Research Article Cite This: ACS Synth. Biol. 2018, 7, 1259−1268
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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*,†
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State Key Laboratory of Bioreactor Engineering, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China ‡ Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China § Center of Translational Biomedical Research, University of North Carolina at Greensboro, Greensboro, North Carolina 27310, United States ∥ State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361102, Fujian, PR China S Supporting Information *
ABSTRACT: Chinese hamster ovary (CHO) cells are the famous expression system for industrial production of recombinant proteins, such as therapeutic antibodies. However, there still remain 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 cells using CRISPR-Cas9 by editing the genome precisely with two genes for improving ER microenvironment and reinforcing antiapoptotic 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 antiapoptotic viability of edited CHO-K1 cells 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 antiapoptotic ability and protein biosynthesis in modified CHO-K1 cells 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, antiapoptotic ability, genome editing by CRISPR-Cas9, high quality protein production
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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 importance.2,4 Genetic horizons in host cell engineering technologies have paved the way for remarkable achievements in CHO cell line development,5 especially the genome sequence and subsequent omics data of CHO-K1,6−8 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
hinese hamster ovary (CHO) cells are the most famous factories of mammalian hosts for recombinant therapeutic protein manufacture since human tissue plasminogen activator (r-tPA) 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 cells.3 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 © 2018 American Chemical Society
Received: October 16, 2017 Published: April 23, 2018 1259
DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
Research Article
ACS Synthetic Biology
Figure 1. Target deletion and NHEJ mediated targeted integration via CRISPR/Cas9. (A) Illustration of the four sgRNAs target sites in CHO-K1 (Qsox1 (upper panel) and Birc5 (lower panel)). (B) Agarose gel image of T7E1 assay to detect the efficiency of targeted deletion with CRISPR/ Cas9. Indels ranged from 9.1% to 19.1%. (C) Schematic diagram of two expression cassette (EC).The heterologous EC#1 contains a HsQSOX1b coding sequence, which was fused with an eGFP and KDEL at the C and N terminal, respectively. The EC#2 was composed of eGFP-HsQSOX1bKDEL and T2A-Survivin. (D) The integration efficiencies were detected by FACS after cells were passaged once or twice, and the integration efficiency was 5.18% and 3.85% for EC#1 and EC#2, respectively. (E) Agarose gel image of 5′/3′ junction PCR on stable transfected EC#1 and EC#2. M1, 2kb DNA marker; M2, 500b DNA marker (Takara). (F) 5′/3′ Junction PCR sequences of EC#1 (upper) and EC#2 (lower). Sanger sequencing of PCR amplicons. Amplicons from stable clones were purified and sequenced, and compared with the reference sequence (RF) at the genome-donor boundaries.
affected by cell-inborn repair system.9 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 precharacterized genomic locus of any target cells in any model organisms.10−14 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 overexpression of antiapoptotic ones.15−17 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 overexpression 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 antiapoptotic ability, elucidate the possible molecular mechanisms and pathways of 1260
DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
Research Article
ACS Synthetic Biology
Figure 2. Expression of QSOX1 and Survivin in Wt CHO-K1 cells and transfected cell lines. (A) QSOX1 expression levels vary at different confluence. QSOX1 was secreted in Wt CHO-K1 and its protein level was associated with cell confluence. (B) Western Blot detection of the protein levels of QSOX1 and Survivin in Wt CHO-K1 cells and EC#1-C1 and EC#2-B6. HsQSOX1b was overexpressed in EC#1-C1 and EC#2-B6 compared to the Wt CHO-K1 cells. Survivin was expressed much lower in Wt CHO-K1 cells than that in EC#2-B6. Survivin expression in EC#1-C1 that only overexpresses HsQSOX1b was similar to that of the Wt CHO-K1 cells. (C) Confocal laser scanning microscope (CLSM) was used to detect the subcellular location of KDEL-tagged HsQSOX1b. CHO-K1 cells stably expressing eGFP-HsQSOX1b-KDEL were fixed and ER-Tracker Red was used to detect ER (upper left panel), the eGFP exhibited a characteristic of ER-like distribution (upper right panel), which was similar to the staining of the ER-Tracker Red dye. Cell nuclei were stained with Hoechst 33342 dye (lower left panel), scale bar, 2 μm. (D) Stable cell lines EC#1C1 (left panel) and EC#2-B6 (right panel) under bright field (upper) and fluorescence microscope (lower), scale bar, 50 μm.
