Study On The Effect And Mechanism Of Reprogrammed CRISPR-Cas9 In Proliferation And Apoptosis Of HPV6/11 E7 Genes-transformed Cells

Background Human papillomaviruses(HPVs) are important human pathogens that cause sexually transmitted diseases. The most common high risk HPV types, 16 and 18,are frequently detected in cervical and penile cancers. The low risk HPV types, 6 and 11,are associated with anogenital warts and laryngeal papillomatosis. The persistence infection of low-risk type(type 6 or type 11) of human papillomavirus(HPV)is the main cause of genital warts. The current treatments for genital warts usually include trichloracetic acid, cryotherapy, and surgical removal. Although there are many approaches for treating this disease, the rate of recurrence after successful clearance is still very high. Further improvements in treatment outcome may derive from a combination of conventional therapy with novel molecular agents.HPV-related lesions require continued production of the oncogenic E7 protein, which has targets in both nuclear and cytoplasm and promotes S-phase induction in differentiated human keratinocytes.Small molecule inhibitors targeting the E7 oncogene can be used for improving the therapeutic effects of current therapy for HPV-related genital diseases.These molecular agents usually include antisense oligodeoxynucleotides, ribozymes,and short interfering RNA(si RNA). However, these approaches have only achieved limited success due to the low accessibility of most sites on HPV6/11 E7 m RNAs.Therefore, treatments based on new molecular agents are still to be developed. DNA interference(DNAi) is a recently described natural phenomenon mediated by the bacterial type II CRISPR(clustered regularly interspaced short palindrome repeats)-Cas9 system. This system can be reprogrammed to induce DNA double strand breaks(DSBs) at specific genomic loci and to create frame shift indel mutations that result in aloss-of-function of the target gene. The potential of CRISPR-Cas9 to treat or prevent human genetic diseases has yet to be proven.Objective(1) To study the molecular mechanism of inactivation of viral E7 gene in keratinocytes induced by reprogrammed CRISPR-Cas9 system.(2) To test the possibility of using the reprogrammed CRISPR-Cas9 system as a novel molecular agent for against E7 genes of low risk HPV types.Methods(1) Primary human keratinocytes were isolated from the foreskin and grown in specfic media that were suitable for keratinocytes growth at 37°C in an atmosphere of5% CO2.(2) The coding sequences for HPV6 E7(Gen Bank: HG793938.1) and HPV11E7(Gen Bank: KC329894.1) were chemically synthesized and individually inserted into pc DNA3.1(+) digested with Hind III and Eco R I to construct the plasmids pc DNA3.1-HPV6-E7 and pc DNA3.1-HPV11-E7.(3) We designed two different sg RNA sequences and inserted them into the sg RNA expression cassettes of p CRISPR-CG01 vector containing U6 promoter to drive the transcription of each sg RNA to construct the reprogrammed CRISPR-Cas9 system that targets E7 genes of HPV 6/11.(4) Human keratinocytes stably expressing either the HPV6 E7 gene or the HPV11 E7 gene were obtained by selecting cells with G418 after transfection with the related plasmids. The transformed keratinocytes were used as the test models for investigating the effects of CRISPR-cas9.(5) Cells were cultured in the plates till they reached 70% confluency and transfected with the Cas9- and dual sg RNAs expressing plasmids by using Lipofectamine 2000(Invitrogen).(6) The expression levels of E7 proteins at 48 hours post-transfection were detected using western-blot analysis. PCR products of HPV 6/11 E7 genes were subjected to validation using Sanger sequencing.(7) The proliferations of keratinocytes were determined using CCK-8 assay at the related time points.(8) At 48 hours post-transfection, morphological assessment of apoptotic cells was performed using Hoechst 33258 staining kit and theactivity of Caspase-3 was determined using the Enzyme-linked immunosorbent assay kit. The rate of cell apoptosis was also calculated by flow cytometry assay.Result Based on the alignment of sequences of E7 genes of HPV6/11, two highly conserved regions were selected as the targets of the dual sg RNAs. We constructed a single plasmid for expressing the human codon–optimized Cas9 protein and dual sg RNAs that can be used to make two cuts simultaneously at designated sites. A CRISPR-Cas9 system that expresses sg RNAs lacking the complementary regions was also used as the negative control.The E7 gene of HPV6 or HPV11 caused significant promotion of cell growth when compared with normal keratinocytes transfected with empty pc DNA3.1(+) vector.Expression of the single CRISPR-Cas9 system resulted in loss of both HPV6 E7 protein and HPV11 E7 protein. The results of sequencing indicated that Cas9 deleted the intervening DNA segments of E7. The proliferations of keratinocytes were decreased when cells were treated with the reprogrammed CRISPR-Cas9 and such effects were not observed in keratinocytes stably transfected with empty pc DNA3.1(+) vector. Cells transfected with the reprogrammed CRISPR-Cas9 exhibited strong blue fluorescence,revealing the typical apoptosis characteristics. An increase of caspase 3 was also observed in E7-transformed keratinocytes when transfected with the reprogrammed CRISPR-Cas9.The rate of apoptosis was increased in E7-transformed keratinocytes transfected with the reprogrammed CRISPR-Cas9. Such effects were also not observed in keratinocytes stably transfected with empty pc DNA3.1(+) vector.Conclusion We have developed a kind of potent CRISPR-Cas9 that suppresses cell growth and induces apoptosis in HPV E7-transformed cells through inactivating E7,while having minimal effect in E7-negative human cells. The reprogrammed CRISPR-Cas9 may be further developed as an adjuvant therapy for genital warts.