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. 2026 Mar 21;20:79. doi: 10.1186/s13036-026-00669-2

A novel chassis for high-quality recombinant protein expression: apoptosis pathway engineering via multi-gene knockout and efficacy improvement of CHO cells

Youzhi Wu 1, Ying Cao 1, Zexuan Guo 1, Yanhua Ma 1, Xiaohui Zan 1, Xingjin Gu 1, Fan Zhang 1, Chunxia Chai 1, Rui Nui 1, Qiang Shang 1, Wei Wang 1,2,3,✉
PMCID: PMC13130613  PMID: 41864927

Abstract

Background

Chinese hamster ovary (CHO) cells are pivotal in biopharmaceutical production, yet balancing high recombinant protein yield with cell survival remains challenging. While previous studies have targeted single apoptotic regulators, the synergistic effects of multi-gene ablation on protein stability are unknown.

Results

This study presents a multi-knockout strategy for CHO cells. Herewithin, CHO-K1 knockout lines were engineered via CRISPR-Cas9 targeting of key triple (TKO, BAK/BAX/CASP3), double (DKO, BAK/BAX), and single (SKO, CASP3) apoptotic nodes. Subsequently, comprehensive analyses of apoptosis, cell viability, doubling time, cell cycle, and mitochondrial membrane potential were conducted for isolated clones. The triple knockout cell lines exhibited the highest overall levels of cell viability, a shortened doubling time, and enhanced resistance to apoptosis. These characteristics directly translated to improved expression of recombinant blue fluorescent protein, with triple knockouts outperforming WT and single/double knockout lines.

Conclusions

These findings establish a robust foundation for engineering apoptosis-resistant CHO cell lines with enhanced protein production capacity, offering a promising approach for improved efficiency and reduced costs in biopharmaceutical manufacturing.

Supplementary information

The online version contains supplementary material available at 10.1186/s13036-026-00669-2.

Keywords: CHO cells, Apoptosis, Doubling time, Knockouts, Recombinant proteins

Introduction

Chinese hamster ovary (CHO) cell lines serve as the cornerstone of the modern biopharmaceutical industry, accounting for the production of over 80% of recombinant therapeutic proteins worldwide, including monoclonal antibodies, enzymes, and hormones [1]. Their predominance stems from an unparalleled ability to perform complex, human-like post-translational modifications and to adapt to large-scale suspension culture [2, 3]. However, during high-density and extended cultivation in industrial bioreactors, cells invariably encounter multiple stress factors, such as nutrient depletion, accumulation of metabolic by-products, osmolality shifts, and hydrodynamic shear forces [4, 5]. These stresses converge to activate the intrinsic apoptotic program, leading to widespread and irreversible cell death during the late stages of culture. This process severely limits final product titers and overall volumetric yield, while increasing process variability and manufacturing costs. Consequently, a deep understanding of, and effective control over, apoptosis in CHO cells remains a central challenge in overcoming productivity bottlenecks and enabling more efficient and economical biomanufacturing.

Apoptosis is a highly conserved, genetically encoded cell death pathway executed by a precise proteolytic cascade, centrally regulated by the Bcl-2 protein family and downstream caspases [6, 7]. Upon apoptotic stimulation, the pro-apoptotic effectors BAK and BAX oligomerize on the mitochondrial outer membrane to form permeabilization pores, an event known as mitochondrial outer membrane permeabilization (MOMP) [8, 9]. MOMP represents the irreversible “point of no return” in apoptosis, triggering the release of cytochrome c and other pro-apoptotic factors into the cytosol [8, 10]. Cytochrome c binds to Apaf-1 to form the “apoptosome,” which activates the initiator caspase-9 [11]. Activated caspase-9 then cleaves and activates the key executioner caspase-3 (CASP3) [12]. Caspase-3, in turn, systematically hydrolyzes hundreds of cellular substrates, ultimately leading to cellular dismantling [13]. This pathway is fully intact and operational in CHO cells, constituting a primary biological barrier limiting culture longevity and protein output.

