Gold Nanocluster-Mediated Efficient Delivery of Cas9 Protein through pH-Induced Assembly-Disassembly for Inactivation of Virus Oncogenes

Abstract

The CRISPR/Cas gene editing system has been successfully applied to combating bacteria, cancer, virus, and genetic disorders. While viral vectors have been used for the delivery of the CRISPR/Cas9 system, the time required for insert cloning, and virus packaging and standardization, hinders its efficient use. Additionally, the high molecular weight of the Cas9 endonuclease makes it not easy for packing into the vehicles. Herein we report the self-assembly of gold nanoclusters (AuNCs) with SpCas9 protein (SpCas9–AuNCs) under physiological conditions and the efficient delivery of SpCas9 into the cell nucleus. This assembly process is highly dependent on pH. SpCas9–AuNCs are stable at a higher pH but are disassembled at a lower pH. Significantly, this assembly–disassembly process facilitates the delivery of SpCas9 into cells and the cell nucleus, where the SpCas9 exerts its cleavage function. As a proof-of-concept, the assembled SpCas9–AuNCs nanoparticles are successfully used for efficient knockout of the E6 oncogene, restoring the function of tumor-suppressive protein p53 and inducing apoptosis in cervical cancer cells with little effect on normal human cells. The SpCas9–AuNCs are useful for sgRNA functional validation, sgRNA library screening, and genomic manipulation.

Graphical Abstract

1. INTRODUCTION

Cluster regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) system has recently evolved to become a powerful and robust gene-editing technique for targeting-specific loci in the genome using a programmable trans-activating CRISPR RNA (crRNA) as a guide to direct the Cas9 enzyme.¹ Benefiting from its versatility, efficiency, and accuracy, the CRISPR/Cas9 system has revolutionized a wide range of fields, including agriculture, biological science, and medicine.² From the perspective of therapy, the CRISPR/Cas9 system has been successfully applied to combating bacteria, cancer, viruses, and genetic disorders.^3-7 Unlike the RNA interference technology that only achieves partial transient knockdown of gene expression, the CRISPR-Cas9 system induces permanent damage of the target genes.⁸ Until now, different strategies have been developed to apply the CRISPR/Cas9 system to edit the genome.⁹ Compared with the plasmid-based CRISPR/Cas9 system or Cas9 mRNA, the use of purified Streptococcus pyogenes Cas9 (SpCas9) endonuclease is the most widely studied strategy, which has several advantages, including rapid action, high gene editing efficiency, and no requirement for codon optimization and promoter selection.¹⁰ Furthermore, this method is transient and therefore has no insertional mutagenesis and only low immunogenicity. Moreover, the ease of large scale production of SpCas9 protein and the excellent clinical track record of protein therapeutics have paved the way for clinical translation. However, significant challenges remain for efficient delivery of SpCas9 endonuclease because of the large size (about 160 kDa), which is not easy for packing into the vehicles.

Synthetic vehicles such as lipid,^11-13 gold nanoparticles,^14-16 DNA nanoclew,¹⁷ hydrogels,¹⁸ metal–organic frameworks,¹⁹ and graphene oxide²⁰ have recently been used for delivery of the CRISPR/Cas9 system. However, incorporation of additional spare DNA or cotranscriptional activator complex for multiple applications, such as gene knock-in and regulation of endogenous gene expression, often complicate these delivery systems. One solution is to deliver the Cas9 protein and other component elements separately, which greatly simplifies the delivery process. For example, Liao et al. generated a dual adeno-associated viral system for separate delivery of Cas9 protein and the modified gRNA to induce epigenetic remodeling of targeted loci.²¹ Although promising, it is limited by cell type specificity, tissue tropism, and inevitable immunogenicity. Therefore, the development of vehicles for separate delivery of CRISPR components to the cell nucleus remains an urgent need for a safe and efficient gene-based therapy.

