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. 2023 May 22;14:2922. doi: 10.1038/s41467-023-38528-z

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Schematic diagram of the IMGZ system for inactivation of multiple genes in one step in embryos. b The editing efficiency of on-target C-to-T base conversion induced by different sgRNAs in Tet1, Tet2, and Tet3. 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. c Representative Sanger sequence chromatograms of one Tet1/2/3-TKO blastocyst. 9 of 16 checked blastocysts show homozygous mutant chromatograms. d Deep sequencing analysis of genome mutant sites of one control and three Tet1/2/3-TKO embryos (E8.5). The results indicated the conversion of CAG codon of Tet1/2/3 into TAG stop codon in resultant embryos. e Frequencies of 5mC and 5hmC modified nucleotides in the genomic DNA of control and Tet1/2/3-TKO 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. f Domain structure of full‑length TET1, TET2, and TET3 proteins. The red arrows indicate amino acid mutation sites produced by the sgRNAs with the highest base conversion efficiency. g RNA in situ hybridization of T (also known as brachyury, a primitive streak marker) in the control and Tet-TKO embryos (E7.5). Three independent embryos were analyzed for each group. Scale bar, 100 μm. h Representative images of IMGZ-derived control and Tet-TKO embryos at E7.5 and E8.5. Green fluorescence indicates the expression of Oct4-EGFP. Three independent embryos were analyzed for each group. Scale bar, 200 μm in the left panel and 500 μm in the right panel. i Representative images of control and Tet-TKO embryos at E9.5 and E10.5 generated by the IMGZ system. Green fluorescence indicates the expression of Oct4-EGFP. Three independent embryos were analyzed for each group. Scale bars, 500 μm in the up panel and 2 mm in the down panel. Source data are provided as a Source Data file.