To enhance the apoptosis resistance of CHO cells, two main strategic approaches have been explored. The first involves the exogenous addition of caspase inhibitors or the overexpression of anti-apoptotic proteins like Bcl-2 or Bcl-xL [14–16]. While effective, this strategy can impose a metabolic burden, lead to genomic instability, or require continuous supplementation of costly chemical inhibitors. The second approach employs gene-editing technologies, such as CRISPR-Cas9, to knockout key pro-apoptotic genes. Previous studies have shown that individual knockout of BAK, BAX, or CASP3 can delay apoptosis induced by specific stressors to varying degrees [17, 18]. However, the apoptotic network is characterized by significant redundancy and compensatory mechanisms. For instance, BAK and BAX share overlapping functions, making single knockouts of either gene often insufficient [19]. Furthermore, even if MOMP is inhibited, alternative pathways (e.g., the death receptor pathway) can still activate downstream caspases [20]. Thus, the protection afforded by existing single-gene knockout strategies is often incomplete and context-dependent. A fundamental question that remains inadequately addressed is whether a synergistic effect can be achieved by systematically ablating multiple critical nodes within the apoptotic cascade—for instance, simultaneously targeting both the initiation (MOMP) and execution (caspase) phases—to engineer cell lines with broader and more robust resistance. The comprehensive impact of such multiplexed gene editing on cellular homeostasis, long-term growth, and the stability of recombinant protein production is currently unknown.

To address this gap, the present study moves beyond conventional single-target strategies by proposing and validating a synergistic multi-gene knockout approach. We utilized CRISPR-Cas9 to engineer an isogenic panel of CHO-K1 cell lines featuring triple (BAK/BAX/CASP3), double (BAK/BAX), and single (CASP3) knockouts of key apoptotic nodes. Through a systematic comparative analysis of these models regarding fundamental growth properties (cell viability, doubling time, cell cycle), mitochondrial function (membrane potential), and resistance to apoptotic inducers, we aimed to elucidate the additive or synergistic benefits of combinatorial gene ablation in enhancing cellular robustness. The central objective of this work is to determine whether this engineered resilient phenotype can be directly and effectively translated into a tangible enhancement of recombinant protein productivity. Our findings provide not only novel insights into the functional redundancies within the apoptotic pathway but, more importantly, offer a new and powerful engineering strategy and experimental framework for designing next-generation, high-performance, and stress-resistant CHO cell platforms.

Materials and methods

Cell culture

CHO-K1 (China Center for Type Culture Collection; CCTCC), HEK293T and CHO-K1 knockout cell lines were cultured in DMEM/F12K medium (Gibco) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2. For routine maintenance, CHO cells were sub-cultured with fresh medium at three-day intervals in 25 cm2 flasks (Corning). The cell concentration and viability were assessed using a Countstar hemocytometer and the trypan blue exclusion method (Solarbio).

Generation of BAK, BAX, and CASP3 knockout CHO-K1 cell lines

CHO-TKO (BAK/BAX/CASP3 triple knockout), CHO-DKO (BAK/BAX double knockout), and CHO-SKO (CASP3 single knockout) cell lines were generated using CRISPR/Cas9-mediated genome editing. The single guide RNAs (sgRNAs) targeting BAK (NM001246795.1), BAX (NM001244020.1), and CASP3 (NM-001244246) genes were designed using the CRISPR Design Tool (crispor.gi.ucsc.edu). For each of the genes, two distinct sgRNAs were designed to ensure efficient targeting (Table S1). The sgRNAs were synthesized and cloned into lentiCRISPRv2-EGFP vector (Addgene, #52961) using BsmBI restriction sites.

Lentiviral particles were produced in HEK293T cells by co-transfecting the lentiCRISPRv2-EGFP vectors containing the sgRNAs with the packaging plasmids psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) using Lipofectamine 3000 (Thermo Fisher Scientific). Six Lentiviral supernatants were collected 48 h post-transfection and filtered through a 0.45 μm pore size filter. CHO-K1 cells were infected with the condensed 50 X lentiviral supernatants in the presence of 8 μg/mL polybrene (Solorbio). After 48 h, single EGFP-positive cells were deposited into 96-well plates using the Sony MA900 cell sorter. Individual clones were expanded and maintained for at least 8 weeks (approximately 20 passages) to generate stable knockout cell lines.

Genomic DNA was isolated from the stable knockout cell lines using the Universal Genomic DNA Kit (Cwbio, CW2298M). The target regions were amplified by PCR using gene-specific primers (Table S2). The PCR products were then analyzed by Sanger sequencing to confirm the presence of indels and other mutations. Cells with confirmed mutations in the target genes were further characterized by Western blot analysis using anti-BAK (Proteintech, #29552–1-AP), anti-BAX (Proteintech, #50599–2-lg), and anti-CASP3 (Proteintech, #19677–1-AP) antibodies to verify the absence of the respective proteins.

