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Biomolecules. 2019 Apr 4;9(4). pii: E136. doi: 10.3390/biom9040136.

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

Author information

1
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, Hungary. palinkas.hajnalka@ttk.mta.hu.
2
Doctoral School of Multidisciplinary Medical Science, University of Szeged, H-6720 Szeged, Hungary. palinkas.hajnalka@ttk.mta.hu.
3
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, Hungary. palinkas.hajnalka@ttk.mta.hu.
4
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, Hungary. racz.gergely@ttk.mta.hu.
5
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, Hungary. racz.gergely@ttk.mta.hu.
6
Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, Hungary. zoltan.gal89@gmail.com.
7
Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, Hungary. hoffmannorsi@gmail.com.
8
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, Hungary. tihanyi@stud.uni-heidelberg.de.
9
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, Hungary. tihanyi@stud.uni-heidelberg.de.
10
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, Hungary. Gergely.Rona@nyulangone.org.
11
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA. Gergely.Rona@nyulangone.org.
12
Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016, USA. Gergely.Rona@nyulangone.org.
13
Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, Hungary. gocza.elen@abc.naik.hu.
14
Department of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, Hungary. hiripi1@gmail.com.
15
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, Hungary. vertessy@mail.bme.hu.
16
Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, Hungary. vertessy@mail.bme.hu.

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.

KEYWORDS:

CRISPR/Cas9-mediated knock-out; blastocyst outgrowth; dUTPase; embryonic development

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