Self-assembly is a facile and efficient way to synthesize large molecular aggregates in the natural process. Multiple copies of subunits such as proteins can spontaneously agglomerate into hierarchical architectures such as spherical virus capsids.²² Inspired by nature, scientists have been devoted to the construction of superstructures of multicomponents self-assembled from different building blocks through entropic effects, van der Waals force, and electrostatic interaction. These new structures have the potential for unique and improved functions for diverse technological applications.^23-28 Especially, the metal nanoclusters provide an ideal platform for exploring self-assembly because of ultrasmall size, customizable and controllable surface composition, and availability of a diverse library of building blocks.^29-32 Herein, we report, for the first time, that gold nanoclusters (AuNCs) can self-assemble with SpCas9 protein, and the complexes (SpCas9–AuNCs) efficiently deliver SpCas9 protein into the cell nucleus (Figure 1). This assembly process is highly dependent on pH. SpCas9–AuNCs are stable at a higher pH but are disassembled at a lower pH. The assembly disassembly process enables the delivery of SpCas9 into cells and the cell nucleus, where SpCas9 exerts its cleavage function. Furthermore, as a proof-of-concept, we used the self-assembled SpCas9–AuNCs nanoparticles to effectively knockout the E6 oncogene following transfection of HPV18 E6 sgRNA into cervical cancer cells, hence restoring the function of tumor-suppressive protein p53 and inducing apoptosis. Importantly, knockout of the E6 gene by SpCas9–AuNCs and E6 sgRNA had little effect on the normal cells. These unique features make SpCas9–AuNCs an exciting biomaterial for gene-based cancer therapy.

Figure 1.

Schematic illustration of pH-induced assembly disassembly of SpCas9–AuNCs. AuNCs self-assemble with SpCas9 at a higher pH through electrostatic interaction whereas the disassembly of SpCas9–AuNCs occurs at a lower pH, which weakens the interaction between AuNCs and SpCas9.

2. EXPERIMENTAL SECTION

2.1. Reagents and Materials.

Gold(III) chloride trihydrate, glutathione, 3-[4,5-dimethylthiazolyl-2-]-2,5-diphenyltetrazolium bromide (MTT), and fetal bovine serums (FBS) were purchased from Sigma-Aldrich. Trypsin was obtained from Genesee. Dulbecco’s modified Eagle’s Medium (DMEM) was purchased from VWR. All chemical reagents were of analytical grade and were directly used without further purification. Deionized water (18.2 MΩ Millipore) was used in all experiments.

2.2. Expression and Purification of Streptococcus pyogenes Cas9 Protein (SpCas9).

Plasmid pET-NLS-Cas9–6xHis (a gift from David Liu, Addgene #62934) encoding the S. pyogenes Cas9 fused with a N-terminal nuclear localization sequences (NLS) and C-terminal His-tag were transformed into E. coli BL21 (DE3) competent cells. The cells were inoculated into Luria–Bertani (LB) broth containing 100 μg of ampicillin and cultured at 37 °C overnight. Then the cells were diluted 1:100 into the same medium and allowed to grow until the OD₆₀₀ reached 0.6. The culture was incubated at 20 °C for 20 min, and isopropyl β-D-thiogalactopyranoside (IPTG) was added at 0.1 mM to induce the expression of SpCas9. After 16 h, the cells were centrifuged and the pellet was suspended in lysis buffer containing 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, 20 mM imidazole, and 1 mM PMSF with 1 mg/mL of lysozyme. The cells were lysed by sonication (3 s pulse on, and 3 s pulse off for a total 10 min at 30% amplitude). The lysate was centrifuged at 16 000g for 30 min, and the supernatant was transferred to a fresh tube. The soluble lysate was then incubated with Hispur Ni-NTA resin (Thermo Fisher #88221) at 4 °C for 45 min. The resin was transferred to a gravity column and washed with 20 column volumes of washing buffer containing 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, and 20 mM imidazole until no protein was detected in washing buffer. Finally, SpCas9 was eluted with the elution buffer containing 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, and 250 mM imidazole, and the fractions containing the SpCas9 protein were determined using Quick Start Bradford Protein Assay (Bio-Rad). After dialyzing against the storage buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20% (v/v) glycerol, and 0.5 mM PMSF overnight, the SpCas9 protein was concentrated using a 100 K centrifugal filter (Millipore, UFC810008) and stored in aliquots at −80 °C. The concentration of the SpCas9 protein was quantified and analyzed by SDS-PAGE.

2.3. Design, Synthesis, And Purification of sgRNA.

Single guide RNA (sgRNA) specific to HPV18 E6 gene was designed using the online tool Benchling. The template for the sgRNA was generated by repeated annealing and extension of complementary oligonucleotide primers with 30 cycles of annealing at 60 °C for 40 s, extension at 72 °C for 30 s, followed by gel extraction (Qiagen). In vitro transcription was performed using the HiScribe Quick T7 High Yield RNA Synthesis Kit (NEB #E2050). After the reaction, DNase was used to remove the DNA template. The transcribed sgRNA was purified by phenol–chloroform extraction followed by ethanol precipitation according to the kit’s manual. The purified sgRNA was analyzed by agarose gel electrophoresis in TBE buffer using RiboRuler Low Range RNA Ladder (SM1833, ThermoFisher) as a reference, and the concentration was quantified with a Synergy Multi-Mode Reader (Biotek). The primers for HPV18 E6 sgRNA was listed as follows:

Forward: 5′-GAAATTAATACGACTCACTATAGG-GACAGTATTGGAACTTACAGGTTTTAGAGCTAGAA-ATAGCAAGTTAAAATAAGGCTAGTCCG-3′. Reverse: 5′-A A A A A A G C A C C G A C T C G G T G C C A C T T T T -TCAAGTTGATAACGGACTAGCCTTATTTTAACTTGC-3′ (the underlined region is the targeted region for HPV18 E6).

2.4. Synthesis of Glutathione (GSH)-Protected Gold Nanoclusters (AuNCs).

GSH-protected AuNCs were prepared. Typically, 2.5 mL of 4 mM aqueous HAuCl4 and 2.5 mL of 8 mM GSH solution were mixed under vigorous stirring at room temperature for 5 min. Then the mixture was allowed to react at 70 °C for 24 h. The solution was first centrifuged at 16 000g for 1 min to remove the insoluble aggregates as well as large nanoparticles. The obtained supernatant was further purified using a dialysis bag with a MW cutoff of 14 kDa in double distilled water for 48 h to remove any free GSH and gold ions. The obtained final AuNCs were stored at 4 °C for further use.

2.5. Formation of AuNCs and SpCas9 Nanoassembly (SpCas9–AuNCs).

The assembly of AuNCs and SpCas9 occurred through electrostatic action. Briefly, the above prepared AuNCs and SpCas9 were first dissolved in a phosphate-buffered saline (PBS) solution (pH at 7.4) at a concentration of 1 mg/mL and 500 μ/mL, respectively. Then 100 μL of the positively charged SpCas9 at 500 μ/mL was added dropwise into 1 mL of the negatively charged AuNCs solution at 1 mg/mL with stirring, and the mixture was incubated at room temperature for 30 min. The prepared SpCas9–AuNCs nanoassembly was used for the in vitro plasmid cleavage assay and transfection experiments. Mixtures at different ratios of SpCas9 and AuNCs were also prepared with the same method as described above.

2.6. Plasmid DNA Cleavage Assay for Endonuclease Activity.

Plasmid pLXSN18E6E7 (Addgene #53459) containing the HPV E6 gene was linearized with PvuI (NEB), purified by QIAquick Gel Extraction Kit (Qiagen), and used as the substrate for spCas9 activity assay. In a typical reaction, the linearized plasmid was treated with SpCas9 complexed with E6 sgRNA with or without AuNCs at a molar ratio of SpCas9:sgRNA:plasmid at 10:10:1 in the Cas9 nuclease reaction buffer containing 20 mM HEPES at pH 6.5, 100 mM NaCl, 5 mM MgCl₂ and 0.1 mM EDTA at 37 °C for 1 h. The final products were analyzed by electrophoresis with a 1.0% agarose gel to confirm the presence and sizes of the DNA fragments.

2.7. Cell Culture.

The human cervical cancer HeLa cells contain integrated HPV18 DNA and express E6 gene. HEK-293T cells were negative for HPV. Cells were maintained in DMEM, supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5.0% CO₂.