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 cotransfected with sgRNA2_Q-
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.
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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 offtarget 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 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, 1261
DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
Research Article
ACS Synthetic Biology
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 residing in the ER. In addition, CHO-K1 cells stably expressing eGFPHsQSOX1b-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 ERTracker Red dye (Figure 2C, upper left panel). These results confirm that KDEL-tag caused the retention of eGFPHsQSOX1b in the ER in modified CHO-K1 cells. Reproducible observations were conducted in CHO-K1 cells cotransfected 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 and Survivin for our further study of HsQSOX1b-KDEL and Survivin on disulfide bond formation within the ER and cell antiapoptotic 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 stress.18 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
SOX1 (pX459) plasmid and the linearized EC#1/2 between CMV (E+P) and BGH polyA. The cotransfection 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 cotransfection experiment (Figure S5) with about 33% and 35.9% for EC#1 and EC#2, respectively. In order to acquire accurate integration efficiency, cotransfected 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 off-target 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/Cas9induced DSBs. Establishment of CHO-K1 Cell Lines Modified by HsQSOX1b and 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 CHOK1 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 cotransfected with EC#2, while the Wt CHO-K1 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
Figure 3. GSH/GSSG ratio of Wild-type and transfected CHO-K1 cells. The GSH/GSSG ratio in EC#1-C1 and EC#2-B6 were decreased to 40.4% and 70.4%, respectively, compared to that of the Wt CHOK1 (Histogram, *p < 0.05, n = 3). The GSH/tGSH ratios in both cell lines were 91.2% and 97.3%, respectively (Line chart, *p < 0.05, n = 3).
GSH/tGSH ratio in each cell type was 91.2% and 97.3%, respectively (Figure 3 line chart, *p < 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 1262
DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
Research Article
ACS Synthetic Biology of negative control (NC) and positive control (PC) of CHOK1 cells was 3.47% and 14.5%, respectively (Figure 4, upper left
Figure 5. Luciferase assay in Wild-type CHO-K1 and transfected cells. The measured RLU values were 2.33 and 5.55 times higher in EC#1C1 and EC#2-B6 than that of control group at 48 h after transfection, respectively (**p < 0.01, n = 3).
Figure 4. Apoptosis assay of each cell lines. Staurosprine induced apoptosis was analyzed by flow cytometry. Values represent the percentage of cells in apoptosis (annexin V-positive, PI-positive). *p < 0.05 versus CHO-K1 (PC), n = 3. NC, negative control, PC, positive control.
panel and upper right panel). In HsQSOX1b-expressing EC#1C1, 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 conformation.19 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 < 0.01). The linear detection range of bioluminescence is shown in Figure S9. Cell Viability during the Serum Deprivation. CHO cells are adapted to growth in serum free medium considering the industrial biotherapeutical production is commonly performed in serum free environment. For this reason, CHO-K1, clone EC#1-C1 and clone EC#2-B6 were subjected to serum deprivation for 96 h, and cell viability were measured by the MTT assay. Results showed that EC#1-C1 and EC#2-B6 maintained relatively higher cell viability than that of Wt CHOK1 following 96 h of serum deprivation. The resistance to serum-deprivation was promoted 10.3% and 11.6%, respectively. (Figure 6; **p < 0.01, n = 6). Overall, we obtained two cell lines CHO-K1/EC#1-C1 and CHO-K1/EC#2-B6 that have a higher cell viability compared with that of Wt CHO-K1
Figure 6. Cell viability during serum deprivation. EC#1-C1 and EC#2B6 maintained relatively higher cell viability than that of Wt CHO-K1 cells after 96 h of serum deprivation. The resistance ability to serum deprivation was promoted 10.3% and 11.6%, respectively. (**p < 0.01, n = 6).
cells, showing a promising potential for biopharmaceutical production.
