Notably, CHO cells have previously been described as a cell type recalcitrant to homology-based integration of large DNA constructs, thus targeted integration in CHO cells has been confined to NHEJ based approaches 16, 17.Ĭompared with protein-based genome editing tools with customizable DNA binding specificities, such as ZFNs and TALENS, the newer CRISPR/Cas9 platform is based on simple base-pairing between an engineered RNA and the targeted genomic site, which enables rapid design, ease of use and low costs 18.

Although HDR is typically observed at a lower or more variable frequency than NHEJ, HDR can be leveraged to generate targeted integrations with engineered nucleases in mammalian cells 11, 12, 13, 14, 15. Compared with the error-prone NHEJ, which can result in efficient indel mutation generation subsequently leading to knockout of target locus, HDR can be used to modify endogenous loci precisely in the presence of homologous pieces of endogenous/exogenous DNA. Upon site-specific DNA double-strand breaks (DSBs) induced by engineered nucleases, the target locus will typically be repaired by one of two major DNA damage repair pathway: nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). Customized nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) RNA guided nucleases are technologies readily available to induce targeted insertion/deletion (indel) mutations or precise sequence changes in a broad range of organisms and cell types (reviewed in Ref.

With draft genomes of several CHO cell lines recently being made available 8, 9, it is now possible to efficiently engineer the genomic sequence of CHO cells with engineered nucleases. However, these systems are limited by the need of a prior establishment of platform cell lines with the insertion of recombination site into random or a limited number of specific genomic regions, which is a prerequisite for retargeting to generate cell lines expressing the GOI. In order to circumvent uncontrollable gene insertion during rCHO cell line construction, different approaches have been applied for site-directed integration of transgenes using site-specific recombinases, which includes Cre/loxP system, Flp/FRT system and phiC31/R4 integrases 5, 6, 7. In this article, we show that targeting of transgenes into specific desirable sites in the CHO genome in a controlled manner reduces the variation in expression, generating a uniform population with stable transgene expression. Due to this variation in expression and genomic composition, subsequent screening of multiple clones is necessary to select proper clones suitable for high and stable expression of recombinant proteins 1, 2. As a result, these cell lines are often unstable and show reduced production over time 4.

In addition, gene amplification methods are traditionally used to increase expression. However, lack of control of gene insertion can give rise to unwanted phenotypic heterogeneity due to the varying accessibility of integration sites for gene expression – termed the position effect variation 2, 3. Traditionally, development of recombinant CHO (rCHO) cell lines relies on random integration of a gene of interest (GOI) into the genome, followed by selection of cells carrying the transgene 1, 2. Chinese hamster ovary (CHO) cells have been used as the predominant workhorses for production of recombinant therapeutic proteins with complex glycoforms 1, 2.

Pfu ultra touchdown pcr