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

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 E7 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 E7 genes was constructed by using a dual guide RNA vector. After transfection of the vector into E7-transfromed keratinocytes, the expression level of E7 protein was measured using western-blot analysis and the sequence of the E7 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 E7 genes can be inactivated by the single CRISPR-Cas9 system. Furthermore, silencing of E7 led to inhibition of cell proliferation and induction of apoptosis in E7-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.