Human Papillomavirus E7 and E6 oncoproteins have been considered as suitable candidate anti-viral targets since they cause malignant conversion in cervical cancers. Transcription Activator-Like Effector Nucleases (TALENs) are recent editing tools to knockout genes by inducing double stranded breaks at specific sites in the genome. In here, we have designed specific TALENs to target E7 and analyzed their efficiency in inducing cell death in cervical cancer cells. We found that designed TALENs could yield about 10–12% editing activity as observed from T7E1 and nuclease resistance assays. Down-regulation of E7 and E6 was further evident at the transcript as well as proteins levels indicating that the selected TALENs were effective. TALEN-mediated E7 editing led to cell death as ascertained by cell cycle and Annexin V assays. Annexin profiling suggested that cell death could be due to necrosis as observed by upregulation of necrotic markers such as LDH A, Rip-1, and Cyclophilin A. Necrosis appears to be a better therapeutic response as it could further activate pro-inflammatory cytokines to attract immune cells to eliminate HPV-integrated cells and therefore TALEN editing strategy has the potential to be a promising tool as an adjuvant therapy in cervical cancer along with surgery.

BackgroundGene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Gene editing using Zinc-finger nucleases (ZFNs), Transcription activator like effectors (TALEs) and CRISPR/Cas systems has been used to create changes in the genome in order to make it non-functional. In the present study, we have looked into the possibility of using six fingered CompoZr ZFN pair to target the E6 gene of HPV 16 genome.MethodsHPV 16+ve cell lines; SiHa and CaSki were used for experiments. CompoZr ZFNs targeting E6 gene were designed and constructed by Sigma-Aldrich. TALENs targeting E6 and E7 genes were made using TALEN assembly kit. Gene editing was monitored by T7E1 mismatch nuclease and Nuclease resistance assays. Levels of E6 and E7 were further analyzed by RT-PCR, western blot as well as immunoflourescence analyses. To check if there is any interference due to methylation, cell lines were treated with sodium butyrate, and Nocodazole.ResultsAlthough ZFN editing activity in yeast based MEL-I assay was high, it yielded very low activity in tumor cell lines; only 6% editing in CaSki and negligible activity in SiHa cell lines. Though editing efficiency was better in CaSki, no significant reduction in E6 protein levels was observed in immunocytochemical analysis. Further, in silico analysis of DNA binding prediction revealed that some of the ZFN modules bound to sequence that did not match the target sequence. Hence, alternate ZFN pairs for E6 and E7 were not synthesized since no further active sites could be identified by in silico analyses. Then we designed TALENs to target E6 and E7 gene. TALENs designed to target E7 gene led to reduction of E7 levels in CaSki and SiHa cervical cancer cell lines. However, TALEN designed to target E6 gene did not yield any editing activity.ConclusionsOur study highlights that designed nucleases intended to obtain bulk effect should have a reasonable editing activity which reflects phenotypically as well. Nucleases with low editing efficiency, intended for generation of knockout cell lines nucleases could be obtained by single cell cloning. This could serve as a criterion for designing ZFNs and TALENs.Electronic supplementary materialThe online version of this article (10.1186/s12935-018-0666-0) contains supplementary material, which is available to authorized users.

Synthetic biology concerns applying engineering principles to biological systems. Engineering properties such as fine tuning, novel specificity, and modularity could be components of a synthetic toolkit that can be exploited to explore various issues in cancer research such as elucidation of mechanisms and pathways, creating new diagnostic tools and novel therapeutic approaches. A repertoire of synthetic biology toolkits involving DNA, RNA and protein bio-parts, have been applied to address the issues of drug target identification, drug discovery and therapeutic treatment in cancer research, thereby projecting a new dimension in oncology research.