2.8. Immunofluorescence Assay.

TO investigate the uptake of SpCas9–AuNCs, HeLa cells were seeded on coverslips in 24-well plates at 5 × 10⁴ cells per well overnight. The cells were incubated with SpCas9–AuNCs (containing 50 nM or ~8 μ/mL SpCas9) for the indicated time (1, 2, or 4 h) and then washed with PBS three times. After that, the treated cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were washed with cold PBS twice and then incubated with 0.25% Triton X-100. Cells were then washed and blocked with 1% BSA in PBS for 30 min and then incubated with the anti-Cas9 antibody for 1 h at room temperature. After washing with PBS three times, cells were further incubated with the corresponding secondary antibody conjugated with Alexa Fluor 488 for 1 h in the dark at room temperature. The cell nuclei were stained with Hoechst 33342 dye for 5 min, and the slides were mounted with FluorSave Reagent (cat. #345789, Calbiochem, Billerica, MA). Samples were then observed with a laser-scanning confocal Nikon Eclipse Ti fluorescence microscope (Nikon Instruments). Alexa Fluor 488 was observed with an excitation wavelength of 488 nm and an emission wavelength of 500–550 nm. Hoechst 33342 was observed with an excitation wavelength of 405 nm and an emission wavelength of 425–475 nm. AuNCs was observed with an excitation wavelength of 561 nm and an emission wavelength of 575–625 nm.

For detecting the expression of HPV18 E6 and p53 proteins, HeLa cells were seeded on coverslips in 24-well plates at 5 × 10⁴ per well overnight. The cells were incubated with SpCas9–AuNCs (containing 50 nM or ~8 μg/mL SpCas9) for 4 h and then washed with PBS three times. After that, sgRNA (1.92 μg/mL) was then transfected into the cells using Lipofectamine RNAiMAX. After 3 days, the treated cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were washed with cold PBS twice and then incubated with 0.25% Triton X-100. After that, the cells were washed and blocked with 1% BSA in PBS for 30 min and then incubated with an anti-HPV18 E6 antibody or anti-p53 antibody for 1 h at room temperature. After washing with PBS three times, cells were further incubated with the corresponding secondary antibody conjugated with Alexa Fluor 488 for 1 h in the dark at room temperature. The cell nuclei were stained with Hoechst 33342 dye for 5 min, and the slides were mounted with FluorSave Reagent. Samples were then observed with a laser-scanning confocal Nikon Eclipse Ti fluorescence microscope. Alexa Fluor 488 was observed with an excitation wavelength of 488 nm and an emission wavelength of 500–550 nm. Hoechst 33342 was observed with an excitation wavelength of 405 nm and an emission wavelength of 425–475 nm.

2.9. Mechanism of Cellular Internalization.

To study the mechanism of cellular internalization, several inhibitors of the cellular uptake pathways were used, including chlorpromazine hydrochloride (an inhibitor of clathrin-dependent endocytosis), nystatin (an inhibitor of caveolin-dependent endocytosis), methyl-β-cyclodextrin (an inhibitor of cholesterol-dependent membrane fusion), and amiloride (an inhibitor of macropinocytosis). HeLa cells were seeded in a 24-well plate at a density of 2 × 10⁵ cells/well. Cells were then washed with PBS and incubated with 0.5 μg/mL of chlorpromazine hydrochloride, 25 μg/mL of nystatin, 50 μg/mL of methyl-β-cyclodextrin, or 20 μg/mL of amiloride in serum-free medium at 37 °C for 1 h. Then the medium was replaced with fresh medium containing the inhibitors and SpCas9–AuNCs. The cells were further incubated for another 4 h at 37 °C. Cells were washed three times with PBS, and the intracellular fluorescence intensities were observed using an Olympus IX 81 confocal laser scanning microscope.

2.10. Western Blot Assay.

Total cell lysates were separated in SDS polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (GE Healthcare). The membranes were incubated sequentially with primary and secondary antibodies, respectively. The signal was detected with Luminiata Crescendo Western HRP substrate (Millipore Sigma, #WBLUR0500). Primary antibodies to β-actin (Santa Cruz, #SC-47778), HPV18 E6 (Santa Cruz, #SC-460), Cas9 (Abcam, #ab191468), p53 (Cell Signaling Technology, #2524s), and p21 (Santa Cruz, #sc-397) were used.

