Rapid Development Of Stable Transgene CHO Cell Lines Via CRISPR/Cas9 Mediated Site- Specific Integration

Background and Purpose:Chinese hamster ovary(CHO)cells are the most widely used mammalian hosts for recombinant protein production,including 7 among the global top 10 best-selling biotech drugs of 2016.There are some significant advantages of CHO cell expression system.Firstly,CHO cell performs human-like post-translational modifications,such as glycosylation.Secondly,it offers superior biological safety since human-specific pathogenic viruses cannot replicate in CHO cells.Finally,CHO cell is easily adaptable to serum-free suspension culture for industrial applications.However,the traditional method to obtain high expressing recombinant CHO(rCHO)cell lines for industrial production is random integration,which is considerably time-and cost-consuming.To obtain a clone with high expressing level and cell growth property,multiple rounds of screening by a selective marker is obligatory.Moreover,due to lack of control of insertion sites in random integration,protein productivity of some selected clones may diminish over time,causing instability of cell lines,which is known as ‘position effect'.Site-specific integration is a promising alternative to develop recombinant CHO cell lines.CRISPR/Cas9 technology has been recently demonstrated to be an efficient gene-editing tool for site-specific integration.The strategy can be realized through CRISPR/Cas9 engineered nucleases to induce a DNA double-strand break(DSB)at a desired site of the genome,triggering the homology-directed repair(HDR)insertion of the exogenous transgene into the DSB location.Using this strategy,we can insert exogenous genes into targeted locus fast and accurately to realize high-level and stable expression,and avoid positive effect at the same time.In this study,we selected three possible candidate sites based on previous reports,and inserted mCherry genes and anti-PD1 genes into these sites through CRISPR/Cas9 mediated site-specific integration technology respectively.After screening and identifying,we obtained targeted integration cell clones and assessed their properties,productivity,stability and product quality.Methods:We selected C12orf35,HPRT and GRIK1 locus as three possible candidate sites based on previous reports.Three pairs of sgRNA targeting each site were designed and the most efficient pair was chosen for further use.The donor plasmids were constructed with targeting loci-specific 5' and 3' homology arms(750 bp),targeted gene expression cassette(mCherry gene,anti-PD1 gene)and puromycin expression cassette.Cells were stably transfected with different donor plasmids and/or sgRNA-Cas9 plasmids respectively.For CHO-S cells,8 ?g/mL puromycin was used for 3 weeks of antibiotic selection.Stably transfected pools were then detected with 5'/3' junction PCR and Western Blot,and further seeded at 0.5-1 cell/ 200 ?L/well in 96-well plates for limiting dilution.After another 3-4 weeks,the colonies were generated and analyzed by 5'/3' junction PCR and Western Blot for protein expression.Only those colonies positive in both junction PCR and Western Blot assessment were selected for further investigation.We further assessed the site-specific integration strategy by evaluation of rCHO cell lines properties as well as their productivities,including growth profile,relative copy number,expression stability,off-target effects of clones with targeted integration and quality assessment of the products produced by targeted integrations.Results:We targeted exogenous genes(mCherry gene,anti-PD1 gene)into C12orf35,HPRT and GRIK1 loci successfully through site specific integration mediated by CRISPR/Cas9 and got targeting efficiency from 25.4% to 66.0%.Compared with wild type CHO-S cells and targeted integration cells,we found that all the 3 studied sites are not essential to cell growth and could be engineered without substantial interference to cell characteristics.Moreover,mRNA expression level of the inserted gene was not dependent on integrated locus or biallelic/monoallelic insertion,indicating all 3 studied loci suitable for stable transcription of the exogenous gene.We further assessed the site-specific integration strategy by evaluation of rCHO cell lines stability as well as their productivities.The cell lines were cultivated for a total of 20 passages and the expressing level was inspected every 5 passages.mCherry cells integrated at C12orf35 locus maintained expression level of 10000~20000 RFU,and anti-PD1 mAb cells integrated at C12orf35 locus exhibited stable productivity of 110~190 mg/L.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 development of recombinant CHO cell lines.