Quantitative assessment of apoptosis in CHO cells

To systematically assess apoptosis quantitatively, we utilized the Annexin V-AbFlour 647/PI apoptosis detection kit (Abbkine). CHO cells were seeded in 6-well plates at a density of 50,000 cells per well. To induce apoptosis, puromycin, a ribosomal protein translation inhibitor, was applied at a concentration of 6 mg/L [21]. A control group without puromycin treatment was processed in parallel. After treatment, the cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in binding buffer. Annexin V-AbFlour 647 and propidium iodide (PI) were subsequently added to the cell suspension, followed by a 15-minute incubation at room temperature in the dark. Apoptotic cells were identified and quantified using a Cytoflex LX flow cytometer (Beckman Coulter), with data analysis performed using CytExpert software. Cells were classified as early apoptotic (Annexin V-positive, PI-negative) or late apoptotic/necrotic (Annexin V-positive, PI-positive) based on their fluorescence profiles.

Cell viability and doubling time analysis

To assess cell viability, we employed the SuperKine Maximum Sensitivity Cell Counting Kit-8 (CCK-8, Abbkin) in accordance with the manufacturer’s guidelines [22]. Cells were seeded at a density of 5.0 × 103 cells per well in eight 96-well plates, with 10 wells per plate for each cell line. After adding CCK-8 reagent, the plates were incubated for 4-h at 37 °C. The absorbance at 450 nm, indicative of cellular metabolic activity, was measured using a microplate reader (SpectraMax i3, Molecular Devices). Daily measurements were recorded over 8 consecutive days at 24-h intervals to track temporal changes in cell viability.

The cell doubling time (DT) was determined via cell proliferation assay. Cells were plated at an initial density of 5.0 × 103 cells per well in 96-well plates. Cell counts were calculated every 24 h for 8 days using a hemocytometer. The formula (1) employed to calculate DT was based on the initial cell number (N0), recorded at the start of the observation period and the change in cell number (ΔN) over a specific time interval (Δt). Each cell division was reflected as an increment of one cell in the count; hence, ΔN also signified the total number of cell divisions that occurred during Δt [23].

graphic file with name d33e497.gif 1

Analysis of the cell cycle distribution

A suspension of CHO knockout and native CHO-K1 cells, each at a concentration of 5 × 105 cells/mL, was seeded into individual wells of a 6-well culture plate. PI staining was employed to assess the cell cycle profile. The cells were collected, subjected to fixation in 70% ethanol, and stained with 50 μg/mL PI plus 100 μg/mL RNase A for 30 minutes at ambient temperature. The cell cycle distribution was determined by flow cytometric analysis using a Cytoflex LX instrument (Beckman Coulter). The resulting data were processed and analyzed using ModiFit LT software [24].

Mitochondrial membrane potential assay

The mitochondrial membrane potential (ΔΨm) was assessed in wild-type CHO-K1 and isogenic knockout lines (CHO-BBC13, CHO-BB1, CHO-C2) using tetramethylrhodamine ethyl ester (TMRE; Beyotime, C2001S). The cells were treated with 6 μg/mL puromycin for 24 h to induce apoptosis-associated mitochondrial stress or 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 1 h as a positive control for complete ΔΨm depolarization. Untreated cells served as baseline controls. After treatment, the cells were incubated with 100 nM TMRE in culture medium at 37 °C for 30 min, washed twice with PBS, and analyzed via flow cytometry (BD Cytoflex LX) using 549/575 nm excitation/emission wavelengths. Live cells were gated based on forward/side scatter profiles.

BFP protein expression analysis

To assess the protein expression capabilities of CHO-K1 and CHO knockout cells, a PTagBFP-N expression plasmid encoding blue fluorescent protein (BFP; 500 ng per well) was transfected in 24-well plates using Lipofectamine 3000 (Thermo Fisher Scientific). BFP expression was monitored at 24-h intervals for three days, employing both fluorescence microscopy for cellular visualization and flow cytometry for quantitative analysis across the cell population. For each measurement, 20,000 cell events were recorded. Data were analyzed using CytCxpert software to quantify fluorescence intensity as a marker of protein expression levels. Experiments were conducted in triplicate to ensure reproducibility.

Statistical analysis

Statistical analyses of the data were performed with OriginPro 2024 and GraphPad Prism 8. Experimental data were represented as mean ± SD using two-way analysis of variance (ANOVA) for three and more groups. Data were considered significant when *p ≤ 0.05.