2.11. T7E1 Assay To Detect Genomic Indel Formation.

Genomic DNA of HeLa cells was harvest using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The targeted HPV18 genome encoding E6 gene was amplified by PCR Then the PCR products were annealed and digested with T7 endonuclease I. Finally, the fragments were analyzed to determine the efficiency of indel induction in the targeted genome.

2.12. Cell Survival Assay.

HeLa cells seeded at a density of 5000 cells/well (100 μL) in 96-well plates for 24 h were incubated with SpCas9–AuNCs for 4 h and washed with PBS three times. sgRNA was then transfected into the cells using Lipofectamine RNAiMAX. Finally, cell cytotoxicity was measured at day 3 using the MTT assay. Briefly, the cell culture medium was removed and the cells were washed with PBS twice. Then 10 μL of MTT solution at 5 mg/mL was added to each well to a final volume of 100 μL. The plate was placed in the CO₂ incubator for an additional 4 h. The medium was removed, and 100 μL of DMSO as added into each well. The plate was then gently swirled for 2 min at room temperature at dark to dissolve all formed precipitate. Absorbance values were determined with a Synergy Multi-Mode Reader (Biotek) at 570 nm with a reference filter of 650 nm.

2.13. Cell Apoptosis Assay.

Apoptosis was examined to determine the effect of SpCas9–AuNCs and sgRNA by staining the cells with Annexin V Alexa Fluor 488 and propidium iodide according to the instructions of the manufacturer (Thermo Scientific). Briefly, HeLa cells and HEK-293T cells seeded in six-well plates at a density of 1 × 10⁵ cells per well overnight were incubated with SpCas9–AuNCs for 4 h and then washed with PBS three times. SgRNA was transfected into the cells. After incubation for 3 days, cells were collected for the detection of apoptotic cells. Quantitative analysis was carried out on flow cytometry.

2.14. Statistical Analysis.

All data were expressed as means ± standard errors of the means (SEMs) from at least three independent experiments, each with three repeats unless stated otherwise. A two tailed t test was performed, and P0.05 (*) and P0.01 (**) were considered statistically significant.

3. RESULTS AND DISCUSSION

3.1. Preparation and Characterization of SpCas9–AuNCs.

Glutathione-capped gold nanoclusters were synthesized through reduction of the Au precursor HAuCl₄ by GSH, which acted as both a reduction and a protection agent.³³ The monodispersed spherical AuNCs had an average diameter of about 2.0 nm (Figure 2A). The ultrasmall controllable size makes AuNCs an ideal platform for self-assembly while unique fluorescence of AuNCs facilitates cellular tracking. Recombinant positively charged SpCas9 fused with N-terminal nuclear localization sequences and C-terminal 6X His-tag was expressed in Escherichia col and purified.¹² Coomassie blue staining and anti-Cas9 antibody confirmed the size of SpCas9 of about 160 kDa (Figure S1). The AuNCs and SpCas9 were mixed at pH 7.4 at different weight ratios. Dynamic light scattering (DLS) indicated that the hydrodynamic diameter of the mixtures of AuNCs and SpCas9 increased with the increasing ratio of AuNCs and SpCas9 (Figure S2). As a lower ratio was not efficient for forming uniform particles while a higher ratio generated some aggregates, the ratio of AuNCs to SpCas9 was chosen at 20:1 in the following experiments.

Figure 2.

(A–C) TEM images of AuNCs (A), SpCas9–AuNC (B) in PBS at pH 7.4, and SpCas9–AuNCs in PBS with the pH adjusted from 7.4 to 4.5 (C). (D) Dynamic light scattering characteristics of AuNCs and SpCas9–AuNCs at different pH.