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DISCUSSION Necessity and Significance of Improving ER Lumen Microenvironment and Reinforcing Antiapoptotic Ability of CHO-K1 Cells. Chinese hamster ovary (CHO) cell and a series of engineered cells derived from cells, such as CHO-K1 cells, are the predominant mammalian hosts for industrial production of recombinant proteins, especially therapeutic antibodies. However, there still remained bottlenecks in the quality and quantity of the protein production to meet the huge demand for clinical applications.2,4 The traditional methods of upgrading and promoting were mainly in media composition optimization, metabolic process control, vector and genetic engineering, and downstream process improvement.20 But ER acting as an important site for protein processing and secretion had not been researched further and regulated or controlled artificially in the CHO cell modification to improve production performance. The ER lumen has an elaborate chaperone and quality control network to maintain protein homeostasis;21 however, any physiological or environmental stimuli such as the redox imbalance, mutant protein accumulation, nutrition 1263
DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
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We further chose GLuc as a sulfhydryl oxidation probe to explore whether the overexpressed HsQSOX1b-KDEL can introduce disulfide into nascent polypeptide translocate into the ER. Luciferase assay results showed that EC#1-C1 and EC#2B6 had significant higher of bioluminesence over time compared to the Wt CHO-K1 cells, the RLU values in EC#2-B6 was 5.55 times higher than that of control group after 48 h culture (Figure 5). These results suggested that HsQSOX1b can function as an oxidase in the ER and can promote the disulfide formation in GLuc, exhibiting a promising prospect for engineering protein folding pathway. Survivin is the strongest inhibitor of apoptosis protein (IAP) that contains a single baculovirus IAP repeat (BIR) and an extended α-helical coiled-coil domain,30 it can not only directly inhibit the activity of Caspase-3/7,31 but also inhibit the apoptotic process induced by Fas (CD95), Bax and anticancer drugs.32 Thus, Survivin was chosen to explore its antiapoptotic performance in the modified CHO-K1 cells considering there was little study about its anti apoptotic ability in the CHO-K1 cells. In this study, the antiapoptotic performance of Survivin in transfected CHO-K1 cells was detected after 18 h staurosporine treatment. Results showed that the antiapoptotic ability of EC#2-B6 was promoted about 34.21% compared to that of the positive control cells (Figure 4). The result suggested that Survivin could be an outstanding antiapoptotic protein in engineering cell factory to enhance its recombination protein production performance and adverse environment resistance competence. Additionally, serum deprivation assay was performed to explore the potential biopharmaceutical production in serum free medium, results showed that the resistance to serum deprivation of EC#1-C1 and EC#2-B6 was promoted 10.3% and 11.6%, respectively, compared to that of the Wt CHO-K1 cells (Figure 6), indicating a promising potential for industrial recombination protein production. Advantages of Precise Editing of the Genome of CHO-K1 Cell with Dual-Genes by CRISPR/Cas9 Technology and Further Improvements. The traditional GOI integration are mainly vector transfection, which results in random insertion and subsequently long-term screening and low efficiency, coupled with unpredictable alterations due to the lack of convenient and efficient genome editing technology.9 The developed CRISPR/Cas9 in recent years is a powerful gene-editing tool in engineering model cells and animals.33 Upon the site-specific cleavation of DNA double strand brakes (DSBs) by Cas9 nuclease, the DSBs will be repaired by one of the two major DNA damage repair pathways in