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CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism

Author

Listed:
  • Jonathan L. Schmid-Burgk

    (Institute of Molecular Medicine, University Hospital, University of Bonn)

  • Klara Höning

    (Institute of Molecular Medicine, University Hospital, University of Bonn)

  • Thomas S. Ebert

    (Institute of Molecular Medicine, University Hospital, University of Bonn)

  • Veit Hornung

    (Institute of Molecular Medicine, University Hospital, University of Bonn
    Ludwig-Maximilians-Universität München
    Center for Integrated Protein Sciences)

Abstract

The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications.

Suggested Citation

  • Jonathan L. Schmid-Burgk & Klara Höning & Thomas S. Ebert & Veit Hornung, 2016. "CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism," Nature Communications, Nature, vol. 7(1), pages 1-12, November.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms12338
    DOI: 10.1038/ncomms12338
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    Cited by:

    1. Evelyn Fessler & Luisa Krumwiede & Lucas T. Jae, 2022. "DELE1 tracks perturbed protein import and processing in human mitochondria," Nature Communications, Nature, vol. 13(1), pages 1-15, December.

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