Results

Generation of apoptosis-resistant single-cell knockout lines

Leveraging CRISPR/Cas9 technology, we generated CHO-K1 single-cell clones with targeted knockouts (KOs) of BAK, BAX, and/or CASP3. To minimize the adverse effects associated with sequential knockouts, we employed a synchronous co-knockout strategy. Specifically, for the construction of a TKO cell line for BAK, BAX, and CASP3, we simultaneously infected CHO-K1 cells with six sgRNA-expressing lentiviral vectors. Additionally, we generated a DKO cell line for BAK and BAX, as well as a SKO cell line for CASP3. Then we isolated single-cell clones from these TKO, DKO, and SKO lines, resulting in 17 BAK/BAX/CASP3 TKOs (designated as CHO-BBC1 to CHO-BBC17), six BAK/BAX DKOs (designated as CHO-BB1 to CHO-BB6), and four CASP3 SKOs which were designated CHO-C1 to CHO-C4. Following eight weeks of continuous passaging, we further verified one TKO (CHO-BBC13), one DKO (CHO-BB1), and one SKO (CHO-C2) cell clone from each parental host (Table S3). The success of the CRISPR/Cas9-mediated knockout was confirmed at the genomic level using Sanger sequencing to identify insertions or deletions within the target gene sequences (Supplementary Figure S1 and Table S1), and at the protein level using Western blot analysis (Fig. 1A and B). All CHO-K1 knockout cell clones exhibited successful knockouts at both at the genomic and proteomic levels.

Fig. 1.

Fig. 1

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Generation of CHO-K1 knockout cell lines that are apoptosis resistant. (A) Experimental setup for knockout experiments and assessment of apoptosis in CHO-K1 cells: CHO-K1 cells were plated on day −16 and infected with lentivirus on day −14. After 48 h, the cells were replated and monoclones were isolated. Apoptosis was induced by treatment with puromycin (pur), and 24 and 48 h later, PI/Annexin V staining was evaluated by fluorescence-activated cell sorting (FACS). (B) Western blot analysis to determine the BAK, BAX and CASP3 levels in CHO-K1 wild-type and knockout cells. (C) Annexin V/PI staining of CHO wild-type and knockout cells treated with vehicle (control) or puromycin for 24 h. Primary FACS plots and corresponding quantification of Annexin V/PI double-positive cells are shown. (D) Annexin V/PI staining of CHO wild-type and knockout cells treated with vehicle (control) or puromycin for 48 h. (Dark green: untreated group without pur processing; (Green) pur-treated group). Primary FACS plots and corresponding quantification of Annexin V/PI double-positive cells are shown. Statistical analysis was performed using two-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001). Experimental values represent the mean of three biological replicates (n = 3) ± SD

To elucidate the differences in apoptosis resistance among CHO-K1 wild-type and CHO-KOs cell lines, we conducted experiments with and without puromycin-induced apoptosis. After 24 h of puromycin treatment, the CHO-K1 wild-type cell line exhibited a significant increase in apoptosis, with approximately 20% of cells being double-positive for Annexin V and PI, which is indicative of late apoptotic or necrotic stage cells (Fig. 1C). In contrast, the CHO knockout cell lines showed negligible changes in apoptosis levels, suggesting that these genetically modified cell lines are resistant to apoptosis induction. Extending the induction period to 48 h revealed a more pronounced apoptotic response in the CHO-K1 wild-type and CHO-C2 cell lines. Specifically, the CHO-K1 wild-type cell line displayed a dramatic increase in apoptosis, with approximately 90% of cells being double-positive for Annexin V and PI (Fig. 1D). The CHO-C2 cell line exhibited approximately 80% double-positivity, while the CHO-BBC13 and CHO-BB1 clones demonstrated a much lower degree of apoptosis after 48 h of puromycin treatment, with only about 15% double-positivity for Annexin V and PI (Fig. 1D). This relatively low level of apoptosis indicates that these cell lines possess robust mechanisms to resist apoptosis induction, even under prolonged exposure to puromycin. The resistance to apoptosis observed in the CHO-TKO and CHO-DKO cell clonogenic lines, both at 24 and 48 h, verifies the impact of the genetic modifications on the apoptotic response.

Growth dynamics and metabolic profiles of CHO-K1 and knockout cell lines

To further evaluate the effect of BAK, BAX and CASP3 knockdown in CHO-K1 cells, we performed a comprehensive analysis of cell viability and doubling time. Representative phase-contrast images of the cell lines captured over the eight-day culture period are shown in Fig. 2A. Although all lines exhibited typical adherent morphology, subtle differences in cell shape and clustering were observed, particularly in the BBC13 triple-knockout line, which appeared more refractive and formed denser cells at later time points. Our cell density analysis showed that the growth dynamics varied significantly among the cell lines (Fig. 2B). During the first 4 days, CHO-BBC13 cells showed comparable or slightly higher growth rates compared with WT cells, while other knockout lines exhibited similar growth kinetics as the WT line. However, by the sixth day, all cell lines except CHO-BB1 reached their maximum growth density, which ranged from 7.7 to 18.9 × 105 cells/mL. CHO-BB1 achieved its maximum growth density of 8.8 × 105 cells/mL on the seventh day, indicating a slightly delayed growth pattern compared to that of the other cell lines. The CHO-BBC13 cell line displayed the highest peak (18.9 × 105 cells/mL), and CHO-C2 peaked at 7.72 × 105 cells/mL.

