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Biomolecules 2019, 9(4), 136; https://doi.org/10.3390/biom9040136

CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality

1
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, Hungary
2
Doctoral School of Multidisciplinary Medical Science, University of Szeged, H-6720 Szeged, Hungary
3
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, Hungary
4
Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, Hungary
5
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA
6
Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016, USA
*
Authors to whom correspondence should be addressed.
Received: 14 March 2019 / Revised: 1 April 2019 / Accepted: 2 April 2019 / Published: 4 April 2019
(This article belongs to the Section Biochemistry)
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Abstract

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2′-deoxyuridine 5′-triphosphate (dUTP) into 2′-deoxyuridine 5′-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/– animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut −/− embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice. View Full-Text
Keywords: dUTPase; CRISPR/Cas9-mediated knock-out; blastocyst outgrowth; embryonic development dUTPase; CRISPR/Cas9-mediated knock-out; blastocyst outgrowth; embryonic development
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    Doi: https://doi.org/10.1101/335422
    Link: https://www.biorxiv.org/content/10.1101/335422v1.supplementary-material
    Description: Figure S1: Sequencing analysis of dut gene from founder mouse #2 and #4, Figure S2: Alignment of the sequencing results from wild type and mouse #4 animals, Figure S3.:Phase contrast images of embryos obtained by crossing D47 heterozygous mice, Figure S4: Outgrowth assay of preimplantation embryos obtained by intercrossing D47 heterozygous mice, Figure S5: Images of embryos at 8.5 dpc obtained by crossing D47 heterozygous mice, Figure S6: Images of embryos at 9.5 dpc obtained by crossing D47 heterozygous mice, Figure S7: Full-length agarose gel of Figure 2b, Figure S8: Full-length membrane of Figure 5a, Table S1: Oligonucleotides used in this study.
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Pálinkás, H.L.; Rácz, G.A.; Gál, Z.; Hoffmann, O.I.; Tihanyi, G.; Róna, G.; Gócza, E.; Hiripi, L.; Vértessy, B.G. CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality. Biomolecules 2019, 9, 136.

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