title: CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism.
abstract: The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on canonical NHEJ (DNA-PKcs, XLF and ligase-4). A large repertoire of modular donor vectors renders CRISPaint compatible with a wide array of applications.
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Schmid-Burgk
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.
authors.forename: Klara
authors.initials: K
authors.lastname: Höning
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Ebert
authors.affiliation: Institute of Molecular Medicine, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.|Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.|Center for Integrated Protein Sciences, Munich, Germany.
authors.forename: Veit
authors.initials: V
authors.lastname: Hornung
mesh_terms: D064113:CRISPR-Cas Systems; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids; D011691:Puromycin; D016364:Reading Frames
chemical_list: D011691:Puromycin