Transmission electron microscopy (TEM) revealed that AuNCs and SpCas9 assembled to compact and spheroid morphology with an average size of 104 nm (Figure 2B). Interestingly, when the pH was adjusted to 4.5, the assembled SpCas9–AuNCs complexes were not stable as shown in TEM images and DLS (Figure 2C and Figure 2D). The surface of AuNCs has carboxylic groups, which would be deprotonated under physiologic conditions, hence endowing the AuNCs with a negatively charged surface and the ability to assemble with positively charged SpCas9 through electrostatic interaction. However, when the pH decreased, the carboxylic groups of AuNCs are reversed to partially protonated form, which decreases the amount of negative charge on the surface of AuNCs, hence greatly weakening the interaction between AuNCs and SpCas9. ³⁴ Intriguingly, SpCas9–AuNCs were not completely disassembled when the pH was adjusted to 4.5, which could be due to the protonated carboxylate–histidine interaction between AuNCs and SpCas9.³⁵ The fact that the mixture of SpCas9 and AuNCs could not assemble at pH 4.5 further proved that the electrostatic interaction played a dominant role (Figure S3). Zeta poteinitials were also measured to further confirm this observation (Figure S4). Furthermore, no plasmon band at ca. 520 nm was observed, indicating there were no gold nanoparticles formed under both assembly and disassembly conditions (Figure S5). The size of SpCas9–AuNCs changed little after storage in 4 °C for 1 week, indicating the assembly of SpCas9–AuNCs was stable during this period (Figure S6). Because the major intracellular trafficking pathway is mediated by endosomes/lysosomes, which have an acidic environment, we hypothesized that this property of SpCas9–AuNCs has the potential to facilitate the efficient delivery of SpCas9 into the cell and cell nucleus through this pathway.

3.2. Efficient Delivery of SpCas9–AuNCs into Cells.

Because efficient cellular uptake and delivery of SpCas9 into the cell nucleus is essential for gene disruption, we investigated the internalization process of SpCas9–AuNCs into HeLa cells by incubating SpCas9–AuNCs with cells for 1, 2, and 4 h. SpCas9 and AuNCs were detected in green and red fluorescences, respectively. As shown in Figure 3A and Figure 3C, native SpCas9 could hardly reach the cell nucleus even after incubation for 4 h. In contrast, cells incubated with SpCas9–AuNCs showed much stronger green fluorescence intensity than those with native SpCas9 alone, indicating that the assembled nanoparticles indeed facilitated the uptake process (Figure 3B). Moreover, by increasing the incubation time, more SpCas9 was localized to the cell nucleus, where endonuclease-mediated cleavage of target DNA would take place. This observation was also quantitatively confirmed by the line scanning profiles of fluorescence intensity (Figure 3D). Confocal z-stacking images and three-dimensional reconstruction images clearly demonstrated that SpCas9 was localized to both cytoplasm and cell nucleus (Figures S7 and S8). In particular, the green fluorescence did not completely overlap with the red fluorescence after 4 h incubation of SpCas9- AuNCs with the cells, indicating the disassembly of the complexes. This result suggested that SpCas9–AuNCs might be internalized into the cells via endocytosis pathway and that the complexes were exposed to the endosomal/lysosomal low pH, which induced the disassembly of SpCas9–AuNCs as well as the proton sponge, resulting in the release of SpCas9 into the cytoplasm. The nuclear localization sequences on SpCas9 could then guide it into the cell nucleus. To confirm this hypothesis, the LysoSensor Green DND-189 dye was used to tracker the lysosome of Hela cells treated with SpCas9–AuNCs. Confocal images showed that the red fluorescence from AuNCs was not colocalized with green fluorescence from LysoSensor, indicating that SpCas9–AuNCs escaped from the lysosome (Figure S9). The high cellular uptake efficiency was further confirmed by flow cytometry (Figure 3E). In addition, SpCas9–AuNCs could also be efficiently internalized by BCP1 cells, which are usually hard to transfect, suggesting the possible application of this approach for a variety of difficult to transfect cell lines (Figure S10). To investigate the uptake mechanism, we treated the cells with different endocytosis inhibitors and then evaluated the cellular uptake of SpCas9–AuNCs. Chlorpromazine, nystatin, and amiloride greatly reduced the uptake of the SpCas9–AuNCs, suggesting that the main internalization was through clathrin-dependent, caveolin-dependent endocytosis and macropinocytosis (Figure S11). Collectively, these results indicated that SpCas9–AuNCs could effectively deliver the SpCas9 protein into the cell nucleus.

