title: Hyper expressing p53 in HPV positive HeLa cells using CRISPRa
Creators: name: Lai, Alan J
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Human Papillomaviruses (HPV) infection
subject: p53 protein
subject: CRISPR
subject: gRNA sequences

description: Human Papillomaviruses (HPV) is the leading cause of cervical cancer, with 95% of cervical cancer being attributed to HPV infection. There is a link between HPV infection of the epithelial cell and uncontrolled cell growth and cancer progression as HPV infection is implicated in 99% of cervical cancer, and approximately 50% of all cervical intraepithelial neoplasia and 70% of all cervical cancers. The National Institute of Health found that cervical cancer has a rate of 6.6 per 10 000 in 2019, down from 9.4 per 10 000 in 1999. Although the rates of cervical cancer has fallen over the years, thanks in part due to HPV vaccinations, increased awareness of cervical cancer and early screenings, the rates of Head and Neck cancers have stayed the same, at 10.8 per 10 000. Current treatment methods for cervical cancer depend on the disease progression at the time of diagnosis, but if the cervical cancer has metastasised to the lymph nodes, combined radical surgery and radiation therapy is required. This treatment has 28% morbidity rate that has not seen significant reduction in recent years despite advancements in modern medicine. Thus, there is an interest in developing novel and innovative treatments that can treat HPV positive cancers with reduced complications. HPV increase the risk of infected cells developing cancer by promoting genetic instability and inhibiting the action of cell-cycle repressor proteins such as p53. The p53 protein is a powerful tumour suppressor that is involved many cell-cycle controls such as cell cycle arrest, DNA repair, apoptosis, senescence, anti-angiogenesis, autophagy, and synthesis of metabolic antioxidants. Thus, the HPV genome contains two viral proteins, E6, and E7. E6 ubiquitinates the p53, marking the p53 for degradation by the host cell’s own ubiquitin proteasome system. This impairs the cell’s ability to regulate its cell-cycle in response to DNA damage and increases the risk of cancer.E7binds to hypophosphorylated Rb and degrades Rb, inhibiting Rb function to control the G1 checkpoint in the cell cycle. Restoring the level of p53 has been proposed as a novel way to treat cancers, and one possible way is by increasing transcription. CRISPR activation (CRISPRa) has been considered as a new way to increase transcription of p53. CRISPRa works by designing a guide RNA that guides a catalytic-deficient cas9 (dcas9) to the transcriptional start site of the desired gene. By attaching transcriptional activators such as Rta, p65 and VP64 to the dcas9, it is possible to promote transcription of the desired gene, with the hope that this system could overcome E6 ubiquitination and restore p53 function in the cell. Two different guide RNA (gRNA) sequences (called gRNA1 and gRNA2) that target two different sequences upstream of the p53 promoter were cloned into the gRNA scaffold of the Doxycycline-dependent plasmid FgH1t_UTG, verified using PshAI restriction enzyme digestion and Sanger sequencing. The gRNA-FgH1t_UTG and Sp-dcas9-VPR was transiently transfected using Fugene into HeLa cells and cultured in media containing 3μg/mL of Doxycycline. Differences in p53 levels were assessed using western blotting while differences in p53 mRNA transcripts was assessed using Q-PCR. Cell viability was then assessed by MTT Assay and Colony Forming Assay after seven days after transfection. The results suggests that transient transfection of HeLa cells with dcas9 and gRNA had successfully increased the p53 levels in the transiently transfected HeLa cells over the WT-HeLa cells. Quantification of the protein extracts from the HeLa cells, transfected with dcas9+gRNA1 showed approximately 2.5x increase in p53 quantity over WT-HeLa while dcas9+gRNA2 showed an increase of 2.4x increase in p53 quantity. In regards to changes in cell viability, gRNA1 was more effective at reducing the cell viability in the MTT assay by 2/3 compared to WT-HeLa while gRNA2 did not have a noticeable impact on cell viability. This is further demonstrated with the Colony Forming Assay where the gRNA1 caused reduced the cell colonies 42% while gRNA2 did not have noticeable impact on cell viability. Downstream effects of increased p53 levels in HeLa was found to be inconsistent, with increases in p21 found in the transiently transfected HeLa cells, but not PARP or Caspase-3. This study has demonstrated that transient transfection of dcas9 and gRNA can increase the level of p53 presence and transcription in HeLa cells and inhibit cell growth. Further research is required to determine the most effective gRNAs in increasing p53 and other cell-cycle control proteins in a broader range of HPV positive cells, along with determining the mechanism of action of this CRISPRa.