Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

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

Nat Commun

Publication Date

17/12/2015

Volume

6

Keywords

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