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 efficienc

SUBMITTER: Schmid-Burgk JL 

PROVIDER: S-EPMC4974478 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature