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. 2026 Sep 14;11(38):57034–57045. doi: 10.1021/acsomega.6c04206

A CS•CBE System Targeting β‑Catenin: Exploring Additional Inhibition Targets for Colon Cancer Proliferation

Min Li †,‡, Qian Ban ‡, Dahai Liu §, Bingang Li ‡, Jintang Xu ‡, Jiaqi Geng ‡, Dakun Chen ‡, Wenqiao Hui †,*
PMCID: PMC13628616  PMID: 42825015

Abstract

Colon cancer ranks among the most prevalent malignant tumors worldwide, with its incidence rising progressively year by year. The identification of novel research tool for therapeutic targets has thus become an urgent priority. Here, we constructed a CS•CBE integrative delivery and editing system via chitosan and cytosine base editor (CBE), which could successfully target and inhibit the phosphorylation of β-catenin. In vitro studies demonstrated that this system achieved a high delivery efficiency of 86.0% and an editing efficiency of 78% in HCT116 cells, and Transwell and wound-healing assays verified that the CBE-β-catenin-HCT116 cells exhibited an increased proliferation rate. Furthermore, a xenograft tumor model was established in nude mice, and we observed that the tumor volume and weight in the experimental group were both significantly increased compared with those in the control group. Subsequently, the candidate genes identified from transcriptomic results, such as AXIN2 and GPR15, which are downstream of β-catenin pathways, validated the feasibility of the CS•CBE system as a research tool. Collectively, our CS•CBE system serves as a valuable research tool for targeted protein modulation, downstream target identification, and probe specific post-translational modifications analysis in colon cancer research.

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1. Introduction

Colon cancer is the third most common cancer worldwide, with an incidence rate only second to lung cancer and breast cancer. − It has become a significant contributor to the mortality caused by malignancies. The pathogenesis of colon cancer is complex; however, early screening and intervention can significantly enhance the cure rates. Therefore, research on its etiology and prevention strategies is vital for public health.

Current treatment methods primarily include surgical resection, chemotherapy, , and targeted therapy. Common targeted agents include bevacizumab − and cetuximab. , While targeted therapies generally have fewer side effects, many of these agents are not suitable for all patients. Consequently, this study proposes a new targeted site to provide further possibilities for targeted drug therapy.

β-catenin is a multifunctional oncogene that regulates cell proliferation, differentiation, and apoptosis. It is well established that the aberrant accumulation of β-catenin drives the uncontrolled proliferation of tumor cells. Under physiological conditions, β-catenin undergoes phosphorylation at the Ser33 locus followed by degradation. Inhibiting this specific phosphorylation can induce the aberrant accumulation of β-catenin and thereby promotes colon tumorigenesis. − Therefore, targeted modification of the Ser33 residue is a necessary focal point for advancing colon cancer research.

Cytosine base editing (CBE) is a precise gene-editing technology that enables specific single-base substitutions by mediating the targeted conversion of cytosine (C) to thymine (T) in DNA sequences. Since CBE does not induce double-stranded DNA breaks (DSBs), − it substantially reduces various erroneous repair events and thus achieves more accurate gene editing. Based on this, we utilized CBE to modify the Ser33 site of β-catenin, converting the original serine codon (TCT) to a phenylalanine codon (TTT) via C•G to T•A substitution, which renders the protein undetectable and refractory to degradation.

Although CBE enables efficient gene editing, delivery efficiency remains a critical limiting factor. Conventional electroporation compromises cell viability, viral vectors pose inherent biosafety concerns, and liposomes exhibit suboptimal delivery efficacy. There is a critical need for efficient alternative nanomaterial delivery platforms.

Chitosan is a natural product that exhibits good biocompatibility and biodegradability, making it relatively safe. , The cationic nature of chitosan allows it to form stable complexes with negatively charged nucleic acids. Therefore, we selected chitosan as a safe alternative delivery vector to achieve a higher delivery efficiency.

Based on this, we established an integrative delivery and editing system via chitosan (CS) and cytosine base editor (CBE), named CS•CBE system, to mediate the targeted modification of β-catenin. We first utilized this system in vitro (HCT116 cells) to investigate the increased cellular proliferation and migration induced by the excessive accumulation of β-catenin. After verifying the biosafety of chitosan-mediated transfection, we performed tumorigenesis assays in nude mice in vivo and identified two target loci for the research of colon cancer through subsequent analyses. (Schematic illustration was shown in Figure .) We hope our study could provide a novel research tool for the targeted protein modulation and downstream target identification in the colon cancer research.

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Schematic illustration of our approach.

An integrative CS•CBE delivery and editing system was constructed via the complexation of chitosan (CS) with cytosine base editor (CBE), which induced β-catenin C → T mutations in HCT116 colon cancer cells, followed by in vivo tumor formation and analysis to evaluate the impact of the editing on tumor progression.

