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Enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9 Wenpeng Wang, Wenyun Zheng, Fengzhi Hu, Xiujuan He, Dong Wu, Wenliang Zhang, Haipeng Liu, and Xingyuan Ma ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00375 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

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ACS Synthetic Biology

Enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9

Wenpeng Wang1#, Wenyun Zheng2#, Fengzhi Hu2, Xiujuan He2, Dong Wu1, Wenliang Zhang3*, Haipeng Liu4*, Xingyuan Ma1*

1

State Key Laboratory of Bioreactor engineering, School of Biotechnology, East China

University of Science and Technology, Shanghai 200237, PR China 2

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, Shanghai 200237, PR China 3

Center of Translational Biomedical Research, University of North Carolina at

Greensboro, NC 27310, USA 4

State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen

361102, Fujian, PR China # Contributed equally to this work. *Correspondence and requests should be addressed to Prof. Xingyuan Ma. Tel./fax:+86 2164250135 (X. MA); E-mail: [email protected]

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Abstract

Chinese hamster ovary (CHO) cells are the famous expression system for industrial production of recombinant proteins, such as therapeutic antibodies. However, there retained still bottlenecks in protein quality and weakness in expression efficiency because of the intrinsic genetic properties of the cell. Here we have enhanced biosynthesis performance of heterologous proteins in CHO-K1 cell using CRISPR-Cas9 by editing the genome precisely with two genes for improving ER microenvironment and reinforcing anti-apoptotic ability. A linear donor plasmid harboring eGFP-HsQSOX1b and Survivin genes was knocked in specific locus in CHO-K1 genome by the CRISPR-Cas9 RNA guided nucleases via NHEJ with efficiencies of up to 3.85% in the CHO-K1 cell pools following FACS, and the hQSOX1 and hSurvivin genes were integrated into expected genome locus successfully. Compared with control, the anti-apoptotic viability of edited CHO-K1 cell was increased by 6.40 times and the yield has been raised by 5.55 times with GLuc as model protein. The possible molecular mechanisms and pathways of remarkable anti-apoptotic ability and protein biosynthesis in modified CHO-K1 cell have been elucidated reasonably. In conclusion, the novel ideas and reliable techniques for obtaining foreign proteins more efficiently in engineered animal cells were very valuable to meet large clinical needs.

Keywords: CHO-K1 mammalian cell, ER microenvironment, Anti-apoptotic ability, Genome editing by CRISPR-Cas9, High quality protein production


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Chinese hamster ovary (CHO) cells are the most famous factories of mammalian hosts for recombinant therapeutic protein manufacture since human tissue plasminogen activator (rtPA) became the first recombinant therapeutics synthesized in CHO cell and obtained market approval for clinical use in 1986 1, 2. During the past 30 years, great progress has been made in the performance of CHO recombinant therapeutic production, mainly in media composition optimization, metabolic process control, and vector and genetic engineering application. However, compared to bacterial or yeast expression system, cellular limitations such as quality and quantity of the target protein and the adverse environmental resistance competence still curb the production of biopharmaceuticals in CHO cells3. Thus, the steady improvement of CHO cell factories for biopharmaceutical production is still a key challenge to meet increasing demands for therapeutic proteins as the percentage of biopharmaceuticals in pharmaceutical market is still rapidly growing and the demand for biologics is expected to further increase. In this context, the development of more efficient productive cell lines for recombinant protein production that meet industrial requirements is of the utmost importance2, 4. Genetic horizons in host cell engineering technologies have paved the way for remarkable achievements in CHO cell line development5, especially the genome sequence and subsequent omics data of CHO-K16-8, which significantly accelerated biotechnological research of CHO cellular engineering for applications and facilitated genetic manipulation in a more rational and feasible way. The traditional DNA integration routes used for modification of cellular genomes are mainly vector transfections, which characterized by high-tense screening, low efficiency, and unpredictable alterations, as the integration of the transgene and the potential locus can be affected by cell-inborn repair system9. However, the newly genome editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) RNA guided nucleases, can overcome these problems. The widely used type II CRISPR/Cas9 nuclease has the benefits of fast design, time efficiency and low cost, and facilitates predictable integration which enables the insertion of an expression cassette at a pre-characterized genomic locus of any target cells in any model organisms10-14.



In recombinant CHO cells, the expression of foreign Gene of interest (GOI) is often driven by viral promoters, which exerts considerable pressure on the Endoplasmic Reticulum (ER), and during the long culture process any stimulus such as anoxia caused by metabolic stresses can disrupt ER protein homeostasis to trigger the unfolded protein response (UPR) in the ER and induce cell apoptosis. Strategies aimed to ameliorate these problems often concentrate on overexpression major molecules in the ER, knockdown/knockout of pro-apoptotic proteins, or over-expression of anti-apoptotic ones15-17. We choose Quiescin-sulfhydryl oxidase1 (QSOX1) and Inhibitor of apoptosis protein (IAP) Survivin to manipulate the protein disulfide formation process and cell apoptosis pathway to promote the protein process ability in the ER and enhance the longevity of CHO cells to break the bottleneck of recombinant protein production, especially the over-expression of mAbs and other intricate proteins in biopharmaceutical manufacturing. Here, we will explore the role and influence on CHO-K1 cell in biosynthesis of heterologous protein by improving ER microenvironment and reinforcing anti-apoptotic ability, elucidate the possible molecular mechanisms and pathways of efficient processing and secreting foreign protein in CHO cells, and create a series of novel methods used for editing and modifying of mammalian cell genome with different functional genes by advanced CRISPR/Cas9 via NHEJ. Through the implementation of this study, we hope to obtain engineered animal cells that could produce foreign proteins more efficiently, and to meet the large demand for therapeutic high-quality antibodies especially in clinical needs.