Fig. 2.

Fig. 2

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Growth dynamics and metabolic profiles of CHO-K1 and knockout cell lines. (A) Representative phase-contrast images of wild-type (WT) and knockout clones (BBC13, BB1, and C2) captured daily for eight days to monitor morphology and confluency. (B) Comparison of the average daily cell density during continuous culture for 8 days. (C) Comparison of the average daily cell metabolic activity as assessed by CCK-8 assay over the course of continuous culture for eight days. (D) Population doubling time (DT, hours) of CHO-K1 and CHO knockout cell lines calculated during the exponential growth phase. Experimental values represent the mean of 10 biological replicates (n = 10) ± SD

For additional verification of these findings, we performed cell metabolic activity analysis using the CCK-8 assay over 8 days (Fig. 2C). The CHO-WT and CHO-C2 cell lines experienced a significant decrease in viability after the seventh day, while other cell lines maintained high viability levels throughout the experimental period. Consistent with its faster proliferation rate, the CHO-BBC13 cell line demonstrated the highest metabolic activity during the middle phase of the experiment, as evidenced by the highest optical density values at 450 nm (1.6–2.0) on the fourth and fifth days, which were significantly higher than the peak values of other cell lines (0.7–1.5; Fig. 2C).

Finally, to better understand the growth characteristics of the different CHO cell lines, we analyzed their average doubling times. The CHO-K1 cells showed a relatively consistent doubling time of 22.23 h, while the CHO-BBC13 and CHO-C2 cells displayed a significantly faster doubling time of 18.7 h. The CHO-BB1 line displayed an intermediate phenotype, achieving a doubling time of 20.16 h, thus bridging the gap between the parental and high-growth variants (Fig. 2D). These insights are valuable for understanding the growth regulation and potential applications of these cell lines in biotechnological and therapeutic contexts.

Cell cycle distribution and mitochondrial membrane potential of CHO-K1 and knockout cell lines

Genetic dissection of apoptotic regulators in CHO cell models has revealed their critical, non-canonical roles in cell cycle surveillance, with CASP3 and BAK/BAX governing distinct checkpoints that directly influence recombinant protein production. Furthermore, the systematic evaluation of the mitochondrial membrane potential (ΔΨm) dynamics across engineered CHO-K1 knockout lines has revealed striking hierarchical dependencies among BAK, BAX, and CASP3 in regulating stress-induced mitochondrial depolarization [24, 25]. Therefore, we sought to investigate the effect of TKO, DKO, and SKO on the cell cycle distribution and ΔΨm of CHO cells.

Under basal conditions, the CHO-C2 cells displayed a striking accumulation of S-phase cells, a phenotype absent in CHO-BBC13 and CHO-BB1 cells. The persistence of this phenotype under puromycin-induced ribosomal stress further suggests that CASP3 ablation enhances translational resilience, enabling sustained protein synthesis even under stress conditions (Fig. 3A). In contrast, BAK/BAX ablation induced constitutive G2/M accumulation, which escalated under stress (up to 38.0% in the TKO cells) (Fig. 3A). Notably, CASP3 deletion normalized puromycin-induced G2/M arrest (28.4% vs. wild-type: 23.8%, p > 0.05), revealing a dynamic balance between CASP3’s dual roles in resolving replication stress and moderating mitotic arrest—a balance critical for optimizing both protein yield (S-phase-driven synthesis) and quality (G2/M-driven maturation).

Fig. 3.

