ORIGINAL ARTICLE
Year : 2016  |  Volume : 18  |  Issue : 3  |  Page : 475-479

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


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 Address:
Dr. Xue-Jun Zhang
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
Dr. Zhi-Ming Cai
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
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1008-682X.157399

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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.


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