Results Target deletion and NHEJ mediated targeted integration via CRISPR/Cas9 SgRNAs were generated by the online tool Optimized CRISPR Design (http://crispr.mit.edu/), and we chose two sgRNAs (Figure 1A) with fewer off-target sites for each of the two genes: quiescin sulfhydryl oxidase 1 (QSOX1) encoding the enzyme QSOX1 and baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) encoding protein Survivin. Previous studies have not shown whether the two genes express or not in CHO-K1 cells, they were predicted


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by computational analysis according to the genome sequence of CHO-K1. The genome of CHO-K1 indicates that QSOX1 consists of 12 exons and the designed sgRNA_QSOX1 constructs target exon 7, BIRC5 consists of 3 exons and the designed sgRNA_BIRC5 constructs target exon 1. To test the activity of the designed sgRNA-Cas9 (pX458) plasmids, adherent CHO-K1 cells were transiently transfection with sgRNA-Cas9 (pX458) plasmids in triplicates. Transfection efficiency was detected by Flow cytometry analysis after 24 h and the results ranged from 28.3%-39.2% (Figure S3). T7E1 assay was conducted to test whether the designed sgRNA_Cas9 constructs can guide Cas9 to introduce DSBs at targeted sites, and a schematic diagram of T7E1 assay on exon7 of QSOX1 was presented in Figure S4. The cleavage efficiency (indel) of each targeting sgRNA was analyzed by ImageJ, and indels for each sgRNA were between 9.1% and 19.1% (Figure 1B). The digested fragments of the amplicons corresponded to the expected sizes. In order to knock-in exogenous Expression Cassette (EC) into a defined locus in the CHO-K1 genome, we chose the gDNA2 (sgRNA2 targeting locus in the exon 7 of CHO-K1 genome) to construct an sgRNA2_QSOX1 (pX459) plasmid and two ECs: EC#1 and EC#2 (Figure 1C). Protein expression was driven by CMV promoter and terminated by BGH polyA, eGFP and an ER resident signal KDEL sequences were added at the 5’ and 3’ end of HsQSOX1b, respectively. The EC#2 was additionally infused with a T2A-Survivin sequences between HsQSOX1b-KDEL and BGH polyA. The construction procedure was presented in Figure S2. To test the NHEJ mediated targeted integration effect via CRISPR/Cas9, adherent CHO-K1 cells were co-transfected with sgRNA2_QSOX1 (pX459) plasmid and the linearized EC#1/2 between CMV (E+P) and BGH polyA. The co-transfection assay was conducted in triplicates and pX458 was used for transfection control to monitor transfection efficiency. Intriguingly, we have observed a high transient transfection rate in co-transfection experiment (Figure S5) with about 33% and 35.9% for EC#1 and EC#2, respectively. In order to acquire accurate integration efficiency, co-transfected cells were passaged once or twice to rule out transient transfection before FACS analysis and no positive cell was detected in the absence of EC#1/2. The integration efficiency results were supplied in Figure 1D. And the schematic diagram of the NHEJ-mediated EC insertion edited by pX459/sgRNA2_QSOX1 was



portrayed in Figure S6. 5’/3’ junction PCR was conducted to detect the NHEJ mediated integration of sorted eGFP positive clones with primers which are designed between the genome-donor boundaries (Table S6). PCR products were subjected to 2% agarose gel electrophoresis and expected bands were found, indicating that the target EC was integrated into the QSOX1 locus (Figure 1E). To further verify the rejoining between the target site and the EC DNA, TA cloning-based sequencing of 5’/3’ junction PCR products was performed. The sequences of junctions were consistent with the expected sequences with indels at the cleavage site (Figure 1F). Indeed, 5’/3’ junction PCR sequences confirmed the integration of the target expression cassette into the QSOX1 locus. To evaluate the off-target effect, potential off-target sites that contain ≤ 3 mismatches to the used sgRNAs throughout the entire Cricetulus griseus genome were listed in Table S2. In NHEJ mediated targeted integration at sgRNA2_QSOX1 locus, two off-targets that targeting two potential genes loci were selected to perform junction PCR analysis on off-target integrations, junction PCR primers of each off-target locus were listed in Table S7. Among the sorted eGFP positive clones, no distinct positive junction PCR bands were found at any offtarget site. These results prove that NHEJ mediated targeted integration is an efficient means to knock in foreign expression cassette into site-specific locus via sgRNA/Cas9-induced DSBs.