Fig. 3

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Cell cycle and mitochondrial membrane potential activity assays for CHO-K1 and knockout cell lines. (A) The cell cycle distribution in TKO (BBC13), DKO (BB1), and SKO (C2) cells as compared to wild-type CHO-K1 cells (WT) was determined by FACS-based, quantification. Puromycin or a vehicle control was added, as indicated. (B) Mitochondrial membrane potential activity assay of puromycin-treated WT, TKO, DKO or SKO cells. CCCP was applied in parallel as a control. TMRE was read out by FACS (left panel), which is quantified (right panel). Statistical analysis was performed by two-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Experimental values represent the mean of three biological replicates (n = 3) ± SD. Pur, puromycin. TMRE, tetramethylrhodamine ethyl ester. CCCP, carbonyl cyanide 3-chlorophenylhydrazone

In the ΔΨm assay, wild-type (CHO-WT) cells exhibited severe collapse under puromycin-induced apoptotic stress, with only 29.69% of cells retaining TMRE fluorescence (Fig. 3B). In stark contrast, the TKO clones (CHO-BBC13) demonstrated profound resistance to puromycin, retaining 90.67%, 77.09%, and 73.09% of the TMRE fluorescence. Notably, the BAK/BAX DKO (CHO-BB1) mirrored the TKO phenotype, preserving 70.83% TMRE fluorescence post-puromycin (p < 0.0001 vs. WT), which implicates BAK and BAX as the dominant executioners of MOMP-driven ΔΨm loss. The partial but significant protection observed in CASP3 SKO cells (CHO-C2; 68.83% retention, p < 0.0001 vs. WT) suggests a secondary role for CASP3 in amplifying mitochondrial dysfunction downstream of MOMP [26, 27]. As a control, CCCP treatment abolished TMRE fluorescence across all lines, confirming assay specificity. Intriguingly, clonal variability among TKO lines hinted at epigenetic effects influencing ΔΨm stability. CHO-BBC13, with near-complete puromycin resistance (90.67%), may represent a clone with optimal CRISPR editing efficiency or compensatory metabolic adaptations. Nevertheless, all TKO clones exhibited statistically indistinguishable protection from ΔΨm collapse compared to CHO-BB1, thus reinforcing the critical role of BAK/BAX in apoptotic signaling.

BFP protein expression in CHO-K1 and knockout cell lines

To evaluate the recombinant protein expression capabilities of the engineered CHO-K1 cell lines, we employed a blue fluorescent protein (BFP) reporter system. CHO-WT, CHO-BBC13 (TKO), CHO-BB1 (DKO), and CHO-C2 (SKO) cells were transfected with the PTagBFP-N plasmid, and BFP expression was monitored at 24, 48, and 72 h post-transfection using both fluorescence microscopy and flow cytometry (Fig. 4A). Quantitative analysis was performed by measuring both the mean fluorescence intensity (MFI) of BFP-positive cells and the percentage of BFP-positive cells (i.e., transfection efficiency).

Fig. 4.

Fig. 4

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BFP expression assay for CHO-K1 and CHO knockout cell lines. (A) Fluorescence microscopy of CHO-K1 and knockout cell lines was performed at 48 h post-transfection with BFP. Simultaneously, the intrinsic GFP fluorescence of the knockout cell lines was monitored to facilitate cellular localization; a merged image is presented. (B, C) Quantitative results corresponding to the mean fluorescence (panel B), BFP fluorescence rate (panel C) of each cell line were determined by flow cytometry at the indicated time points (24, 48, and 72 h) post-transfection. Statistical analysis was performed by two-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. Experimental values represent the mean of three biological replicates (n = 3) ± SD

At 24 h post-transfection, significant differences in BFP expression were observed among the cell lines. The CHO-BBC13 TKO cells exhibited the highest MFI at 35,380.7, representing a 2.05-fold increase over CHO-WT cells (17,246.1; p < 0.001). Similarly, the CHO-BB1 DKO cells showed elevated MFI (28,770.8; 1.67-fold vs. WT cells; p < 0.01), while the CHO-C2 SKO cells demonstrated the highest MFI among all groups (26681.83; 1.55-fold vs. WT cells; p < 0.001) (Fig. 4B). Notably, the transfection efficiency (percentage of BFP-positive cells) was markedly higher in the CHO-BBC13 cells (34.26%) compared with the CHO-WT (12.92%), CHO-BB1 (26.01%), and CHO-C2 (11.80%) cells (Fig. 4C). These results suggest that the TKO background may enhance both transfection efficiency and initial protein expression.

By 48 h, the expression patterns diverged significantly, revealing the impact of apoptosis resistance on sustained protein production. While the CHO-WT cells exhibited a substantial decline in MFI (8,366; 51.5% decrease from 24 h), the CHO-BBC13 cells maintained robust expression (30,867.5; only 12.8% decrease from 24 h), resulting in a 3.69-fold higher MFI compared with the WT cells (p < 0.0001). The CHO-BB1 cells also demonstrated superior maintenance of expression (23,233.4; 19.3% decrease from 24 h; 2.78-fold vs. WT cells; p < 0.001). In contrast, the CHO-C2 cells showed a dramatic decline (13,833.9; 62.3% decrease from 24 h), reaching a level comparable to that of the WT cells (p > 0.05) (Fig. 4B). Remarkably, the transfection efficiency of the CHO-BBC13 cells increased substantially to 53.72%, while that of WT cells declined to 2.85%, suggesting that the TKO cells not only maintain higher per-cell expression but also exhibit enhanced cell survival and continued protein production (Fig. 4C).