2. Materials and Methods

2.1. Materials

Dulbecco’s Modified Eagle Medium (DMEM), nonessential amino acid solution, phosphate-buffered saline (PBS), fetal bovine serum (FBS), β-mercaptoethanol, penicillin/streptomycin (PS, 10,000 U/mL), and trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) were purchased from Gibco (Waltham, MA, USA). Chitosan and 1% sodium pentobarbital were obtained from Merck KGaA (Darmstadt, Germany). Human colon cancer cell line (HCT116/SW480) was obtained from the American Type Culture Collection (Manassas, VA, USA). Liposome was purchased from Beyotime Biotechnology called Lipo8000 Transfection Reagent (C0533, Shanghai, China). Cell Counting Kit-8 (CCK-8) Plus was purchased from APExBIO (Houston, TX, USA). Methanol and crystal violet were purchased from Abcam (Cambridge, UK). Total RNA Extractor Kit (Trizol) was purchased from Sangon Biotech (Shanghai, China).

2.2. Cell Culture

HCT116 and SW480 were obtained from the Stem Cell and Translational Medicine Research Center at Anhui University. Cells were cultured in MES medium, which was formulated by supplementing DMEM medium with 1% non-essential amino acid solution, 1% penicillin/streptomycin mixture, 10% FBS, and 0.1% β-mercaptoethanol. A total of 2 × 105 cells were seeded into each well of a 6-well plate, followed by the addition of 2 mL of culture medium per well. Cells were maintained in a humidified incubator with 5% CO2 at 37 °C.

2.3. Plasmid Construction

Two plasmids were constructed, namely β-catenin-CBE and β-catenin-sgRNA-mCherry. The latter harbored an mCherry tag, a red fluorescent marker that emits detectable red fluorescence under a fluorescence microscope, thus facilitating subsequent experimental detection. The maps of both plasmids are presented in Figure S1.

2.4. Preparation of Chitosan Nanoparticles

Chitosan (2 mg, 4 mg, 6 mg, and 8 mg) samples were dissolved individually in 20 mL of 5 mM sodium acetate buffer, yielding chitosan solutions with concentrations of 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, and 0.4 mg/mL. The pH values of the solutions were adjusted to 5.5, followed by preheating in a 55 °C water bath for 15 min.

Then, the two plasmids were divided into four groups as follows:

Group A: 2 μg sgRNA + 2 μg CBE

Group B: 1.5 μg sgRNA + 2 μg CBE

Group C: 2 μg sgRNA + 1.5 μg CBE

Group D: 1.5 μg sgRNA + 1.5 μg CBE

(Note: For the physicochemical characterization of chitosan nanoparticles, plasmids from Group A were used for encapsulation.)

Subsequently, the plasmids were added to 500 μL of chitosan solution, vigorously vortexed for 30 s, and incubated for 30 min to form nanoparticles with a nitrogen/phosphorus (N/P) ratio ranging from 1 to 20.

Dynamic light scattering (DLS) measurements of the synthesized chitosan-plasmid nanoparticles were performed using a Zetasizer Lab (Malvern, England). Nanoparticles formed by encapsulating plasmids with chitosan at different concentrations were loaded into disposable cuvettes for the measurements. Scanning electron microscopy (SEM) imaging was conducted using a Supra 40VP SEM instrument (Zeiss, Germany). Chitosan nanoparticles were cast onto a silicon wafer, air-dried overnight at room temperature, and sputter-coated with gold (Ted Pella, USA). The morphology of the nanoparticles was then observed and recorded at multiple magnifications under SEM.

2.5. Chitosan Delivery

When the cell confluency in 6-well plates reached 70%, the culture medium was aspirated and replaced with 2 mL of serum-free DMEM per well. The plates were incubated in a cell culture incubator (37 °C, 5% CO2) for 3 h to induce cell starvation. After starvation, the serum-free DMEM was replaced with 2 mL of MES medium per well, and 5 μL of the pre-prepared chitosan/plasmid mixture was added to each well for co-cultivation over 6 h. Finally, the medium was discarded, and 2 mL of fresh MES medium was added to each well, with the plates returned to the incubator for further culture under the same conditions (37 °C, 5% CO2).

2.6. Liposome Delivery

Upon reaching 70% confluency in 6-well plates, 500 μL of DMEM medium was mixed with 8 μL of LIP transfection reagent and incubated at room temperature for 5 min. Subsequently, 1 μg β-catenin-sgRNA-mCherry plasmids and 1 μg β-catenin-CBE plasmids were added to the mixture, followed by incubation at room temperature for 20 min in the dark. The spent medium in the culture plates was aspirated and discarded. Then, 2 mL of DMEM medium and 500 μL of the transfection mixture were added to each well for a 6 h co-cultivation period. Afterward, the medium was discarded, 2 mL of MES medium was replenished per well, and the plates were placed back into the incubator (37 °C, 5% CO2) for continuous culture.

2.7. Flow Cytometry

Transfected cell suspensions (both chitosan- and liposome-mediated groups) were harvested, with untransfected HCT116 cells serving as the blank control group. Cells were centrifuged at 1000 × g for 3 min, and the resulting cell pellets were resuspended in 500 μL of 1 × PBS and adjusted to a concentration of 1 × 106 cells/mL. The cell suspensions were transferred to flow cytometry tubes, and transfection efficiency was measured using a BD FACS Calibur flow cytometer (BD Biosciences, USA). Owing to the mCherry red fluorescent tag carried by the plasmids, transfected cells could be discriminated and sorted. Fluorescence-based transfection efficiency was analyzed using FlowJo v10 software, and corresponding histograms were generated.

2.8. PCR and Sanger Sequencing

Transfected cell suspensions from the two delivery groups were collected, and genomic DNA was extracted using a TIANGEN genomic DNA extraction kit following the manufacturer’s instructions. The PCR reaction system (50 μL total volume) was composed of 1 μL Phanta Max Super-Fidelity DNA Polymerase, 25 μL 2× Phanta Max buffer, 2 μL 10 μM primer mix, 1 μL 10 mM dNTP mixture, 2 μL genomic DNA template, and ddH2O to make up the final volume. The forward primer was 5′-ATGGCTACTCAAGCTGATTTGATG-3′ and the reverse primer was 5′-TGAGAAAATCCCTGTTCCCAC-3. The PCR amplification program was set as follows: initial denaturation at 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 15 s, annealing at 52 °C for 15 s, and extension at 72 °C for 30 s; and a final extension step at 72 °C for 5 min. The PCR products were then stored at 4 °C.

PCR products were separated via electrophoresis on 1.5% agarose gel. Target bands were excised from the gels and sent to Sangon Biotech (Shanghai) Co., Ltd., for Sanger sequencing.

2.9. Monoclonal Cell Line Selection and Validation

CBE plasmids and sgRNAs were delivered into HCT116 cells. After 48 h of culture, cells exhibiting robust growth were selected for further processing. The cells were subjected to 1:10 serial gradient dilution, and on the third day postdilution, the diluted cell suspensions were seeded into 96-well plates at a density of one cell per well. During the incubation period, the formation of mCherry-positive clones was monitored daily with a fluorescence microscope (Leica Microsystems, Germany).

At 7 days after delivery, distinct red-fluorescent clones were carefully picked using a sterile pipette tip and transferred to new culture dishes for expansion culture. After expansion, genomic DNA was extracted from the cloned cells, and Sanger sequencing was performed to evaluate the editing efficiency. Clones with 100% editing efficiency were deemed qualified and designated as CBE-β-catenin-HCT116 cells. Qualified monoclonal cell lines were cryopreserved for long-term storage and subsequent experimental use.

2.10. CCK-8 Proliferation Assay

Untreated HCT116 cells were used as the wild-type control group (WT), while CBE-β-catenin-HCT116 cells served as the experimental group (MT). 100 μL of cell suspension in MES medium was seeded into each well of a 96-well plate at a density of 2000 cells per well. The 96-well plate was incubated in a humidified incubator (37 °C, 5% CO2) for 90 min. After incubation, 10 μL of CCK-8 solution (APExBIO) was added to each well, followed by further 1 h incubation. The absorbance at 450 nm was measured using a SpectraMax i3x microplate reader (Molecular Devices) and recorded as the 0 h time point, with three technical replicates per group.

Subsequently, 10 μL of CCK-8 solution was added to each well at 12, 24, 48, and 72 h post-seeding, with a 1 h incubation period after each addition. The absorbance values at each time point were measured and recorded. A cell proliferation curve was plotted using GraphPad Prism 8.

2.11. Wound Healing Assay

When CBE-β-catenin-HCT116 cells reached 90% confluency in culture dishes, parallel linear scratches were made on the cell monolayer using a sterile 10 μL pipette tip. The dishes were rinsed gently with PBS to remove detached cells, and fresh cell culture medium was added. The scratch area was photographed under a microscope (Leica Microsystems, Germany) and recorded as the 0 h time point. The culture dishes were then returned to the incubator, and images of the scratch area were captured again at 24 and 48 h post-scratching. The wound healing rate was quantified using ImageJ software.

2.12. Transwell Migration Assay

The migratory capacity of CBE-β-catenin-HCT116 cells was evaluated using a 24-well Transwell chamber system. The lower chamber was filled with 600 μL of DMEM supplemented with 20% fetal bovine serum, while the upper chamber was loaded with 8 × 104 cells resuspended in 200 μL of serum-free DMEM. The Transwell chambers were incubated in a cell culture incubator for 48 h.

After incubation, the Transwell inserts were removed, and the culture medium was discarded. Non-migratory cells on the upper surface of the membrane were gently wiped off with a cotton swab, followed by three gentle rinses with PBS. Then, 600 μL of methanol was added to each well for 15 min at room temperature to fix the migrated cells. The methanol was aspirated, and the wells were rinsed three times with PBS. Subsequently, 600 μL of 0.5% crystal violet staining solution (in methanol) was added to each well for 15 min. Finally, the staining solution was discarded, and the wells were rinsed thoroughly with PBS, air-dried at room temperature, and imaged under a microscope (Leica Microsystems, Germany).

2.13. RNA Sequencing

Total RNA was extracted from the CBE-β-catenin-HCT116 cells and the HCT116 cells. DNase I, free of RNase, was used to eliminate genomic DNA contamination. The RNA samples were then submitted to Sangon Biotech (Shanghai) Co., Ltd. for preparation and RNA sequencing.

