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CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism

2016; Nature Portfolio; Volume: 7; Issue: 1 Linguagem: Inglês

10.1038/ncomms12338

2041-1723

Jonathan L. Schmid‐Burgk, Klara Höning, Thomas S. Ebert, Veit Hornung,

Animal Genetics and Reproduction

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.