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.

在胚胎发育过程中，DNA 甲基化由 DNMT3A/3B 建立，随后由 DNMT1 维持。尽管在这一领域已经进行了大量研究，但 DNA 甲基化在胚胎发生中的功能意义仍然未知。在这里，我们通过筛选能够有效引入终止密码子的碱基编辑器，建立了受精卵中多个内源基因同时失活的系统。可以使用 IMGZ 一步生成具有Dnmts和/或Tets突变的胚胎。 Dnmt缺失胚胎在 E7.5 时显示原肠胚形成失败。有趣的是，尽管不存在 DNA 甲基化，但Dnmt缺失胚胎中原肠胚形成相关途径却被下调。此外，DNMT1、DNMT3A 和 DNMT3B 对于原肠胚形成至关重要，它们的功能独立于 TET 蛋白。 DNMT1 或 DNMT3A/3B 在某些启动子上可以维持高甲基化，这与 miRNA 的抑制有关。六个 miRNA 和父本IG -DMR 的单一突变等位基因的引入部分恢复了Dnmt缺失胚胎中的原条伸长。因此，我们的结果揭示了启动子甲基化与原肠胚形成的 miRNA 表达抑制之间的表观遗传相关性，并证明 IMGZ 可以加速破译体内多个基因的功能。