Establishment of CHO-K1 cell lines modified by HsQSOX1b & Survivin and its ER location identification. Sequence analysis of 5’/3’ junction PCR amplicons of GFP positive cells confirmed the EC was integrated at the cleavage locus induced by sgRNA/Cas9. To identify that GOI was overexpression together with the eGFP, Western Blotting (WB) was performed to detect expression levels of HsQSOX1b and Survivin. We observed that QSOX1 was secreted in CHO-K1 and its protein level was associated with cell confluence (Figure 2A), however, Survivin was expressed in a very lower level (Figure 2B). HsQSOX1b protein levels in cotransfected positive cells were higher than that in the Wild-type (Wt) CHO-K1 cells. Survivin was significantly overexpressed in cells that co-transfected with EC#2, while the Wt CHO-K1


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cells expressed much less Survivin (Figure 2B). All these results indicated that HsQSOX1b and Survivin were efficiently expressed in modified CHO-K1 cells. Considering that protein biosynthesis and processing is mainly occurred in eukaryotic ER, an ER retention signal KDEL was added at the C-terminal of HsQSOX1b. To ascertain its ER location, we first determined the subcellular location of the KDEL-tagged eGFP-HSQSOX1b protein by fluorescence microscope in live cells (Figure S7). ER-Tracker Red was used to profile the ER (Figure S7-3), and eGFP was observed in the ER under fluorescence microscope (Figure S7-2). Cell nuclei were stained with Hoechst 33342 dye (Figure S7-4). These results suggested that KDEL-tagged HsQSOX1b was resided in the ER. In addition, CHO-K1 cells stably expressing eGFP-HsQSOX1b-KDEL were fixed for confocal laser scanning microscope (CLSM), and the KDEL-tagged HsQSOX1b exhibited a characteristic of ER-like distribution, (Figure 2C. upper right panel), which was similar to the staining of the ER-Tracker Red dye (Figure 2C. upper left panel). These results confirm that KDEL-tag caused the retention of eGFP-HsQSOX1b in the ER in modified CHO-K1 cells. Reproducible observations were conducted in CHO-K1 cells co-transfected with EC#2. Finally, we selected two stable clones transfected with each EC: EC#1-C1 (Figure 2D, left panel) that overexpress HsQSOX1b and EC#2-B6 (Figure 2D, right panel) that overexpress HsQSOX1b & Survivin for our further study of HsQSOX1b-KDEL and Survivin on disulfide bond formation within the ER and cell anti-apoptotic ability, respectively.

GSH/GSSG ratio in the cells that expressing HsQSOX1b Glutathione is a ubiquitous molecule found in all parts of the cell, which provides the main redox buffer for cells. It has been implicated to play a role in the thiol-disulfide relay between enzymes containing disulfide and substrates, and in protecting cells from ER-generated oxidative stress18. Considering HsQSOX1b was an active oxidase, we explored the GSH levels in the KDEL-tagged-HsQSOX1b-expressing cells. GSH and tGSH (GSH+GSSG) levels were detected by the GSH and GSSG Assay Kit, and GSSG levels was calculated based on detected tGSH and GSH. The GSH/GSSG ratios of EC#1-C1 and EC#2-B6 were ＊

decreased to 40.4% and 70.4%, respectively (Figure 3 histogram, p < 0.05). However, the ＊

GSH/tGSH ratio in each cell type was 91.2% and 97.3%, respectively (Figure 3 line chart, p



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< 0.05). Standard curve for the measurement of tGSH and GSSG level was supplied in Figure S8.

Enhanced resistance to apoptosis Annexin V-PE/PI was used to detect the apoptosis after 18 h staurosporine treatment, and as shown in flow cytometry histogram, apoptosis of negative control (NC) and positive control (PC) of CHO-K1 cells was 3.47% and 14.5%, respectively (Figure 4, upper left panel and upper right panel). In HsQSOX1b-expressing EC#1-C1, the result (13.9%) was similar to that ＊

of the PC (Figure 4, lower left panel; p < 0.05 versus CHO-K1 (PC)) In contrast, cells that overexpressing both HsQSOX1b and Survivin exhibited significantly reduced apoptosis ＊

(4.96%) (Figure 4, lower right panel; p < 0.05 versus CHO-K1 (PC)).

Effects of overexpression of KDEL-tagged HsQSOX1b on the folding of

Gaussia Luciferase Gaussia luciferase (GLuc) was chosen as sulfhydryl oxidation probe to explore whether the overexpressed HsQSOX1b-KDEL could introduce disulfide into nascent polypeptides when translocate into the ER. The GLuc protein contains 11 cysteine residues, and its luciferase activity can be detected when it is completely oxidized to form its native conformation19. Therefore, luciferase assay was performed to evaluate the disulfide formation properties in cells stably expressing HsQSOX1b-KDEL. RLU values in clone EC#1-C1 and clone EC#2-B6 were 2.33- and 5.55-times higher than that of the Wt CHO-K1, respectively (Figure 5,

＊＊

p

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