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Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of <t>pH2AX</t> (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.
Ph2ax Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of <t>pH2AX</t> (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.
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Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of <t>pH2AX</t> (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.
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Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of <t>pH2AX</t> (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.
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Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of <t>pH2AX</t> (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.
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Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of pH2AX (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.

Journal: Molecular Therapy. Nucleic Acids

Article Title: Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

doi: 10.1016/j.omtn.2023.102035

Figure Lengend Snippet: Analysis of the guide-free Cas9-induced genome damage in PA porcine embryos (A) Schematic of the DNA damage test in Cas9-T2A-tdTomato mRNA-injected PA porcine embryos, with tdTomato mRNA-injected embryos as controls. We selected two-cell (day 1), four-cell (day 2), morula (day 4), and blastocyst (day 6) stage embryos to test DNA damage. (B–E) Fluorescence microscopic images at different embryo stages of pH2AX (green), tdTomato (red), and DAPI (blue) in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA- and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. Scale bar, 20 μm. (F–I) Statistical results of the number of pH2AX foci per nucleus in tdTomato mRNA-, Cas9-T2A-tdTomato mRNA-, and Cas9-T2A-tdTomato with sgRNA mRNA-injected embryos. For (H) and (I), we counted the number of pH2AX foci in the damaged nuclei. For (F), day 1 tdTomato-injected blastomeres, n = 10; Cas9-injected blastomeres, n = 8; Cas9 with sgRNA-injected blastomeres, n = 10. For (G), day 2 tdTomato-injected blastomeres, mean ± SEM = 0.4211 ± 0.2572, n = 19; Cas9-injected blastomeres, mean ± SEM = 0.35 ± 0.1094, n = 20; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 0.8 ± 0.2218, n = 30. For (H), day 4 tdTomato-injected blastomeres, mean ± SEM = 7.588 ± 1.32, n = 17; Cas9-injected blastomeres, mean ± SEM = 34.96 ± 5.067, n = 26; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 20.27 ± 2.22, n = 22. For (I), day 6 tdTomato-injected blastomeres, mean ± SEM = 6.13 ± 0.6177, n = 54; Cas9-injected blastomeres, mean ± SEM = 15.64 ± 0.9625, n = 115; Cas9 with sgRNA-injected blastomeres, mean ± SEM = 9.908 ± 0.6563, n = 109; ∗∗∗p = 0.0003, ∗∗∗∗p < 0.0001, unpaired t test.

Article Snippet: Then, the embryos were incubated overnight at 4°C in a 1:200 solution of the primary pH2AX antibody (CST, 9718) or Cas9 antibody (Huabio, ET1703-85) in an antibody diluent buffer.

Techniques: Injection, Fluorescence