mammalian cells: the nonhomologous end-joining (NHEJ) or the homology-directed repair (HDR). Studies showed that the CRISPR/Cas9 genome editing technology can integrate GOI harboring short-homology arms into specific locus by HDR,34 and many researchers have devoted to enhance the HDR integration efficiencies.35−37 However, HDR is inefficient and observed at a lower or more variable frequency. In contrast, NHEJ is a predominate repair mechanism when cells face the DSBs,38,39 and studies have proved that NHEJ pathway can promote high-efficient rejoining of genome and plasmids in restoring the CRISPR/Cas9-induced DSBs in various human cell types.40−42 Thus, we introduced two functional genes, HsQSOX1b and Survivin, into the specific locus in CHO-K1 cells via NHEJ. The integration efficiency was up to 3.85%. WB assay proved that both HsQSOX1b and Survivin were reinforced in the modified CHO-K1 cells. Additionally, live cell image and
deprivation, or constitutive expression recombinant biopharmaceuticals leading to the accumulation of misfolded proteins can trigger the ER stress response.22 Cells react to ER stress by activating a series of sensors termed the unfolded protein response (UPR), which leads to a temporary inhibition of protein synthesis and an increase in synthesis of ER chaperone proteins, which promote protein folding, secretion and degradation to reduce the unfolded protein load in the ER.23 Meanwhile, constant or extended activation of the UPR may trigger the cell apoptosis pathway resulting in cell apoptosis.24 Thus, manipulation protein processing and cell apoptotic pathway are typical strategies to improve the biopharmaceutical production. Therefore, in the study, to improve ER lumen microenvironment and reinforce antiapoptotic ability of CHOK1 cells were very necessary and meaningful for the cell to enhance environment resistance competence and viability in biosynthesis of heterologous protein. The novel design idea of editing the genome of CHO-K1 cell factory with dual genes for high quality production of heterologous proteins was shown in the graphical abstract. Biological Activities of Human Derived Protein QSOX1 and Survivin and Their Functions in Modified CHO Cells. In the study, we have selected two genes (HsQSOX1b and Survivin) to edit the genome of CHO-K1 cells for improving the ER microenvironment and reinforcing antiapoptotic ability of the animal cell. This approach achieved our intended two goals of high quality production of heterologous proteins in CHO-K1 as we have obtained from the experimental results in the research. The two genes played their original biological activities in improving ER lumen microenvironment and reinforcing antiapoptotic ability in modified CHO cells, and this was very important for the experiment to achieve the desired results. The QSOX1 is a chimeric enzyme that contains a disulfideexchange (thioredoxin-like) domain and an oxidase (Erv-like) domain.25 It can introduce disulfides into substrate proteins using molecular oxygen as an electron acceptor, which is independent of the well-established endoplasmic reticulum oxidase 1 (Ero1)-protein disulfide isomerase (PDI) oxidation of disulfide bond pathway within the eukaryotic ER.26 Here, we observed that QSOX1 was expressed in Wt CHO-K1 cells and its protein level was associated with cell confluence (Figure 1A). The constitutive expression of QSOX1 in Wt CHO-K1 cells at different confluence interferes the detecting of our knocked in HsQSOX1b. Replacing the Wt CHO-K1 QSOX1 with HsQSOX1b would be better for our further research of HsQSOX1b’s effect in the ER. In vitro enzymatic studies of QSOX1 have demonstrated that it is able to both catalyze disulfide bonds formation of a large array of monothiol substrates (such as glutathione) and reduce proteins and peptides.27 However, enzymatic studies revealed that the avian QSOX1 have a relatively high KM for GSH and lower kcat/KM value in catalyzing GSH compared with unfolded substrate proteins.28,29 Thus, in our study, the decreased GSH in modified CHO-K1 cells (Figure 3) may not be the direct substrate of HsQSOX1b, instead of undertaking the reducing force to balance the overoxidized reside proteins in the ER. GSH and GSSG Assay results indicated that HsQSOX1bKDEL functioned as an oxidase in the ER, decreased reduced glutathione (GSH), turned ER into a more oxidative environment and provided an environment favorable for protein folding. 1264
