Fig. 3: One-step inactivation of Dnmt1/3a/3b mediated by IMGZ reveals critical roles of DNA methylation in mouse gastrulation.

a The editing efficiencies of on-target C-to-T base conversion induced by different sgRNAs in Dnmt1, Dnmt3a, and Dnmt3b. Detailed data are presented in Supplementary Data 4. Data are mean ± s.e.m for the indicated numbers of blastocysts. P values were determined by Student’s unpaired two-sided t-test. b Domain structure of full‑length DNMT1, DNMT3A, and DNMT3B proteins. The red arrows indicate the amino acid mutation site produced by the sgRNAs with the highest base conversion efficiency. c Expression of DNMT1, DNMT3A, and DNMT3B in Dnmt1-KO and Dnmt3a/3b-DKO embryos. GAPDH is the control. Proteins were obtained from E9.5 embryo lysis. Two control, two Dnmt1-KO, and three Dnmt3a/3b-DKO embryos were analyzed. d Frequencies of 5mC and 5hmC modified nucleotides in the genomic DNA of control, Dnmt1-KO, Dnmt3a/3b-DKO, and Dnmt-null embryos (E8.5) were determined by quantitative mass spectrometry. Data are mean ± s.e.m of three biological replicates. P-values were determined by Student’s unpaired two-sided t-test. e Representative images of IMGZ-derived control, Dnmt1-KO, Dnmt3a/3b-DKO, and Dnmt-null embryos at E7.5 and E8.5, showing developmental retardation at E7.5 in Dnmt-null embryos. Green fluorescence indicates the expression of Oct4-EGFP. Three independent embryos were analyzed for each group. Scale bars, 200 μm. f RNA in situ hybridization of T in the control, Dnmt1-KO, Dnmt3a/3b-DKO, and Dnmt-null embryos (E7.5), showing primitive streak elongation failure in Dnmt-null embryos. Three independent embryos were analyzed for each group. Scale bar, 100 μm. g Representative images of E7.5 and E8.5 Dnmt-null embryos obtained through crossing germline-specific conditional knockout parents. Scale bars, 200 μm. Three independent embryos were analyzed for each group. Source data are provided as a Source Data file.