The extracted RNA was quality evaluated with FastQC and trimmed via Trimmomatic to obtain high-quality clean reads, which were aligned to the reference genome by using HISAT2. Mapping metrics were analyzed with RSeQC (for redundancy and insert size distribution), Qualimap (for uniformity and genomic structure distribution), and BEDTools (for gene coverage and chromosomal read distribution). Gene structural analyses included SNP calling and annotation with BCFtools and SnpEff, transcript assembly and novel region identification with StringTie and GffCompare, alternative splicing analysis with ASprofile, and fusion gene detection with EricScript. Gene expression levels were quantified using StringTie, followed by weighted gene co-expression network analysis (WGCNA) and multidimensional sample-wise statistical exploration based on the gene expression matrix. Differentially expressed genes (DEGs) were identified via DESeq2, with results visualized as heat maps, Venn diagrams, and cluster analyses; DEG-encoded proteins were mapped to the STRING database for protein–protein interaction network construction. Functional enrichment analyses of DEGs were performed using topGO for gene ontology (GO) terms and clusterProfiler for KEGG pathways and KOG classifications, with results visualized as directed acyclic graphs and functional association networks.

2.14. Construction of Nude Mice Bearing Tumor Model

CBE-β-catenin-HCT116 cells in the logarithmic growth phase were digested with 0.25% trypsin and adjusted to a concentration of 2 × 107 cells/mL. A 1 mL aliquot of the cell suspension was centrifuged at 800 rpm for 3 min; the supernatant was discarded, and the cell pellet was collected. The cells were resuspended in 150 μL of normal saline and stored on ice. Nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Five-week-old male mice were selected and housed in the specific pathogen-free (SPF) immunocompromised mouse facility at Anhui University. Eight nude mice were randomly divided into two groups: a control group (WT) and a chitosan injection experimental group (MT), with four mice per group.

To immobilize the mice, the skin between the ears was held (palm facing upward), while gentle pressure was applied to the base of the tail. Prior to injection, the cell suspension was gently aspirated with a micropipette to ensure homogeneity, and 150 μL of the suspension was drawn into a 1 mL syringe. The needle was inserted slightly above the flank of the mouse, and the cell suspension was slowly injected into the axillary subcutaneous region. After inoculation, the health status of the nude mice was closely monitored, and palpable tumors developed after 10 days.

2.15. Hematoxylin and Eosin (H&E) and Immunohistochemistry (IHC) Staining

The mouse was restrained with the left hand, with its head oriented downward, and then 150 μL of 1% sodium pentobarbital was intraperitoneally injected using a syringe held in the right hand. Anesthesia was confirmed via a toe-pinch reflex test. Upon successful anesthesia, the mouse was euthanized by cervical dislocation. Tumors were excised subsequently, with the surface skin and adherent nontumor tissues carefully removed. Tumor weight and volume from each group were measured and documented precisely. Each tumor was then divided into two aliquots: one was fixed in 4% paraformaldehyde solution, and the other was preserved in RNA storage solution (Mei5 Biotech, MF150-01).

Thereafter, tumor tissues fixed for 24 h were thoroughly rinsed and subjected to graded ethanol dehydration at 37 °C. After complete dehydration, samples were cleared in xylene and then embedded in molten paraffin wax. Once cooled, the solidified paraffin blocks were sectioned into 5-μm-thick slices using a microtome. The sections were mounted on glass slides and dried in a 65 °C oven.

For hematoxylin–eosin (H&E) staining, sections were rehydrated through a graded ethanol series and rinsed gently with distilled water. The sections were stained with a hematoxylin solution for 3 min and rinsed thoroughly with water after staining. Subsequently, eosin staining was performed for counterstaining, followed by another water rinse. Finally, sections were dehydrated with gradient ethanol, cleared in xylene, and mounted with a neutral mounting medium. Tumor tissue morphology and structural features were then observed under a light microscope (Leica Microsystems).

For immunohistochemistry (IHC), 5-μm-thick paraffin sections were baked at 60 °C for 30 min to eliminate residual moisture and enhance section adhesion to slides. The sections were then dewaxed and rehydrated using a graded ethanol series. Nonspecific binding was blocked by incubating the sections with 3% bovine serum albumin (BSA) blocking buffer. Sections were then incubated with specific primary antibodies at 37 °C for 30 min to allow antigen–antibody binding. After washing, a universal horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG secondary antibody complex was applied and incubated at 37 °C for 20 min. Following another round of washing, 3,3’-diaminobenzidine (DAB) chromogenic substrate was added. Once positive signals appeared, sections were mounted with coverslips, and staining results were observed under a microscope (Leica Microsystems, Germany).

Additionally, tumors from the experimental and control groups were sent to Sangon Biotech (Shanghai) Co., Ltd. for RNA sequencing, with procedures performed as described in Section .

2.16. Statistical Methods

The data were expressed as mean ± standard deviation (SD). Statistical significance was analyzed by one-way ANOVA and Student’s t-test using GraphPad Prism 7 software. Statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001.

3. Results

3.1. Synthesis and Characterization of Chitosan

To establish the integrative CS•CBE system for efficient delivery and precise editing performance, which facilitated the exploration of the regulatory relationship between β-catenin and colon cancer progression, we first conducted a study on the chitosan delivery system. By using an ion-crosslinking method, chitosan-coated plasmids were formulated into nanoparticles. To begin with, the physical characterization of the prepared nanoparticles was observed.

Figure A shows that the nanoparticles formed by encapsulation with chitosan at different concentrations exhibited a spherical morphology, as observed by scanning electron microscopy (SEM). Figure B,C presents the particle diameter and polydispersity index (PDI) measured via dynamic light scattering (DLS). The particle size of the nanoparticles varied with the change in the chitosan concentration, with all sizes being almost 100 nm. The ζ-potential of the nanoparticles we measured was around 2 mV. However, when the chitosan concentration reached 0.2 and 0.3 mg/mL, the nanoparticles showed a relatively high polydispersity index (PDI = 0.4377) (Figure D), indicating that excessively high chitosan concentrations tended to induce nanoparticle aggregation. In contrast, a chitosan concentration of 0.1 mg/mL resulted in a low delivery efficiency (44.1%). Therefore, chitosan at a concentration of 0.2 mg/mL was selected for DNA encapsulation in the subsequent experiments.

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Physicochemical characterization of chitosan nanoparticles prepared at different concentrations. A: Scanning electron microscopy (SEM) images of chitosan nanoparticles synthesized at concentrations of 0.1–0.4 μg/mL, showing particle morphology and surface characteristics (scale bar: 100 nm). B: Particle size distribution curves of chitosan nanoparticles at each concentration, as measured by dynamic light scattering (DLS). C: Quantitative analysis of nanoparticle size at different chitosan concentrations. D: Polydispersity index (PDI) of chitosan nanoparticles at varying concentrations.

Meanwhile, we constructed the β-catenin-CBE plasmids using Cytosine Base Editor (CBE) editing, which specifically targets and edits the Ser33 site of β-catenin. The plasmid maps are provided in Figure S1.

3.2. Chitosan Outperforms Liposomes in Delivery and Editing Efficiencies

3.2.1. Enhanced Chitosan Delivery Efficiency with 2 μg β-Catenin-sgRna-mCherry Plasmids and 1.5 μg β-Catenin-CBE Plasmids

Following the determination of 0.2 mg/mL as the optimal chitosan concentration for carrier preparation, we further determined the optimal transfection time for the prepared chitosan. Prior to transfection, the HCT116 cells were revived and cultured. We performed transfection using group A plasmid as a representative. After transfection, we measured the percentage of fluorescent cells at five time points: 12, 24, 36, 48, and 60 h. We found that the percentage of fluorescent cells was highest at 48 h posttransfection (Figure S2). Therefore, subsequent experiments will use 48 h posttransfection as the time point for fluorescence observation.

Next, we turn attention to investigate the plasmid dosage that yields the highest delivery efficiency. HCT116 cells were subjected to recovery and culture prior to delivery. The four groups of plasmids were individually delivered into HCT116 cells via chitosan. After 48 h of incubation, fluorescence efficiency was visualized (Figure A) and quantitatively analyzed by flow cytometry (Figure B). The results demonstrated that the combination of 2 μg β-catenin-sgRNA-mCherry plasmids and 1.5 μg β-catenin-CBE plasmids resulted in the strongest fluorescence intensity and the highest delivery efficiency, reaching 81.9%.

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Chitosan delivery achieves highly delivery efficiency than that of conventional liposomes in HCT116 cells. A: Fluorescence microscopy images of HCT116 cells treated with chitosan delivering CBE plasmids and sgRNA at different groups, showing HCT116 cell morphology (bright field), mCherry fluorescence signal (red), and merged images (scale bar: 100 μm). B: Flow cytometry quantification of delivery efficiency in HCT116 cells at different groups, with positive cell percentages labeled in each histogram (Group A: 44.1%, Group B: 81.9%, Group C: 63.3%, Group D: 32.6%). C: Fluorescence microscopy images of HCT116 cells delivered with CBE plasmids via chitosan (CS) or liposome (Lipo), showing HCT116 cell morphology (bright field), mCherry fluorescence signal (red), and merged images (scale bar: 100 μm). D: Flow cytometry quantification of delivery efficiency in HCT116 cells with chitosan- or liposome-mediated CBE plasmid delivery (CS: 89.0%, Lipo: 66.7%). E: Sanger sequencing analysis of β-catenin editing efficiency at the target locus in HCT116 cells after chitosan or liposome delivery, with the editing efficiency indicated as C-to-T conversion rates (CS: C 22%, T 78%; Lipo: C 58%, T 42%).

Accordingly, the CS•CBE integrative delivery and editing system was constructed using 0.2 mg/mL chitosan solution, 2 μg β-catenin-sgRNA-mCherry plasmids, and 1.5 μg β-catenin-CBE plasmids for subsequent experiments.

3.2.2. Chitosan Outperforms Liposomes in Delivery and Editing Efficiencies

Having established the CS•CBE system, we then headed to investigate its delivery and editing efficiency by using liposomes as the control.

Plasmids were separately delivered into HCT116 and SW480 cells via chitosan and liposome carriers. Fluorescence intensity was visualized and compared between the two groups under a fluorescence microscope at 48 h (Figures C and S3A), with the findings revealing similar initial delivery efficiencies. For a more precise comparison, flow cytometry assays were implemented (Figures D and S3B). This approach quantifies the transfection efficiency by detecting fluorescent signals derived from the interaction of fluorescently labeled antibodies with target cell-associated molecules. The flow cytometry data demonstrated a transfection efficiency of 86.0% (HCT-116) and 73.2% (SW480) in the chitosan group (CS) versus 66.7% (HCT-116) and 60.9% (SW480) in the liposome group (Lipo), confirming the superior plasmid delivery efficiency of chitosan over liposomes.

