Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35

Abstract

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

An Efficient Exogenous Gene Insertion Site in CHO Cells with High Transcription Level to Enhance AID‐Induced Mutation

“…They found that C12orf35 locus (a telomeric region of chromosome 8) [ 34 ] was the most advantageous among these three genes. [ 35 ] T site of ywhae gene that we found in this study is the potential genome hotspot to construct cell lines for recombinant protein production. Further study will be needed for this information.…”

A Site‐Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9‐Mediated Genome Editing Strategies in CHO Cells

“…In prior works, the use of HDR-mediated SSI with linear donor DNA, sometimes referred to as homology-mediated end joining (HMEJ), was accomplished via nuclease-mediated cleavage and release of circular plasmids in vivo [21,37,50,51]. Nonetheless, we observed a 2.3fold improvement in SSI efficiency when using a PCR-amplified linear HDR donor compared with a plasmid HDR donor (both with similar homology arm lengths), which matches previously documented efficiency gains when comparing the two donor formats [21,50,51]. Together, our findings show the functionality of the reporter system and promote the use of linearized HDR donors to improve SSI efficiency relative to other DSB repair strategies.…”

Key Challenges in Designing CHO Chassis Platforms

“…Direct silencing of the CMV promoter used for most transgenes, either by DNA-methylation or by changes in the histone modifications has also been shown [197][198][199][200]. These different strategies to silence the gene of interest are most likely due to the cell's drive to remove the stress of high productivity [201][202][203], by which ever means possible. Therefore, a frequent cause of loss of productivity is also the complete deletion of the recombinant genes from the genome [204,205].…”