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ACS Synthetic Biology
Plus-Neo (TOYOBO). The HsQSOX1b30−604 was amplified through PCR from pETsumo expression vector,27 and added signal sequence through PCR to form a full HsQSOX1b coding sequence. Primers used are listed in Table S3. The HsQSOX1b sequence obtained was inserted into Hind III- and BamH Idigested pcDNA3.1/V5-His B (Invitrogen, Shanghai, China). For EC#1 construction, CMV enhancer and promoter (E+P) and signal peptide of HsQSOX1b, BGH polyA parts were directly amplified from pcDNA3.1/V5-His B, but the eGFP was amplified from preassembled pcDNA3.1/eGFP expression vector constructed in our lab. Primer sequences are listed in Table S4. PCR products were resolved by 1% agarose gel and purified by UltraClean 15 DNA Purification Kit (MO BIO, Carlsbad, CA, USA). The purified PCR fragments were assembled into the linearized pUC19 between Hind III and EcoR I sites supplied by EasyGeno Assembly Cloning Kit and were transformed into DH5α Competent Cells. Positive transformants were selected on Lysogeny broth (LB) agar plates with ampicillin. For EC#2 construction, Thosea asigna virus “self-cleaving” 2a sequence44 was added before Survivin sequences from pET24a/Survivin expression vector constructed in our lab via PCR. EC#1 was linearized between HsQSOX1b+KDEL and BGH poly A, the two parts were assembled together using EasyGeno Assembly Cloning Kit. Primer sequences are listed in Table S4. The construction procedures of ECs were shown in Figure S2. All constructs were fully sequenced to verify and ensure that no additional changes in the sequence had been introduced. Cell Culture, Transfection, and Isolation of Clones by FACS. CHO-K1 cells [ATCC(CCL-61)] (Cell Bank: CAS, Shanghai, China) were grown in Ham’s F-12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Penicillin-Streptomycin in T25 cell culture flasks with vented cap (Coring) at 37 °C in a humidified incubator with 5% CO2. Cells were passaged by 0.25% trypsin (Gibco) at 80− 90% confluence and seeded at 1:2−3 dilutions onto tissue culture dishes or flasks. Culture medium was changed every 2 or 3 d. Cells were transfected with Lipofectamine2000 (Thermo Fisher Scientific) according to manufacturer’s protocols. Briefly, cells at a density of 1−2 × 105/mL were plated to a 24-well plate without antibiotics to grow for 16−24 h. 500 ng sgRNACas9 plasmids and equal amounts linear EC donor through PCR amplification (primers were listed in Table S4) were added into each well for cotransfection. The transfection experiments were conducted in triplicates and pX458 plasmid was used as control to monitor transfection efficiency. Clonal cell lines were obtained by fluorescence activated cell sorting (FACS) using a BD FACS Jazz cell sorter (BD Biosciences, San Jose, CA, USA) and eGFP positive cells were selected from the cell pool and collected into 96-well plates. To rule out transient transfection, transfected cells were passaged once or twice before FACS analysis. Sorted cells were maintained in Ham’s F-12 medium for 1−2 weeks to allow colony formation. Expansions of isolated clones were carried out sequentially in 24-well plate and 25 cm2 flasks to get genetically stable and homogeneous cell lines. Genomic DNA Extraction, PCR Amplification, and T7E1 Assay. Genomic DNA was extracted from cells using QuickExtract DNA extraction solution (Epicenter) 72 h post transfection. Cells were washed twice with PBS, and 50 μL QuickExtract solutions was added to each well of 24-well plates to lyse the cells with the following procedures, 65 °C, 15 min,
