Antiproliferative effects of AAV-delivered CRISPR/Cas9-based degradation of the HPV18-E6 gene in HeLa cells

Zahra Noroozi,Zahra Azizi,Elahe Valipour,Sahar Esfandyari,Amir Monfaredan,Elahe Motevaseli,Mehdi Shamsara,Alireza Ehghaghi,Mohammad Hossein Modarressi

doi:10.1038/s41598-022-06025-w

Abstract

Human papillomavirus infections are associated with most cervical cancers, which are the fourth most common cancer in women. HPV-E6 protein binds to protein p53 and inhibits its function, leading to the switching of normal cells toward cancer cells. Here, we disrupted the HPV-E6 gene and investigated its effects on the proliferation and apoptosis of HeLa cells. The HPV18-E6 gene was targeted with two designed sgRNAs cloned into an AAV-CRISPR-based plasmid. The AAV-E6-CRISPR/Cas9 virions were prepared and titrated in HEK293t cells. The cleavage created in the HPV-E6 gene was detected using the T7E1 assay. Cell cycle profiling, MTT assay, and annexin V/PI staining were performed. Also, the p53 protein level was measured by Western blotting. Our data showed that disruption of the HPV-E6 gene led to increased cell apoptosis and decreased cell proliferation. A significant accumulation of infected cells in sub-G1 phase was observed in the cell profiling assay. Also, HPV-E6 gene disruption resulted in a significant increase in the level of P53 protein. Our findings indicated that AAV-mediated delivery of CRISPR/Cas9 can effectively target the HPV-E6 gene in HeLa cells, and its antiproliferative effects may provide therapeutic benefits of local administration of this gene-editing system for HPV-related cervical cancers.

Highlights

Cervical cancer is the fourth most common cancer in women and the fourth leading cause of cancer death, with an estimated 604,000 new cases and 342,000 deaths in 20201
In order to investigate the effect of the associated viruses (AAVs)-mediated delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] editing tool on the viral E6 gene, two single guide RNAs (sgRNAs) were designed and cloned into the pX601 AAV transfer vector encoding SaCas[9] protein fused with mCherry
The HeLa cells were transduced with a multiplicity of infection (MOI) of about 30

Summary

Introduction

Cervical cancer is the fourth most common cancer in women and the fourth leading cause of cancer death, with an estimated 604,000 new cases and 342,000 deaths in 20201. One of the main functions of these proteins is to inhibit tumor suppressor proteins such as p53 and Rb, which cause host cells to switch toward cancerous cells[6,7]. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system is a new evolving technology that has made it possible to modify genes in a variety of s pecies[8]. This powerful genome-editing tool consists of single guide RNAs (sgRNAs) and the effector protein Cas[99,10]. Under the guidance of sgRNAs, Cas[9] endonuclease creates double-strand breaks (DSBs) at a specific site of targeted DNA complementary to guide RNA. Cas[9] has been used for various genetic disorders that have been associated with satisfactory consequences of gene editing[13,15,16,17]

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