Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

Here, we establish a system to simultaneously inactivate Dnmts in one step through screening for base editors that can efficiently introduce a stop codon endogenously. Dnmt-null embryos display primitive streak elongation failure at E7.5. Interestingly, although DNA methylation is absent, gastrulation-related pathways are down-regulated in Dnmt-null embryos. Moreover, DNMT1 or DNMT3A/3B are indispensable 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 mainly related to miRNAs. Strikingly, majority of hypermethylated promoter-related miRNAs are overexpressed and located at the Dlk1-Dio3 imprinted region. The predicted targets of these miRNAs tend to be down-regulated and enriched in the gastrulation-related pathways in Dnmt-null embryos. Finally, introduction of a single mutant allele of six miRNAs partially restores primitive streak elongation in Dnmt-null embryos. To analyze the influence of DNA methylation on miRNAs expression, we used CAS-seq to analyze different Dnmt mutant embryos, including Control, Dnmt1-KO, Dnmt3a/3b-DKO, and Dnmt1/3a/3b-TKO Exe and Epi of E6.5 and E7.5. To analyze the influence of IG-DMR methylation on Dlk1-Dio3 miRNAs expression, we used CAS-seq to analyze WT- and- DKO AG-haESCs, and WT-and DKO semi-cloned embryos at E6.5. Besides, we also analyzed DKO- and Mir-6KO semi-cloned embryos at E7.5 to describe the miRNAs expression in these samples.