CLSM illustrated KDEL-tagged HsQSOX1b was residing in the ER of modified CHO-K1 cells. Through FACS, junction PCR detection, WB and ER location analysis, two cell lines, EC#1C1 that eGFP-HsQSOX1b-KDEL and EC2-B6 that eGFPHsQSOX1b-KDEL-T2A-Survivin were constructed rightly and introduced a linear expression cassette harboring two functional GOI into exon7 of QSOX1 gene in CHO-K1 genome via NHEJ using the CRISPR/Cas9. The experiment proved that NHEJ was a feasible integration method for host cell modification, and the efficient method can accelerate the efficiency of genomic editing process for obtaining improved CHO cells with high quality production of heterologous proteins. On the basis of two genes HsQSOX1b and IAP Survivin knocked in specific location of CHO-K1 genome via NHEJ by the CRISPR/Cas9 technology precisely, we have obtained modified CHO-K1 cell successfully with dual-genes. The modified CHO-K1 could express the two genes efficiently, and not only showed remarkable antiapoptotic ability from IAP Survivin, but also had excellent characteristics of promoting the disulfide formation of sulfhydryl oxidation model protein GLuc by constructing a more oxidative surroundings in the ER. Compared with the control, the antiapoptotic viability of edited CHO-K1 by dual-genes was increased by 6.40 times and the yield has been raised by 5.55 times with GLuc as model protein. The novel engineered CHO-K1 were contributed to elucidate the role and influence on CHO-K1 cell in biosynthesis of heterologous protein by improving ER microenvironment and reinforcing antiapoptotic ability, and explore the possible molecular mechanisms and pathways of efficient processing and secreting foreign protein in CHO cells. In addition, our research validated that NHEJ can mediated exogenous DNA integration into specific DNA DSBs induced by CRISPR/Cas9, and further demonstrated that NHEJ is an efficient tool for host cell engineering, which offers huge potential to accelerate generation of stable cell lines that meet the industrial requirements. To summarize, this research on the genome editing of CHO-K1 cells has laid an important foundation theoretically and technically on updating of mammalian cell factories to meet the large demand for engineered proteins, especially therapeutic high-quality antibodies in clinical needs.
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MATERIAL AND METHODS SgRNA Target Design and sgRNA-Cas9 Plasmid Construction. The pSpCas9 (BB)-2A-GFP (pX458) (Addgene plasmid #48138) and pSpCas9 (BB)-2A-Puro (pX459) V2.0 (Addgene plasmid # 62988) were gifts from Feng Zhang.43 They were all-in-one plasmids, which contain a cloning backbone for sgRNA that can be designed for targeting a sequence in the CHO-K1 genome and a mammalian expressfriendly pSpCas9 enzyme. sgRNAs were generated by the online tool Optimized CRISPR Design (http://crispr.mit.edu/ ), and the sgRNAs with fewer off-target sites were chosen for further analysis. Target sequences of sgRNAs were listed in Table S1, and the potential off-target sites of each sgRNA were shown in Table S2. SgRNA-Cas9 plasmids were constructed as previously described,43 and the plasmid construction procedure was shown in Figure S1. Expression Cassette Construction. The Expression cassette (EC) plasmids were constructed with EasyGeno Assembly Cloning Kit according to the manufacturer’s instruction (Tiangen, Beijing, China). Different parts of the plasmid were amplified with primers and polymerase KOD1265
DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