At 72 h, the differential impact of apoptosis resistance on sustained protein expression became even more pronounced. The CHO-WT cells exhibited near-complete loss of BFP expression (MFI: 1462.6; 91.5% decrease from 24 h), reaching a level consistent with apoptosis-mediated cell death and protein degradation. In contrast, the CHO-BBC13 cells maintained detectable expression (MFI: 1811.1), with an MFI 1.24-fold higher than that of the WT cells (p < 0.05). The CHO-BB1 cells showed the highest relative expression (MFI: 2226.3; 1.52-fold vs. WT cells; p < 0.01), while the CHO-C2 cells declined to levels comparable with those of the WT cells (MFI: 1418.7; p > 0.05) (Fig. 4B). The transfection efficiency at 72 h further underscored the functional survival advantage: The CHO-BBC13 cell line maintained 11.94% BFP-positive cells, whereas the WT line maintained 2.44%, the CHO-BB1 cell line maintained 2.07%, and the CHO-C2 line maintained 1.22% (Fig. 4C).

Collectively, these results demonstrate that individual cells of the CHO-BBC13 TKO cell line possess strong recombinant protein expression capabilities, characterized by both enhanced transfection efficiency and extended protein production duration. The combination of faster proliferation, apoptosis resistance, and sustained metabolic activity in TKO cells creates an optimal cellular environment for high-yield recombinant protein production, confirming the multi-gene knockout strategy as an effective approach for engineering next-generation CHO cell factories.

Discussion

This research offers novel insights into optimizing recombinant protein expression in CHO-K1 cells by targeting key apoptosis regulatory factors. Previous work has shown that apoptosis modulation can optimize growth and metabolism [28]. However, no prior studies have used a triple-knockout strategy to boost anti-apoptotic capacity and improve recombinant protein expression. Here, our comprehensive approach elucidates differences in the individual and combined effects of BAK/BAX/CASP3 knockout, with profound implications for enhancing protein production in a biopharmaceutical context. The findings presented here represent a paradigm shift in how we perceive and manipulate cellular mechanisms to achieve higher yields of recombinant proteins.

Apoptosis, a tightly regulated process, plays a dual role in biopharmaceutical manufacturing [29, 30]. On one hand, it serves as a protective mechanism against cellular stress and damage; on the other hand, it compromises cell viability and protein expression levels [31]. The Bcl-2 family of proteins, including BAK and BAX, along with downstream effectors like CASP3, are central mediators of apoptosis [32, 33]. By targeting these key players simultaneously, we hypothesized that we could modulate the apoptotic pathway to create a more favorable environment for protein production. Consequently, we constructed multiple knockout cell lines, including CHO-TKO (BAK/BAX/CASP3), DKO (BAK/BAX), and SKO (CASP3) cell lines, which allowed us to systematically evaluate the effects of apoptosis inhibition at different levels. The TKO cell lines emerged as the most promising candidates, exhibiting significantly higher cell viability, prolonged doubling time, and enhanced resistance to apoptosis. These characteristics directly translated to improved expression levels of recombinant BFP, outperforming both wild-type and other knockout lines. The enhanced performance of the TKO cell lines could potentially be attributed to the synergistic effects of BAK, BAX, and CASP3 knockouts. BAK and BAX are essential for MOMP, a critical step in apoptosis [34, 35]. Therefore, our results are consistent with the possibility that BAK/BAX knockout effectively blocked the intrinsic apoptotic pathway via suppression of MOMP, while CASP3 knockout further amplified these effects by eliminating the executioner caspase responsible for cell disassembly and death.

Our comprehensive analyses included the assessment of cell viability, doubling time, cell cycle, apoptosis, and MOMP, thus providing a detailed understanding of the cellular responses to BAK/BAX/CASP3 knockout. The DKO and TKO cell lines demonstrated a remarkable ability to resist apoptosis, as evidenced by the significantly lower levels of PI/Annexin V staining at both 24-h and 48-h under puromycin-induced stress conditions. However, the growth dynamics and metabolic profiles of the TKO and DKO cell lines suggest distinct characteristics that could be leveraged for specific production goals. For example, the DKO cell line exhibited a slightly delayed growth pattern but maintained high viability levels throughout the experimental period, while the triple knockout cell lines demonstrated robust growth and metabolic activity, making them ideal candidates for large-scale protein production.