Cell suspensions from both groups were harvested, and genomic DNA was extracted. The target gene fragment was amplified via polymerase chain reaction (PCR), followed by nucleotide sequence analysis using Sanger sequencing (Figures E and S3C). The results demonstrated that the chitosan-mediated C-to-T mutation rates reached 78% (HCT-116) and 82% (SW480), which were markedly higher than the 42% (HCT-116) and 56% (SW480) rate achieved with liposome-mediated delivery, thus further validating the superior β-catenin editing efficiency of chitosan.

At last, we evaluated the viability of HCT116 and SW480 cells after transfection by using the CCK-8 assay. The optical density (OD) values were measured at five distinct time points. The results of the CCK-8 proliferation assay showed that, following transfection, the survival rates of HCT-116 and SW480 cells first decreased and then increased over time, indicating that this transfection protocol slightly inhibited cell activity in the early stages of transfection but that the activity gradually returned to normal over time. This suggests that the cytotoxic effect caused by the CS transfection method we used is transient and that cells can recover their normal activity approximately 60 h after transfection (Figure S3D).

3.3. Chitosan Delivery Does Not Affect Cell Viability and Promotes Cell Migration and Invasion

Upon the successful establishment of our highly efficient delivery system, we then proceeded to detect its safety. Monoclonal cell lines exhibiting 100% editing efficiency were screened. First, monoclonal cell lines were obtained through limiting dilution, and their genomic DNA was extracted. The target fragment was amplified by PCR and identified via agarose gel electrophoresis. Cell lines with a 100% editing efficiency, as determined by Sanger sequencing, were defined as qualified monoclonal cell line CBE-β-catenin-HCT116 (Figure A).

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Editing of β-catenin in HCT116 cells does not reduce cell viability but enhances migration and invasion capacities. A: Screening of monoclonal cell lines with 100% editing efficiency at the β-catenin Ser33 locus. B: CCK8 assay for evaluating the viability of wild-type (WT) HCT116 cells and CBE-β-catenin-HCT116 cells (MT) at 0 h, 12 h, 24 h, 48 h, and 72 h. C: Wound healing assay for detecting the migratory ability of WT and MT cells at 0 h, 24 h, and 48 h, with the right panel showing the quantitative analysis of migration area (scale bar: 100 μm). D: Transwell assay for determining the invasive potential of WT and MT cells (representative images of invasive cells stained with crystal violet) (scale bar: 100 μm). E: Transcriptome sequencing analysis revealing differentially expressed genes between CBE-β-catenin-HCT116 cells (MT) and WT cells (red: upregulated genes, green: downregulated genes, black: non-differentially expressed genes).

Second, we evaluated the viability of CBE-β-catenin-HCT116 cells by using the CCK-8 assay. The optical density (OD) values were measured at five distinct time points. Compared with the control group, the experimental group exhibited a slight but non-significant reduction in OD values (P>0.05), demonstrating that chitosan-mediated transfection exerts negligible effects on cell viability (Figure B).

Given that unphosphorylated β-catenin accumulation can modulate cell growth, we further performed wound healing assays and Transwell assays (Figure D) to assess the impact of β-catenin modification on the migratory and invasive potential of HCT116 cells. In the wound healing assay, the scratch closure was observed and documented under a microscope at 0, 24, and 48 h. Results showed that the scratch healing rate of the experimental group was significantly higher than that of the control group (Figure C). Consistently, in the Transwell assay, the number of migrated cells in the experimental group was markedly greater than that in the control group when counted in the same microscopic field at 48 h post-seeding (Figure D). The concordant results of these two assays validate that the inhibition of β-catenin accumulation can enhance the proliferation, migration, and invasion of colon cancer cells, thereby accelerating tumor progression.

Additionally, transcriptomic profiling was conducted on the monoclonal cell line and the parental HCT116 cells. A scatter plot depicting differential gene expression between the two groups (Figure E) revealed significant transcriptional divergence.

Taken together, these results indicate that the delivery of β-catenin plasmids using the CS•CBE system exerts no significant effect on HCT116 cell viability but enhances cell proliferation and migration rates.

3.4. Construction of Nude Mice Bearing Tumor Model

After validating the regulatory effect of the CS•CBE system on HCT116 cells in vitro, we further established a nude mouse tumor-bearing model to edit the β-catenin gene in vivo using this system. This approach allowed us to investigate the correlation between β-catenin editing and tumor proliferation, thereby identifying novel potential therapeutic targets for colon cancer.

3.4.1. CBE-β-Catenin-HCT116 Group Exhibits Larger Tumor Volume, Higher Weight, and Faster Proliferation Rate Than Control Group

First, we subcutaneously inoculated nude mice in the control and experimental groups with 2 × 107 HCT116 cells and CBE-β-catenin-HCT116 cells, respectively. Ten days post-inoculation, the xenograft tumors were excised, and their weights and volumes were measured and documented (Figure B,C). The results demonstrated that tumors derived from the CBE-β-catenin-HCT116 group exhibited significantly increased weight and volume compared with the control group, indicating that excessive β-catenin accumulation promotes tumor progression.

