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A generic strategy for CRISPR-Cas9-mediated gene tagging

Author

Listed:
  • Daniel H. Lackner

    (Horizon Genomics)

  • Alexia Carré

    (Horizon Genomics)

  • Paloma M. Guzzardo

    (Horizon Genomics)

  • Carina Banning

    (Horizon Genomics)

  • Ramu Mangena

    (Horizon Discovery)

  • Tom Henley

    (Horizon Discovery)

  • Sarah Oberndorfer

    (Horizon Genomics)

  • Bianca V. Gapp

    (Ludwig Cancer Research, University of Oxford)

  • Sebastian M.B. Nijman

    (Ludwig Cancer Research, University of Oxford)

  • Thijn R. Brummelkamp

    (Netherlands Cancer Institute)

  • Tilmann Bürckstümmer

    (Horizon Genomics)

Abstract

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.

Suggested Citation

  • Daniel H. Lackner & Alexia Carré & Paloma M. Guzzardo & Carina Banning & Ramu Mangena & Tom Henley & Sarah Oberndorfer & Bianca V. Gapp & Sebastian M.B. Nijman & Thijn R. Brummelkamp & Tilmann Bürckst, 2015. "A generic strategy for CRISPR-Cas9-mediated gene tagging," Nature Communications, Nature, vol. 6(1), pages 1-7, December.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms10237
    DOI: 10.1038/ncomms10237
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    Cited by:

    1. Zsolt Bodai & Alena L. Bishop & Valentino M. Gantz & Alexis C. Komor, 2022. "Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    2. Patrizia Tornabene & Rita Ferla & Manel Llado-Santaeularia & Miriam Centrulo & Margherita Dell’Anno & Federica Esposito & Elena Marrocco & Emanuela Pone & Renato Minopoli & Carolina Iodice & Edoardo N, 2022. "Therapeutic homology-independent targeted integration in retina and liver," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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