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ACS Synthetic Biology 68 °C, 15 min, followed by 98 °C, 10 min. 1−2 μL of the extract was used as PCR template. For cell pools in tissue culture dishes or flasks, genomic DNA was purified by Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s directions. DNA extracts were stored at −20 °C. KOD FX (TOYOBO) was used for T7E1 assay, sgRNA target region was amplified by touchdown PCR (94 °C, 2 min; 10×: 98 °C, 10 s, 65−55 °C (−1 °C/cycle) 30 s, 68 °C 45s; 20×: 98 °C, 10 s, 55 °C, 30 s, 68 °C, 45 s; 68 °C, 7 min). PCR products were subjected to 2% agarose gel electrophoresis and purified by UltraClean 15 DNA Purification Kit. 200 ng of purified PCR products were denatured and annealed in 20 μL NEBuffer 2 using a thermocycler. Hybridized PCR products were digested with T7 endonuclease 1 (New England Biolabs) for 1 h at 37 °C and separated by 2% agarose gel electrophoresis. The indel (deletion/insertion) occurrence was quantified using ImageJ as described previously.45 For detecting NHEJ-mediated integration event, 5′/3′ junction PCR was performed using KOD-FX. The NHEJ-mediated integration efficiency was estimated by the FACS analysis. Primers used for T7E1 assay and the detection of NHEJmediated integration were listed in Table S5 and Table S6, respectively. TA Cloning and Sanger Sequence. Junction PCR fragments were purified by QIAquick Gel Extraction Kit (QIAGEN) incubated with DNA A-Tailing Kit (Takara) for Atailing, and ligated into the pMD 19-T Vector (Takara) for TA cloning according to the manufacturer’s instructions. Positive clones were verified by sequencing. Western Blot and Live Cell Imaging. Cells were lysed in RIPA Lysis Buffer I (Sangon) and total protein concentration was measured using BCA Protein Assay kit (Sangon). 50 μg total proteins were separated on SDS-PAGE gels (12%) and transferred onto PVDF membrane (Millipore), and then blocked and probed with rabbit polyclonal anti-Survivin antibody or rabbit polyclonal anti-QSOX1 antibody (Proteintech Group). β-Actin was used as a loading control. Membranes were washed and incubated with HRP-conjugated goat anti rabbit or goat antimouse (Santa Cruz) antibodies, and visualized using the pro-light HRP chemiluminescent kit (Tiangen). For live cell imaging, eGFP positive cells were cultured in sixwell plate and rinsed with HBSS/Ca/Mg (Sangon), and then stained by prewarmed ER-Tracker Red (Thermo Fisher Scientific) and monitored with fluorescence microscope (Olympus, Japan). For confocal laser scanning microscope (CLSM; Nikon, Japan) observation, cells were seeded in Nunc Glass Bottom Dish (Thermo Fisher Scientific), stained by ER-Tracker Red, and fixed by 4% formaldehyde. Nuclei were counterstained by Hoechst 33342 dye (Thermo Fisher scientific). Fluorescent images were obtained by CLSM and analyzed with Nikon NISElements. Measurement of GSH and GSSG. GSH and GSSG in cells were measured by using the GSH and GSSG Assay Kit (Beyotime, Shanghai, China). In brief, cells were lysed by repeated freezing in liquid nitrogen and thawing in water bath at 37 °C. Thirty minutes after incubation at 0 °C, the lysates were centrifuged at 1000g for 10 min at 4 °C. GSSG and tGSH (GSH + GSSG) in the supernatant were directly determined by the GSH and GSSG Assay Kit at OD412 nm with a microplate reader (Thermo Fisher Scientific, LF-1504007, USA). The concentration of GSH was calculated by subtracting the
measured levels of GSSG (multiplied by 2, because one molecule of GSSG is reduced to two molecules of GSH) from the levels of tGSH. FACS Analysis To Detect Apoptotic Cells. The measurement of apoptosis was conducted by Annexin V-PE apoptosis detection kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells at a density of 2 × 105/mL were seeded into 6-well plates and cultured for 24 h. Staurosporine (0.2 nM) was added to each well. After 18 h, cells were harvested and suspended in 190 μL 1 × binding buffer, and 5 μL Annexin V-PE was added. Cells were gently vortexed and incubated in the dark for 15 min at RT, then 10 μL PI Staining Solution (Ready to use) (Yeasen, Shanghai, China) was added to the mixture and incubated in the dark for 5 min at RT. After incubation, 300 μL 1 × binding buffer was added and cells were analyzed directly by flow cytometry (BD FACScan, San Jose, CA, USA). Methylthiazoletetrazolium Assay. Cell viability was measured by methylthiazoletetrazolium (MTT) assay (Sangon). For serum deprivation assay, cells at a density of 5 × 104/ mL were seeded in 96-well plate with six repeats and cultured with serum free F-12 medium. 