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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.

Original publication

DOI

10.1038/ncomms10237

Type

Journal article

Journal

Nature communications

Publication Date

17/12/2015

Volume

6

Pages

10237 - 10237

Addresses

Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.

Keywords

Cell Line, Humans, Deoxyribonuclease I, Endonucleases, Luciferases, Bacterial Proteins, Green Fluorescent Proteins, DNA, RNA, Guide, Microscopy, Fluorescence, Genetic Engineering, Reverse Transcriptase Polymerase Chain Reaction, Genes, Reporter, Genome, Human, Plasmids, CRISPR-Cas Systems