Figure 3.

Confocal laser scanning microscopy (CLSM) images of HeLa cells treated with SpCas9 (A) and SpCas9–AuNCs (B) at 1, 2, and 4 h, respectively. SpCas9 protein was stained with an anti-Cas9 antibody and an antimouse secondary antibody coupled with Alexa Fluor 488 to track the SpCas9 distribution inside the cells while AuNCs were shown in red fluorescence. CLSM line-scan profiles of fluorescence intensity of Hela cells incubated with SpCas9 (C), and SpCas9–AuNCs (D), corresponding to the yellow arrows in Figure 3A and 3B. (E) Analysis by flow cytometry of HeLa cells treated with SpCas9–AuNCs for different lengths of time. Scale bars represent 50 μm.

3.3. Knockout of Viral Oncogene.

Encouraged by the high efficiency of delivery, we then evaluated the utility of SpCas9–AuNCs for in vitro gene disruption. We designed a single guide RNA molecule (sgRNA) that targets the E6 gene of HPV18 flanked by an NGG PAM (Figure 4A). Because E6 gene is one of the high-risk viral oncogenes and has no obvious homology to the human genome, it is regarded as an ideal therapeutic target for cervical cancers.³⁶ As shown in Figure 4B, the sgRNA was successfully synthesized with a size of ~100 nucleotides. To examine the ability of sgRNA to guide the SpCas9 protein to induce double-strand cleavage, we applied linearized plasmid encoding the E6 gene as the substrate (Figure S12). We confirmed that both SpCas9 and sgRNA were functional in vitro as shown by the appearance of smaller new fragments (Figure 4C). We also tested the cleavage activity of SpCas9 and sgRNA in the presence of AuNCs and found the appearance of the predicted fragments. However, SpCas9 and AuNCs in the absence of sgRNA could not induce the generation of any new fragments. These results indicated that AuNCs did not influence the cleavage of the target DNA by SpCas9 and sgRNA.

Figure 4.

(A) Schematic diagram of sgRNA targeting HPV18 E6 gene. PAM sequence and sgRNA targeting site were highlighted in orange and blue, respectively. (B) Gel electrophoresis of the HPV18 E6 sgRNA synthesized by in vitro transcription. (C) Cleavage assay using linearized HPV18E6E7 plasmid to examine the function of sgRNA and SpCas9 with or without AuNCs. (D, E) Examination of the HPV18 E6 protein in HeLa cells treated with SpCas9–AuNCs or SpCas9–AuNCs with E6 sgRNA or SpCas9 with E6 sgRNA by Western blot analysis (D) and immunofluorescence assay (E). Scale bar represents 20 μm.

We next investigated whether SpCas9-AuNCs were able to edit the E6 gene in the HPV 18 genome in HeLa cells by transfection with an E6 sgRNA. We first incubated HeLa cells with SpCas9–AuNCs for 4 h to allow the cells to uptake the nanoparticles. We then transfected sgRNA into the cells. The expression level of HPV18 E6 protein was examined 3 days later. As expected, the expression of HPV18 E6 protein detected by Western blotting was significantly inhibited after treatment with SpCas9–AuNCs and E6 sgRNA compared with the untreated control, and cells treated with AuNCs, SpCas9–AuNCs, or SpCas9 and E6 sgRNA without vehicles (Figure 4D). These results were confirmed by immunofluorescence assay (Figure 4E). Furthermore, the E6 gene locus in the HPV18 genome was amplified by PCR and the genomic indel formation was detected by the T7E1 assay. The mutation frequency in the cells treated with SpCas9–AuNCs reached 34% (Figure S13), which was comparable with that of lipid-based delivery of ribonucleoproteins. Additionally, the genomic indel formation also confirmed that the decrease of HPV18 E6 protein in Hela cells treated with SpCas9–AuNCs and E6 sgRNA was caused by E6 gene knockout other than knockdown. However, complete elimination of E6 protein expression was not observed, which may be attributed to the lack of gene editing efficiency or delivery efficiency. Taken together, SpCas9–AuNCs could effectively disrupt the viral oncogene in the presence of sgRNA.

