CRISPR‐Cas targeted plasmid integration into mammalian cells via non‐homologous end joining

Mammalian cells are widely used for the production of therapeutic recombinant proteins, as these cells facilitate accurate folding and post‐translational modifications often essential for optimum activity. Targeted insertion of a plasmid harboring a gene of interest into the genome of mammalian cells for the expression of a desired protein is a key step in production of such biologics. Here we show that a site specific double strand break (DSB) generated both in the genome and the donor plasmid using the CRISPR‐Cas9 system can be efficiently used to target ∼5 kb plasmids into mammalian genomes via nonhomologous end joining (NHEJ). We were able to achieve efficiencies of up to 0.17% in HEK293 cells and 0.45% in CHO cells. This technique holds promise for quick and efficient insertion of a large foreign DNA sequence into a predetermined genomic site in mammalian cells. Biotechnol. Bioeng. 2015;112: 2154–2162. © 2015 Wiley Periodicals, Inc.