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CRISPR‐Cas targeted plasmid integration into mammalian cells via non‐homologous end joining
Author(s) -
Bachu Ravichandra,
Bergareche Iñigo,
Chasin Lawrence A.
Publication year - 2015
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25629
Subject(s) - plasmid , crispr , cas9 , non homologous end joining , genome editing , biology , genome engineering , homologous recombination , genome , recombinant dna , hek 293 cells , computational biology , gene , microbiology and biotechnology , dna , genetics
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.