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

Here, we first establish an easy Multi-Stop system to simultaneously inactivate genes involved in DNA methylation and demethylation in zygotes through introduction of the stop codon by hA3A-eBE-Y130F-mediated base editor (BE). While Multi-Stop-derived Dnmt-null embryos display embryonic lethal due to gastrulation failure. Moreover, mutation combinations between Tet and Dnmt families show severe embryonic lethal and Dnmt1 or Dnmt3a/3b is indispensable for mouse gastrulation. Then WGBS and RNA-seq analysis of different mutant embryos reveals genes jointly maintained by Dnmt1 and Dnmt3a/3b that are critical for gastrulation. We used WGBS (Whole Genome Bisulfite Sequencing) and RNA-seq to analyze the change of DNA methylation and transcription in DNMT and TET mutant embryos. WGBS data of E7.5 whole embryos from eight groups of 32 samples, including E7.5-control, E7.5-Dnmt1-KO, E7.5-Dnmt3a/3b-DKO, E7.5-Dnmt1/3a/3b-TKO, E7.5-Tet1/2/3;Dnmt1/3a/3b-6KO, E7.5-Tet1/2/3; Dnmt3a/3b-5KO, E7.5-Tet1/2/3; Dnmt1-4KO, E7.5-Tet1/2; Dnmt3a/3b-4KO. WGBS data of E6.5 and E7.5 embryo from 16 groups of 48 samples, including Ex-E6.5-control, Ex-E6.5-Dnmt1-KO, Ex-E6.5-Dnmt3a/3b-DKO, Ex-E6.5-Dnmt1/3a/3b-TKO, Em-E6.5-control, Em-E6.5-Dnmt1-KO, Em-E6.5-Dnmt3a/3b-DKO, Em-E6.5-Dnmt1/3a/3b-TKO, Ex-E7.5-control, Ex-E7.5-Dnmt1-KO, Ex-E7.5-Dnmt3a/3b-DKO, Ex-E7.5-Dnmt1/3a/3b-TKO, Em-E7.5-control, Em-E7.5-Dnmt1-KO, Em-E7.5-Dnmt3a/3b-DKO, Em-E7.5-Dnmt1/3a/3b-TKO. RNA-seq data of E6.5 and E7.5 embryo from 16 groups of 48 samples, including Ex-E6.5-control, Ex-E6.5-Dnmt1-KO, Ex-E6.5-Dnmt3a/3b-DKO, Ex-E6.5-Dnmt1/3a/3b-TKO, Em-E6.5-control, Em-E6.5-Dnmt1-KO, Em-E6.5-Dnmt3a/3b-DKO, Em-E6.5-Dnmt1/3a/3b-TKO, Ex-E7.5-control, Ex-E7.5-Dnmt1-KO, Ex-E7.5-Dnmt3a/3b-DKO, Ex-E7.5-Dnmt1/3a/3b-TKO, Em-E7.5-control, Em-E7.5-Dnmt1-KO, Em-E7.5-Dnmt3a/3b-DKO, Em-E7.5-Dnmt1/3a/3b-TKO.本研究首先构建了一套简便的Multi-Stop系统，通过hA3A-eBE-Y130F介导的碱基编辑器（base editor, BE）引入终止密码子，同时灭活合子中参与DNA甲基化与去甲基化的相关基因。经Multi-Stop系统构建的DNA甲基转移酶（Dnmt）敲除胚胎因原肠胚形成失败而出现胚胎致死现象。此外，Tet家族与Dnmt家族的突变组合可导致严重的胚胎致死，且Dnmt1或Dnmt3a/3b对小鼠原肠胚形成不可或缺。对不同突变胚胎开展全基因组亚硫酸氢盐测序（WGBS, Whole Genome Bisulfite Sequencing）与RNA测序（RNA-seq）分析后，本研究发现由Dnmt1与Dnmt3a/3b共同维持的、对原肠胚形成至关重要的基因集。 本研究采用WGBS与RNA-seq分析DNMT与TET突变胚胎的DNA甲基化水平与转录组变化。其中，包含32个样本的8组E7.5全胚胎WGBS数据涵盖以下组别：E7.5对照组、E7.5-Dnmt1敲除组、E7.5-Dnmt3a/3b双敲除组、E7.5-Dnmt1/3a/3b三敲除组、E7.5-Tet1/2/3;Dnmt1/3a/3b六敲除组、E7.5-Tet1/2/3;Dnmt3a/3b五敲除组、E7.5-Tet1/2/3;Dnmt1四敲除组、E7.5-Tet1/2;Dnmt3a/3b四敲除组。 另有包含48个样本的16组E6.5与E7.5胚胎WGBS数据，涵盖以下组别：胚外E6.5对照组、胚外E6.5-Dnmt1敲除组、胚外E6.5-Dnmt3a/3b双敲除组、胚外E6.5-Dnmt1/3a/3b三敲除组、胚胎本体E6.5对照组、胚胎本体E6.5-Dnmt1敲除组、胚胎本体E6.5-Dnmt3a/3b双敲除组、胚胎本体E6.5-Dnmt1/3a/3b三敲除组、胚外E7.5对照组、胚外E7.5-Dnmt1敲除组、胚外E7.5-Dnmt3a/3b双敲除组、胚外E7.5-Dnmt1/3a/3b三敲除组、胚胎本体E7.5对照组、胚胎本体E7.5-Dnmt1敲除组、胚胎本体E7.5-Dnmt3a/3b双敲除组、胚胎本体E7.5-Dnmt1/3a/3b三敲除组。 同时包含48个样本的16组E6.5与E7.5胚胎RNA-seq数据，涵盖以下组别：胚外E6.5对照组、胚外E6.5-Dnmt1敲除组、胚外E6.5-Dnmt3a/3b双敲除组、胚外E6.5-Dnmt1/3a/3b三敲除组、胚胎本体E6.5对照组、胚胎本体E6.5-Dnmt1敲除组、胚胎本体E6.5-Dnmt3a/3b双敲除组、胚胎本体E6.5-Dnmt1/3a/3b三敲除组、胚外E7.5对照组、胚外E7.5-Dnmt1敲除组、胚外E7.5-Dnmt3a/3b双敲除组、胚外E7.5-Dnmt1/3a/3b三敲除组、胚胎本体E7.5对照组、胚胎本体E7.5-Dnmt1敲除组、胚胎本体E7.5-Dnmt3a/3b双敲除组、胚胎本体E7.5-Dnmt1/3a/3b三敲除组。