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.
Department of Biological Sciences, Columbia University, New York, New York, 10027.
Ravichandra
R
Bachu
Department of Biological Sciences, Columbia University, New York, New York, 10027.
Iñigo
I
Bergareche
Department of Biological Sciences, Columbia University, New York, New York, 10027. lac2@columbia.edu.
Lawrence A
LA
Chasin
D000818:Animals; D064113:CRISPR-Cas Systems; D002460:Cell Line; D059766:DNA End-Joining Repair; D006801:Humans; D008322:Mammals; D060847:Metabolic Engineering / Q000379:methods*; D010957:Plasmids; D011995:Recombination, Genetic
CHO; CRISPR-Cas; HEK293; NHEJ; site-directed insertion