A generic strategy for CRISPR-Cas9-mediated gene tagging

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 and Tilmann Bürckstümmer ()
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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

Nature Communications, 2015, vol. 6, issue 1, 1-7

Abstract: 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.

Date: 2015
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DOI: 10.1038/ncomms10237

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