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CBE-β-catenin-HCT116 cells exhibit enhanced tumorigenic potential in nude mouse xenograft models. A: Procedure for cancer cell injection. B: Representative images of tumors derived from HCT116 cells (WT) and CBE-β-catenin-HCT116 cells (MT) (scale bar: ruler with centimeter markings), with quantitative analysis of tumor volume (left) and tumor weight (right) shown in the bar graphs (n = 3; data presented as mean ± SD). C: Hematoxylin and eosin (H&E) staining of tumor tissues from WT and MT groups, showing histological characteristics of tumor tissues (scale bar: 100 μm). D: Immunohistochemical (IHC) staining of tumor tissues from WT and MT groups (scale bar: 100 μm).

It is acknowledged that tumor cell nuclei are often enlarged and irregularly shaped. Then, we fixed tumor sections from both groups and performed HE staining (Figure D) and immunohistochemical analysis (Figure E). After HE staining, the nuclei appeared blue-purple in color and the cytoplasm appeared pink. Microscopic observations showed that the CBE-β-catenin-HCT116 group had deeper staining and irregularly distributed nuclei compared to the control group. Additionally, KI67 expression was increased in the CBE-β-catenin-HCT116 group. This further validates the conclusion that in the CBE-β-catenin-HCT116 group, the excessive accumulation of β-catenin causes abnormal activation of the Wnt/β-catenin signaling pathway, resulting in a faster tumor proliferation rate compared to the control group.

3.4.2. Two Potential Target Sites Revealed by RNA Sequencing

To identify target genes associated with colon cancer, we conducted an RNA sequencing analysis on the two groups of tumors preserved in RNA preservation solution.

First, differentially expressed genes (DEGs) between the two groups were screened. The screening criteria were |log2 Fold Change| ≥ 1 and p < 0.05. A total of 7024 DEGs were identified (Figure A), among which the top 20 genes with the most significant differential expression were selected for subsequent analysis (Figure B). Compared with the control group, 11 genes showed upregulated expression, including AXIN2, SPTSSB, ZNF704, TMEM98, GPR15, C9orf3, PROM2, CEMIP, INPP4B, ABCC2, and KRT23, whereas 9 genes exhibited downregulated expression, including STC2, CD44, HILPDA, WNT16, ITGB, TNFRSF6B, DDIT4, NFKB2, and SDC4.

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RNA-seq profiling of nude mouse xenograft tumors from CBE-β-catenin- HCT-116 cells. A: Volcano plot displays differentially expressed genes (DEGs) between wild type cells and β-catenin-mutant. B: Heatmap shows the expression profile of the top 20 most significantly altered DEGs. C,D: GO and KEGG pathway enrichment analysis of DEGs was presented as a bubble plot, with bubble size indicating gene count and color representing the adjusted p value. E,F: GO and KEGG functional classification histograms of DEGs were constructed, with the y-axis denoting DEG number and proportion across respective functional categories for both plots.

Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. GO enrichment analysis (Figure C,E) indicated that the DEGs between the control and experimental groups were primarily annotated to GO terms, including cell proliferation, multicellular organism development, integral component of plasma membrane, plasma membrane part, and cell–cell adhesion. The KEGG database integrates and interprets molecular interactions and metabolic pathways in biological systems, thereby facilitating the understanding of life processes and advancing biomedical research. The DEGs were mapped to the KEGG database to identify significantly enriched signaling pathways (Figure D,F), which included some key pathways such as the NF-κB signaling pathway, Wnt signaling pathway, MAPK signaling pathway, cytokine–cytokine receptor interaction, PI3K-Akt signaling pathway, and IL-17 signaling pathway. All these pathways play critical roles in tumor proliferation.

Combined with the screened DEGs, we noted that AXIN2 was enriched in multiple pathways and its expression in the CBE-β-catenin-HCT116 group was significantly upregulated compared with the control group. AXIN2 is a vital regulator of the Wnt/β-catenin signaling pathway, participating in the modulation of critical cellular functions, including cell proliferation, transformation, migration, and apoptosis. , Given its close association with colon cancer progression, AXIN2 may serve as a potential biomarker for monitoring colon cancer progression and prognosis, as well as a promising target.

In addition, the GPR15 gene was enriched in multiple pathways. As a G protein-coupled receptor (GPCR) localized on the plasma membrane, GPR15 mediates diverse biological processes, including inflammatory responses and cell migration, via ligand binding. The upregulation of GPR15 induced by colon cancer proliferation may be linked to tumor immune escape and tumor microenvironment remodeling. −

Collectively, these identified findings verified the feasibility of our CS•CBE system in β-catenin downstream target identification and probe specific post-translational modifications analysis.

4. Discussion

In the present work, we first identified the optimal chitosan concentration and plasmid dosage for efficient delivery. Then, we assessed that chitosan exhibits superior delivery efficiency and editing efficiency compared with liposomes, while validating its biosafety. Furthermore, through the analysis of in vivo editing outcomes, we identified two novel target loci, which validate the feasibility of CS•CBE system as a research tool in colon cancer research.

