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CRISPR/Cas12a-assisted PCR tagging of mammalian genes

Julia Fueller, Matthias Meurer, Konrad Herbst, Krisztina Gubicza, Bahtiyar Kurtulmus, Julia D. Knopf, Daniel Kirrmaier, Benjamin Buchmuller, Gislene Pereira, Marius K. Lemberg, Michael Knop
doi: https://doi.org/10.1101/473876
Julia Fueller
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Matthias Meurer
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Konrad Herbst
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Krisztina Gubicza
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Bahtiyar Kurtulmus
2Center for Organismal Studies (COS), University of Heidelberg and German Cancer Research Centre (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Julia D. Knopf
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Daniel Kirrmaier
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
3Cell Morphogenesis and Signal Transduction, DKFZ-ZMBH Alliance and German Cancer Research Center (DKFZ), Heidelberg, Germany
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Benjamin Buchmuller
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
4Technische Universität Dortmund, Chemische Biologie, Otto-Hahn-Straße 4a, 44227 Dortmund
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Gislene Pereira
2Center for Organismal Studies (COS), University of Heidelberg and German Cancer Research Centre (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Marius K. Lemberg
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
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Michael Knop
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany
3Cell Morphogenesis and Signal Transduction, DKFZ-ZMBH Alliance and German Cancer Research Center (DKFZ), Heidelberg, Germany
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  • For correspondence: m.knop@zmbh.uni-heidelberg.de
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Abstract

Here we describe a simple strategy for tagging of genes in mammalian cells. The method enables efficient creation of endogenously expressed protein fusions. Only PCR for the generation of a DNA fragment is required. This avoids the handling of RNAs, recombinant proteins or cloning of plasmids. The fragment, termed ‘PCR cassette’, is then transfected into cells along with a CRISPR/Cas12a helper plasmid and integrates into the target locus specified by sequences provided by the oligonucleotides used for PCR. The method is robust and works in all cell lines tested with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used.

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Posted November 20, 2018.
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CRISPR/Cas12a-assisted PCR tagging of mammalian genes
Julia Fueller, Matthias Meurer, Konrad Herbst, Krisztina Gubicza, Bahtiyar Kurtulmus, Julia D. Knopf, Daniel Kirrmaier, Benjamin Buchmuller, Gislene Pereira, Marius K. Lemberg, Michael Knop
bioRxiv 473876; doi: https://doi.org/10.1101/473876
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CRISPR/Cas12a-assisted PCR tagging of mammalian genes
Julia Fueller, Matthias Meurer, Konrad Herbst, Krisztina Gubicza, Bahtiyar Kurtulmus, Julia D. Knopf, Daniel Kirrmaier, Benjamin Buchmuller, Gislene Pereira, Marius K. Lemberg, Michael Knop
bioRxiv 473876; doi: https://doi.org/10.1101/473876

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