRNA expression for
gastrulation and demonstrate that IMGZ can accelerate deciphering the functions of multiple genes in vivo.