3.4. Cancer Therapy through Inactivation of Viral Oncogene.

By inhibiting the key tumor-suppressive protein p53, HPV E6 gene is crucial for the malignant transformation and survival of cervical cancer cells.³⁷ We further tested whether p53 was activated after the cleavage of the HPV E6 gene. As shown in Figure 5A, the level of p53 and that of its downstream target p21 was increased following treatment with SpCas9–AuNCs and E6 sgRNA. Immunofluorescence assay further confirmed that p53 expression in the cells treated with SpCas9–AuNCs and sgRNA was higher than those of the untreated and SpCas9–AuNCs treated cells (Figure 5B). These results indicated that the disruption of the E6 gene reactivated the tumor-suppressive protein p53. Meanwhile, MTT assays showed that cell proliferation was reduced in Hela cells treated with SpCas9–AuNCs and sgRNA in a dose-dependent fashion (Figure 5C). We further examined the induction of apoptosis by dual staining with Alexa Fluor 488- Annexin V and propidium iodide. Treatment with SpCas9- AuNCs and sgRNA induced early and late apoptosis in 13.81% of cells and necrosis in 19.3% of cells, respectively. In contrast, there was no significant change of apoptotic cells in the untreated cells, and cells treated with AuNCs or SpCas9–AuNCs. These results were consistent with the results of cell proliferation. In addition, there was no increase of apoptosis in HEK293T cells treated with SpCas9–AuNCs and E6 sgRNA, indicating the lack of disruption of the human genome (Figure S14). Taken together, disruption of oncogenic E6 gene with SpCas9–AuNCs and sgRNA had excellent specificity for HPV18-transformed cells with minimal cytotoxic effect for other cells. Considering the significant advancements in developing safe and clinically advanced delivery systems of oligonucleotides such as liposome and polymeric nanoparticles, it would be worthwhile to test the efficiency of SpCas9–AuNCs combined with nanoparticles-based delivery of sgRNA. Nevertheless, there are numerous challenges to use this system in the preclinical animal tumor models. First, it would be necessary to evalute the delivery efficiency of SpCas9–AuNCs to the tumor site, which is highly correlated with the final therapeutic effect. Secondarily, the optimal choice of a type of nanoparticles is important to deliver and release the sgRNA in the tumor site. Lastly, it is important to consider the immunogenicity of SpCas9–AuNCs and its clearance from the body.³⁸

Figure 5.

(A) Expression levels of p53 and p21 proteins in HeLa cells examined by Western blotting assay after different treatments. (B) CLMS showing the expression of p53 in HeLa cells after different treatments. The cellular nuclei were stained with Hoechst 33342 dye. (C) The proliferation of HeLa cells after different treatments for 3 days examined by MTT assays. The control groups were set as 100%. *P<0.05 and **P<0.01 were calculated by comparing with the control groups. (D) Detection of apoptotic HeLa cells after treatment with AuNCs, SpCas9–AuNCs and SpCas9–AuNCs with E6 sgRNA for 3 days. The representative results from three independent experiments are shown. Scale bar represents 20 μm.

4. CONCLUSION

We demonstrated that AuNCs could assemble with SpCas9 under physiological conditions. The stability of the assembled complexes was highly dependent on pH. The complexes were stable at a higher pH but were disassembled at a lower pH. Moreover, the assembly–disassembly process could facilitate the delivery of SpCas9 into cells and cell nucleus, where the SpCas9 exerted its cleavage function. Furthermore, as a proof-of-concept, the assembled SpCas9–AuNCs was an effective system for knockout of the oncogenic E6 gene when used together with the HPV18 E6 sgRNA, which triggered the expression of tumor-suppressive protein p53 and restored its function, inducing apoptosis of the cervical cancer cells. Importantly, knockout of the E6 gene by SpCas9–AuNCs and E6 sgRNA had little effect on other human cells without the HPV E6 gene. The high efficiency and specificity make SpCas9–AuNCs a powerful tool for disrupting viral oncogenes for gene cancer therapy. Additionally, the SpCas9–AuNCs can make it easier for sgRNA functional validation, sgRNA library screening, and genomic manipulation.