title: A generic strategy for CRISPR-Cas9-mediated gene tagging.
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.
authors.affiliation: Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Lackner
authors.affiliation: Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.
authors.forename: Alexia
authors.initials: A
authors.lastname: Carré
authors.affiliation: Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.
authors.forename: Paloma M
authors.initials: PM
authors.lastname: Guzzardo
authors.affiliation: Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.
authors.forename: Carina
authors.initials: C
authors.lastname: Banning
authors.affiliation: Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge CB25 9TL, UK.
authors.forename: Ramu
authors.initials: R
authors.lastname: Mangena
authors.affiliation: Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge CB25 9TL, UK.
authors.forename: Tom
authors.initials: T
authors.lastname: Henley
authors.affiliation: Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.
authors.forename: Sarah
authors.initials: S
authors.lastname: Oberndorfer
authors.affiliation: Ludwig Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, UK.
authors.forename: Bianca V
authors.initials: BV
authors.lastname: Gapp
authors.affiliation: Ludwig Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, UK.
authors.forename: Sebastian M B
authors.initials: SMB
authors.lastname: Nijman
authors.affiliation: Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, Netherlands.
authors.forename: Thijn R
authors.initials: TR
authors.lastname: Brummelkamp
authors.affiliation: Horizon Genomics, Campus Vienna Biocenter 3, 1030 Vienna, Austria.
authors.forename: Tilmann
authors.initials: T
authors.lastname: Bürckstümmer
mesh_terms: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D003850:Deoxyribonuclease I; D004720:Endonucleases; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003850:Deoxyribonuclease I