96 h later, 20 μL MTT stock (5 mg/mL) was introduced to each well. Four h later, medium was replaced with 100 μL DMSO. Plates were gently shaken for 10 min at RT in the dark, and OD490 nm was recorded with a microplate reader (BioTek ELX808, USA). Gaussia Luciferase Assay. For Gaussia luciferase assay, cells were seeded in 96-well plates at a density of 5 × 104/mL in triplicate. Twenty-four h later, the cells were transiently transfected with 100 ng pGluc-Basic with cytomegalovirus (CMV) promoter inserted between Hind III and BamH I restriction site and cultured in serum free medium. After 48 h, the cell culture supernatant was collected, and Gaussia luciferase activity was measured by BioLux Gaussia Luciferase Assay Kit according to the manufacturer’s instructions (New England Biolabs). The GLuc activity was measured in relative luminescence units (RLU) with an integration time of 5 s after a 35 s delay by luminometer at 475 nm (BioTek ELX800, USA). The linear range of the luminometer was assayed by serial dilution of sample EC#2-B6 48 h post transfection. Statistic Analysis. Unless otherwise stated, data were presented as means ± SD, n = 3. Statistical analysis was performed by paired student t test between the colonial cell lines and the control Wt CHO-K1 cells. The value of *p < 0.05 and **p < 0.01 were regarded as statistically significant.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00375. Supplementary tables that list the sequences included sgRNA sequences, primer sequences for constructing the expression cassettes and verifying indels and chromosomal integrations; Supplementary figures that illustrate the construction of plasmid and expression cassettes, and additional experiments described in the text (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. *Tel./Fax: +86 2164250135. E-mail:
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DOI: 10.1021/acssynbio.7b00375 ACS Synth. Biol. 2018, 7, 1259−1268
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Xingyuan Ma: 0000-0002-8740-7748 Author Contributions
# Wenpeng Wang and Wenyun Zheng contributed equally to this work. Wenpeng Wang designed and conducted the experiments totally and wrote the manuscript; Wenyun Zheng designed the contents on protein folding in CHO-K1 and helped perform the analysis with constructive discussions; Fengzhi Hu and Xiujuan also performed the experiments partly; Dong Wu performed the data analyses and drawn table; Wenliang Zhang and Haipeng Liu revised the manuscript and gave some insightful suggestions; Xingyuan Ma conceived the experiments on the whole and approved the final version.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation (31670944, 81673345), Science and Technology Innovation Action Plan of Shanghai (14431904300, 17431904600), Shanghai Pujiang Program (13PJD012), and supported by the National Science Research Project “Significant New Drugs Created” of Eleventh Five-Year Plan (2009ZX09103-693).
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ABBREVIATIONS CHO, Chinese hamster ovary; CRISPR/Cas9, Clustered regularly interspaced short palindromic repeat/CRISPRassociated 9; DSBs, Double strand breaks; EC, Expression Cassette; ER, Endoplasmic reticulum; Ero1, Endoplasmic reticulum oxidoreductase 1; FACS, Fluorescent-activated cell sorting; GLuc, Gaussia luciferase; GSH/GSSG, Glutathione; HDR, Homology directed repair; IAP, Inhibitor of apoptosis protein; NHEJ, Nonhomologous end-joining; PDI, Protein disulfide isomerase; QSOX1, Quiescin-sulfhydryl oxidase1; sgRNA, Single guide RNA; UPR, Unfolded protein response
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