title: Reprogrammed CRISPR-Cas9 targeting the conserved regions of HPV6/11 E7 genes inhibits proliferation and induces apoptosis in E7-transformed keratinocytes.
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-transformed 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-transformed 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.
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Liu
authors.affiliation: 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.
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Cai
authors.affiliation: 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.
authors.forename: Xue-Jun
authors.initials: XJ
authors.lastname: Zhang
mesh_terms: D017209:Apoptosis / Q000235:genetics*; D015153:Blotting, Western; D064113:CRISPR-Cas Systems; D049109:Cell Proliferation / Q000235:genetics*; D003218:Condylomata Acuminata / Q000628:therapy*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020868:Gene Silencing; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D030361:Papillomavirus Infections / Q000628:therapy*; D014162:Transfection
chemical_list: C493190:E7 protein, human papillomavirus 11; D009856:Oncogene Proteins, Viral; C062495:oncogene protein E7, Human papillomavirus type 6