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Disruption of human papillomavirus 16 E6 gene by clustered regularly interspaced short palindromic repeat/Cas system in human cervical cancer cells

High-risk human papillomavirus (HPV), especially HPV16, is considered a main causative agent of cervical cancer. Upon HPV infection, the viral oncoprotein E6 disrupts the host tumor-suppressor protein p53, thus promoting malignant transformation of normal cervical cells. Here, we used the newly developed programmable ribonucleic acid-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system to disrupt the HPV16 E6 gene. We showed that HPV16 E6 deoxyribonucleic acid was cleaved at specific sites, leading to apoptosis and growth inhibition of HPV16-positive SiHa and CaSki cells, but not HPV-negative C33A or human embryonic kidney 293 cells. We also observed downregulation of the E6 protein and restoration of the p53 protein. These data proved that the HPV16 E6 ribonucleic acid-guided CRISPR/Cas system might be an effective therapeutic agent in treating HPV infection-related cervical malignancy.


Introduction
Cervical cancer is the second-most common cancer in women worldwide. 1 Human papillomavirus (HPV) has long been considered to be a major risk factor for cervical cancer, especially HPV16. 2 During the process of cervical carcinogenesis, the HPV oncogenes E6 and E7 promote and maintain the malignant phenotype of cervical cancer cells, 3,4 making these genes attractive targets for cancer gene therapy.
Being able to target any site of the genome in the form of 5′-N 20 NGG-3′, 5 the newly discovered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has emerged as a powerful genome-editing tool in many organisms, including prokaryotes, Caenorhabditis elegans, and zebrafish. [6][7][8] It consists of a Cas9 enzyme and a guidance ribonucleic acid (gRNA) for a specific deoxyribonucleic acid (DNA) sequence. 5 Under the guidance of gRNAs, Cas9 enzyme will induce double-strand breaks (DSBs) at a determined DNA sequence complementary to the gRNA sequence ( Figure 1). The breaks in the DNA are repaired by the mutagenic nonhomologous end joining (NHEJ) repair pathway, which leads to the disruption of the targeted gene. 9,10 In this study, we applied the CRISPR/Cas system to target the E6 oncogene in HPV16-infected cervical cancer cell lines and investigated the feasibility of HPV16 E6-guided CRISPR/Cas as a potential intervention strategy for treatment of cervical cancer.

Plasmids
Plasmid-encoding Cas9 was kindly provided by Addgene. As recent in vitro work has shown that the specificity of RNAguided endonuclease is guaranteed simply by a short synthetic RNA molecule with no Cas9 restriction, 11 we constructed three gRNAs targeting the HPV16 E6 gene following the protocol of Mali et al, 5 and proved their effect in SiHa and CaSki cells. Sequences of the customized gRNAs are described in Table 1.

T7 endonuclease 1 assay
The T7 endonuclease I (T7E1) assay was performed as previously described. 13 Briefly, the region containing the targeting site of HPV16 E6 was polymerase chain reaction (PCR)-amplified. The primers used are described in

Detection of apoptosis
At 48 hours' posttransfection, cells were trypsinized and costained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide using an annexin V-FITC apoptosis-detection kit (KeyGen Biotech) according to the manufacturer's instructions. Double-positive cells were analyzed using a FACSCalibur™ (BD Biosciences, San Jose, CA, USA) to calculate CRISPR/Cas system-induced HPV16-positive cell death. C33A and HEK293 cells were also transfected with the same amount of plasmids. The data were analyzed using BD CellQuest™ software.

statistical analysis
Statistical analysis among different groups was conducted using one-way analysis of variance. All analyses were performed with SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Data are presented as means ± standard deviation. P,0.05 was considered statistically significant.

hPV16 e6 grnas induced DsBs at specific sites of HPV16 E6 gene
We first synthesized three gRNAs targeting the HPV16 E6 gene ( Figure 1). We tested whether the three synthesized gRNAs could induce DSBs at specific sites by using a mammalian cell-based SSA assay in HEK293 cells. When gRNAinduced DSBs occurred, SSA homologous recombination led to the formation of an active luciferase gene. The Renilla luciferase plasmid was used as a control to monitor CRISPRinduced cytotoxicity. Compared with the negative control, which was transfected with only Cas9 plasmid (Figure 2A), the gRNA-2/Cas9 and gRNA-3/Cas9 group showed higher activity, while the gRNA-1/Cas9 group did not. Meanwhile, measurement of the cotransfected Renilla luciferase revealed no significant change of signal ( Figure 2B), indicating that the gRNAs used in this study had minimal cytotoxicity. We performed the T7E1 assay to further confirm the activity of the three gRNAs. Each of the gRNA/Cas9 plasmids was introduced into SiHa and CaSki cells. Cellular DNA of gRNA/Cas9-transfected cells were PCR-amplified and treated with T7E1. In SiHa and CaSki cells, after the T7E1 treatment of heteroduplex DNA, the gRNA-2/Cas9 and gRNA-3/Cas9 groups gave rise to cleaved products at about 200 and 300 bp, while the products of the gRNA-1/Cas9 group were uncleaved. The CRISPR/Cas system induced mutations at the HPV16 E6 site with frequencies up to 22% (gRNA-2/Cas9 group) and 21% (gRNA-3/Cas9 group) in SiHa cells, and 17% (gRNA-2/Cas9 group) and 19% (gRNA-3/Cas9 group) in CaSki cells ( Figure 2C and D). Therefore, gRNA-1/Cas9 was not effective, and the gRNA-2/Cas9 and gRNA-3/Cas9 groups demonstrating cleavage activities were selected for further study. Taken together, these results indicate that the gRNA-guided CRISPR/Cas system can effectively induce DSBs in the E6 oncogene.