First, our study demonstrated that chitosan possesses high delivery efficiency. In our study, encapsulation of 2 μg of β-catenin-sgRNA-mCherry plasmids and 1.5 μg of β-catenin-CBE plasmids in a 0.2 mg/mL chitosan solution resulted in a delivery efficiency of 81.9% in HCT116 cells. For comparison, the delivery efficiency of liposomes reached 66.7%, which is consistent with observations reported in previous studies, with the value of 60%. Notably, the delivery efficiency of chitosan reached 86.0% in our study, which was significantly higher than that of liposome (66.7%). Surprisingly, for chitosan-mediated DNA delivery cellular assays, the high delivery efficiency of 86.0% exceeded the 62–74% range previously reported by Giulia Anderluzzi Giulia et al., and the 68.8% maximum recorded by Jiayu He et al. , Collectively, the higher delivery efficiency of chitosan further helps enhance the editing efficiency of CBE editing.

Second, combined with CBE, the gene-editing tool, we achieved high editing efficiency of targeted gene. We utilized CBE to target the Ser33 site of β-catenin and constructed the CS•CBE system to achieve both high delivery efficiency and editing efficiency of Ser33 of β-catenin. In HCT116 cells, the editing efficiency of CBE at the target site reached 78%, which was significantly higher than the value of 42% by liposome delivery system. Similarly, this observation is consistent with the findings reported by Michael S. Packer et al. The significant difference between our CS•CBE system and traditional liposomal delivery of CBE plasmids highlights the synergistic advantages of the CS•CBE system, where the optimized chitosan carrier not only facilitates efficient plasmid delivery but also enhances the intracellular editing performance of CBE, laying a solid foundation for subsequent in vivo functional validation of the edited β-catenin locus.

Third, the CS•CBE system established in this study possesses high biosafety. As a precision base-editing tool, CBE catalyzes efficient C•G-to-T•A conversion without triggering DNA double-strand breaks (DSBs), which effectively eliminates errors derived from DNA mismatch repair. Unlike conventional CRISPR/Cas9 that mediates gene manipulation via DSB induction and frequently causes unwanted mutations and cellular damage, CBE functions by creating single-stranded DNA nicks. This working mechanism greatly reduces indels and off-target events, making the system well-suited for fine mapping of amino acid sites linked to protein post-translational modifications. It should be acknowledged that CBE also has some limitations. Its editable base spectrum is relatively narrow, and it cannot achieve large-scale gene fragment manipulation. ,

Consequently, CBE has been widely applied in gene therapy, animal model establishment, and gene function research. − In our previous work, CBE was also utilized for the treatment of β-thalassemia. Additionally, chitosan demonstrates favorable biosafety, which is presumably attributed to its outstanding membrane permeability, targeting capability, biocompatibility, and inherent high biosafety profile. ,,

Finally, we validated the feasibility of the CS•CBE system in HCT116 cells and demonstrated that the aberrant accumulation of β-catenin promotes the proliferation and migration of colon cancer cells, thereby enhancing their proliferative capacity. We then established a colon cancer xenograft model in nude mice; consistent with our expectations, the edited tumors exhibited marked increases in volume, weight, and proliferation rate. Subsequent bioinformatics analysis identified two target genes related to proliferation: AXIN2 and GPR15, which validated the feasibility of our CS•CBE system as a research tool, offering novel insights for colon cancer research.

5. Conclusion

Taken together, our strategy provides novel insights into the therapeutic exploration of colon cancer. CS•CBE integrative delivery and editing system enables efficient and precise modification of the Ser33 locus of β-catenin; once the edited cells from tumors in nude mice, target loci associated with colon tumorigenesis can be elucidated. Collectively, we believe our strategy and findings will offer new research tool for colon cancer research.

Supplementary Material

ao6c04206_si_001.pdf (733.1KB, pdf)

Acknowledgments

This work was supported by the Youth Elite Project of the Anhui Academy of Agricultural Sciences (QNYC 202511).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c04206.

  • Figure S1: plasmids map of sgRNA targeting β-catenin and cytosine base editor; Figure S2: demonstrating time-dependent systematic fluorescence visualization experiment of HCT-116 cells after transfection; Figure S3: chitosan delivery achieves highly delivery efficiency than that of conventional liposomes in SW480 cells and assessment of cell viability after transfection; Figure S4: functional annotation of RNA-seq data from CBE-β-catenin-HCT116 and HCT116 cells (PDF)

#.

M.L., Q.B., and D.L. contributed equally to this work. M.L.: Methodology, Investigation, Data curation, Writingoriginal draft. Q.B.: Writingoriginal draft, Project administration. D.L.: Writingreview and editing, Funding acquisition, Supervision. B.L.: Investigation, Validation. J.X.: Methodology, Validation. J.G.: Methodology, Validation. D.C.: Formal analysis, Validation. W.H.: Writingreview and editing, Funding acquisition, Validation.

All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the Anhui Academy of Agricultural Sciences and Anhui University and carried out in compliance with relevant animal care protocols (AAAS2025-3).

The authors declare no competing financial interest.

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