Our cell cycle distribution analysis results provided additional valuable insights into the non-canonical roles of BAK, BAX, and CASP3 in cell cycle surveillance. CASP3 knockout triggered a significant accumulation of S-phase cells, suggesting an extended window for recombinant protein transcription and translation. This phenotype was further exaggerated under puromycin-induced ribosomal stress, highlighting the potential of CASP3 knockout to enhance translational resilience. S-phase prolongation, driven by CASP3’s role in restraining replication stress, correlates with an extended window for recombinant protein transcription and translation, and S-phase is a critical stage for active mRNA synthesis and ribosome engagement [36, 37]. In contrast, our results suggest that BAK/BAX ablation induces constitutive G2/M accumulation, which could favor prolonged post-translational modification (e.g., glycosylation, folding) of secreted proteins, as mitotic exit delays extend the endoplasmic reticulum processing window [38]. The hierarchical regulation of the ΔΨm across these engineered CHO-K1 knockout lines further suggests the fundamental role of BAK and BAX in apoptotic signaling, highlighting the potential of their knockout to preserve functional mitochondrial integrity under stress.

Most importantly, our BFP protein expression analysis provided compelling evidence of the differential regulation of protein expression efficiency governed by BAK, BAX, and CASP3. The CHO-BBC13 TKO cell line exhibited a dramatic surge in mean fluorescence intensity and fluorescence rate, highlighting its superior protein production capabilities. This sustained expression of BFP aligns with MOMP stabilization in BAK/BAX-deficient cells, suggesting that delayed apoptotic signaling prolongs plasmid-driven protein synthesis.

Conclusion

This study presents a comprehensive evaluation of the effects of BAK, BAX, and CASP3 knockouts on CHO-K1 cell characteristics and recombinant protein expression, which provides a foundation for engineering apoptosis-resistant cell lines with enhanced protein production capacity. The KO cell lines demonstrated significant improvements in cell viability, resistance to apoptosis, and recombinant protein expression capability. The TKO cell CHO-BBC13, in particular, exhibited enhanced cell viability, shortened doubling time, and resistance to apoptosis, which resulted in improved stability and yield of recombinant protein expression. These findings contribute to the fundamental understanding of apoptosis regulation in CHO cells, with practical implications for the development of more efficient and robust CHO cell lines for biopharmaceutical applications. The insights gained from this study could pave the way for innovative strategies to optimize protein production, ultimately benefiting the biopharmaceutical industry by improving the efficiency and scalability of therapeutic proteins.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (136.6KB, doc)
Supplementary Material 2 (3.5MB, doc)

Abbreviations

BAK

BCL2 antagonist/killer

BAX

BCL2 associated X, apoptosis regulator

BFP

Blue fluorescent protein

CCCP

Carbonyl cyanide m-chlorophenylhydrazone

CASP3

Caspase-3

CCK-8

Cell Counting Kit-8

CHO

Chinese hamster ovary

CRISPR

Clustered regularly interspaced short palindromic repeats

DKO

Double knockout

DMEM

Dulbecco’s Modified Eagle Medium

DT

Doubling time

EGFP

Enhanced green fluorescent protein

FACS

Fluorescence-activated cell sorting

MFI

Mean fluorescence intensity

MOMP

Mitochondrial outer membrane permeabilization

PCR

Polymerase chain reaction

PI

Propidium iodide

PBS

Phosphate-buffered saline

SKO

Single knockout

sgRNA

Single guide RNA

TKO

Triple knockout

TMRE

Tetramethylrhodamine ethyl ester

WT

Wild type

ΔΨm

Mitochondrial membrane potential

Author contributions

Conceptualization, investigation, reviewing and revising papers, WW, QS, RN, CC, and FZ; data curation, formal analysis, writing – original draft, XG, ZX, YM and ZG; investigation, writing – original draft, YW and YC. All authors have read and agreed to the published version of the manuscript.

Funding

This research was sponsored by The National Natural Science Foundation of China (NSFC) with grant number (32260879); Natural Science Foundation of Inner Mongolia Autonomous Region with grant number (2025ZD016). The National Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock (Jointly Built by the Province and Ministry) - Identification of Specific Target Points for Important Pathogens in Cattle and Sheep and Development of Novel Diagnostic Technologies (2025KYPT0066).

Data availability

Cell lines generated in this study are available upon request.

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (136.6KB, doc)
Supplementary Material 2 (3.5MB, doc)

Data Availability Statement

Cell lines generated in this study are available upon request.

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