effects of e6 grnas on apoptosis
To determine whether gRNA-guided CRISPR could specifically induce apoptosis of HPV16-positive human cervical cancer cells, we treated SiHa, CaSki, C33A, and HEK293 cells with E6-specific gRNA/Cas9 and calculated the apoptotic rates of these cells by flow-cytometry analysis. Compared with controls, HPV16-positive SiHa and CaSki cells showed dramatically increased apoptotic rates when treated with gRNA-2/Cas9 and gRNA-3/Cas9 ( Figure 3A and B). On the other hand, HPV-negative C33A and HEK293 cells demonstrated no apparent apoptosis on receiving the same gRNA/Cas9 ( Figure 3C and D). The corresponding apoptotic rates of these four cell lines that were treated with gRNA-2/Cas9 and gRNA-3/Cas9 are shown in Figure 3E and F.

cellular proliferation affected by E6 disruption
The inhibitory effects of CRISPR gRNA/Cas9 on cell proliferation were detected by CCK-8 assay. At 72 and 96 hours posttransfection, the absorbance values of SiHa and CaSki cells treated with gRNA-2/Cas9 and gRNA-3/Cas9 were significantly lower than those of gRNA-1/Cas9-transfected cells and the untreated cells ( Figure 4A and B). However, in HPV16-negative C33A and HEK293 cells, the absorbance rate showed no significant difference between transfected and nontransfected wells, indicating that E6 gRNA/Cas9 inhibited cellular proliferation only in HPV16-positive cell lines ( Figure 4C and D).

effects of e6 grnas on protein levels of e6 and p53
The effects of gRNA-guided CRISPR on E6 and p53 protein expression were detected by Western blot analysis. HPV16 E6 protein levels were downregulated in both cell lines, and p53 protein levels were upregulated correspondingly ( Figure 5). As p53 is a tumor-suppressor gene, increasing its expression may contribute to reversal of the malignant phenotype of HPV16-positive cell lines.

Discussion
HPV oncoprotein E6 plays important roles in maintaining the malignant phenotype of cervical cancer cells. 3 Through forming E6/E6AP complex in HPV-infected cells, E6 oncoprotein consequently leads to proteosomal degradation of tumor suppressor p53. 14, 15 We speculated that when oncoprotein E6 is disrupted, the function of p53 protein will be recovered, leading to cellular apoptosis ( Figure 3) and growth inhibition ( Figure 4) in HPV16-positive cells. 16 Therefore, cancer gene therapy targeting the E6 gene may increase the expression level of the p53 protein and contribute to reversal of the malignant phenotype in cervical cancer cells.
Previous studies have shown that small interfering RNA (siRNA) targeting HPV E6 messenger RNA can effectively knock down E6 expression and induce apoptosis in HPVpositive cervical cancer cells. 16 However, the effect of siRNA is transient and only lasts for a few days. 17 Moreover, it was also reported that human cells can counteract long-term silencing of critical genes disrupted by siRNA. 18 Therefore, in this study, instead of targeting E6 RNA, we applied CRISPR endonuclease to directly disrupt viral E6 DNA (Figure 1). We showed that gRNA-2 and gRNA-3 can efficiently cleave viral E6 DNA and cause DSBs in corresponding areas ( Figure 2). These DSBs are then repaired by the error-prone NHEJ repair pathway, creating point mutations or frameshift mutations in the E6 gene. 19 Compared to the transient effects of siRNA, one advantage of the CRISPR/Cas system-induced DNA mutation is that it is permanent and could be passed

43
Disruption of hPV16 e6 gene by crisPr/cas system down to the next generation. 20 In addition, another advantage of CRISPR-based antiviral therapy is that gRNAs are easy to construct, making this technology easily adopted by less equipped laboratories or companies. One important concern of CRISPR-based gene therapy for cervical cancer is its specificity toward the HPV DNA sequence. Although the CRISPR/Cas9 system can recognize sequences in the form of 5′-N 20 NGG-3′, 5 the targeting specificity is mainly determined by the 3′-end of the recognition sites, while mismatches at the 5′-end are tolerable. 21 In our study, we observed that the activities of HPV16 E6-targeted gRNA/Cas9 were specific enough to induce apoptosis only in HPV16-positive SiHa and CaSki cell lines, but not in HPV16-negative C33A and HEK293 cells (Figure 3). Disruption of the E6 oncogene in these cells led directly to restoration of the tumor suppressor p53, as expected ( Figure 5). These data suggested that at least to some extent, our CRISPR/Cas system targeting HPV16 E6 gene is feasible for application as antiviral therapy. We speculate that paired gRNA-guided double nicking of the CRISPR system might further improve the specificity of the HPV16 E6-directed CRISPR/Cas system. 22 In conclusion, our study demonstrated for the first time that the customized CRISPR gRNA/Cas9 could specifically cleave the E6 oncogene, reverse the malignant phenotype, and increase the expression of p53 in HPV16-positive cervical cancer SiHa and CaSki cell lines. Therefore, the HPV16 E6-targeted CRISPR/Cas system might be used as a valuable strategy for the treatment of HPV-related cervical cancer.

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