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. Researchers have shown 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). They 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
The potential advantages of the CRISPR-Cas technology over ZFNs and TALENs are that it is relatively low cost, less time consuming, and does not require complicated protein engineering.
The results demonstrate the feasibility of CRISPR-Cas mediated gene insertion in HEK293 and CHO cells without the need of homologous sequence arms. While the efficiency (colonies per treated cell) of integration was not very high, it is high enough to make recombinant clone isolation without a selective marker feasible; these data encourage us to seek conditions that may improve on this frequency. Even as is, this system may prove useful for the genetic engineering of cultured mammalian cells for the production of high levels of recombinant proteins. For instance, one could target a highly active endogenous gene so as to expropriate its promoter for the production of a protein of interest. Although these ends could be achieved by homologous recombination, the use of NHEJ is simpler to implement as the plasmid vectors would be more easily constructed, or indeed extant plasmids could be used.
Promoter complementation selection. gRNAs were designed to target the region upstream of the promoter-less Puro gene provided by pFW (grey rectangles) in the HEK293 genome (solid line) and the region downstream of the CMV promoter on the pIC3 plasmid (white rectangles). The gRNA target sequences in the genome and in the plasmid are shown by thick vertical black lines representing the two different sequences at each of the two target sites. This assay ensures that only site-specific integration in proper orientation can give rise puromycin resistance. Protospacer adjacent motif (PAM) bases are in grey and the expected DSBs 3 nucleotides upstream to the PAM are shown as dotted lines.
Biotechnology and Bioengineering - CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining