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Abstract

Volume 18, Issue 3 (May 2016) 18, 475–479; 10.4103/1008-682X.157399

Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes

Yu-Chen Liu1, Zhi-Ming Cai2, Xue-Jun Zhang1

1 Institute of Dermatology, Department of Dermatology, The First Affiliated Hospital, Anhui Medical University, Hefei; Key Laboratory of Dermatology, Ministry of Education, State Key Laboratory of Dermatology Incubation Center, Department of Dermatology, Anhui Medical University, Hefei, China
    2 Key Laboratory of Medical Reprogramming Technology, Department of Urology, The Genitourinary Institution of Shenzhen University, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, China

Correspondence: Dr. XJ Zhang (ayzxj@vip.sina.com) or Dr. ZM Cai (caizhiming2000@163.com)

2015-7-31

Abstract

The persistence infection of low-risk type (type 6 or type 11) of human papillomavirus (HPV) is the main cause of genital warts. Given the high rate of recurrence after treatment, the use of a new molecular agent is certain to be of value. The aim of this study was to achieve targeted inactivation of viral E 7 gene in keratinocytes using the reprogrammed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system. To accomplish this, a universal CRISPR-Cas9 system for targeting both HPV6/11 E 7 genes was constructed by using a dual guide RNA vector. After transfection of the vector into E 7-transfromed keratinocytes, the expression level of E 7 protein was measured using western-blot analysis and the sequence of the E 7 gene was determined using Sanger sequencing. Cell proliferation was analyzed by CCK-8 assay, and cell apoptosis was evaluated by Hoechst 33258 staining, flow cytometry analysis and ELISA assay. The results indicated that both HPV6/11 E 7 genes can be inactivated by the single CRISPR-Cas9 system. Furthermore, silencing of E 7 led to inhibition of cell proliferation and induction of apoptosis in E 7-transfromed keratinocytes but not in normal keratinocytes. Our data suggested that the reprogrammed CRISPR-Cas9 system has the potential for the development of an adjuvant therapy for genital warts.

Keywords: Keywords: apoptosis; CRISPR-Cas9; HPV E 7; proliferation

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