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

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&am...

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Main Authors:	Hajnalka Laura Pálinkás, Gergely Attila Rácz, Zoltán Gál, Orsolya Ivett Hoffmann, Gergely Tihanyi, Gergely Róna, Elen Gócza, László Hiripi, Beáta G. Vértessy
Format:	Article
Language:	English
Published:	MDPI AG 2019-04-01
Series:	Biomolecules
Subjects:	dUTPase CRISPR/Cas9-mediated knock-out blastocyst outgrowth embryonic development
Online Access:	https://www.mdpi.com/2218-273X/9/4/136

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spelling	doaj-21208895c4914bc08d0e99ddbb79ab792020-11-25T01:14:54ZengMDPI AGBiomolecules2218-273X2019-04-019413610.3390/biom9040136biom9040136CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic LethalityHajnalka Laura Pálinkás0Gergely Attila Rácz1Zoltán Gál2Orsolya Ivett Hoffmann3Gergely Tihanyi4Gergely Róna5Elen Gócza6László Hiripi7Beáta G. Vértessy8Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, HungaryInstitute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, HungaryDepartment of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, HungaryDepartment of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, HungaryInstitute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, HungaryDepartment of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, H-1111 Budapest, HungaryDepartment of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, HungaryDepartment of Animal Biotechnology, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, H-2100 Gödöllő, HungaryInstitute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, HungarySanitization 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 <i>dut</i> +/– animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early <i>dut</i> −/− 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.https://www.mdpi.com/2218-273X/9/4/136dUTPaseCRISPR/Cas9-mediated knock-outblastocyst outgrowthembryonic development
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Hajnalka Laura Pálinkás Gergely Attila Rácz Zoltán Gál Orsolya Ivett Hoffmann Gergely Tihanyi Gergely Róna Elen Gócza László Hiripi Beáta G. Vértessy
spellingShingle	Hajnalka Laura Pálinkás Gergely Attila Rácz Zoltán Gál Orsolya Ivett Hoffmann Gergely Tihanyi Gergely Róna Elen Gócza László Hiripi Beáta G. Vértessy CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality Biomolecules dUTPase CRISPR/Cas9-mediated knock-out blastocyst outgrowth embryonic development
author_facet	Hajnalka Laura Pálinkás Gergely Attila Rácz Zoltán Gál Orsolya Ivett Hoffmann Gergely Tihanyi Gergely Róna Elen Gócza László Hiripi Beáta G. Vértessy
author_sort	Hajnalka Laura Pálinkás
title	CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality
title_short	CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality
title_full	CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality
title_fullStr	CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality
title_full_unstemmed	CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality
title_sort	crispr/cas9-mediated knock-out of dutpase in mice leads to early embryonic lethality
publisher	MDPI AG
series	Biomolecules
issn	2218-273X
publishDate	2019-04-01
description	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 <i>dut</i> +/– animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early <i>dut</i> −/− 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.
topic	dUTPase CRISPR/Cas9-mediated knock-out blastocyst outgrowth embryonic development
url	https://www.mdpi.com/2218-273X/9/4/136
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CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality

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