Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

Abstract

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.

Human Papillomavirus E6 and E7: The Cervical Cancer Hallmarks and Targets for Therapy

“…They were further proved to be clinically more efficient as they could also establish their therapeutic effect in xenograft mouse model (Ding et al, 2014). Shankar et al (2018) reported that they could not successfully inhibit E6 expression and activity using ZFNs as no matching target sequence could be obtained using publicly available computer software. Thus, they used TALENs instead of the same purpose.…”

“…It resulted in a corresponding increase in the amount of pRb and a decrease in p14ARF, the downstream targets following a necrotic pathway of cell death as shown through the upregulation of RIP-1, Cyclophilin A, and LDH-A. In the due course, another study by Shankar et al (2018) reported that TALEN-mediated editing of HPV16 E6 did not yield any editing activity, while E7 could be successfully knocked down in HPV16 infected CasKi cells. E6-targeted TALEN was composed of 18 modules on both the arms spaced by a 19 nucleotide region, while the E7 targeting TALEN contained 18 modules on both the ends separated by a 21 nucleotide gap.…”

Strategies for Targeting Gene Therapy in Cancer Cells With Tumor-Specific Promoters

“…On the other hand, there are transcription activatorlike effector nucleases (TALENs); these ones arise from the binding of a DNA binding domain and a nuclease catalytic domain of Fok1 (47). Using gene editing techniques such as ZFNs and specific TALENs, has been specifically inhibiting cervix cancer cell growth (48)(49)(50) and acute lymphoblastic leukemia (45,51). As well, ZFNs have been used for fighting the resistance to therapeutic agents in breast cancer cells (52).…”

Designer nucleases to treat malignant cancers driven by viral oncogenes

“…A preprint manuscript that targeted HPV18 E6 and E7 genes with CRISPR/Cas explored the anti-tumor mechanisms [ 47 ], and, contrary to other studies, apoptotic features were no present but instead markers of senescence were observed. Shankar et al [ 48 , 49 ], tested TALENs to HPV16 E7, and observed a distinct lack of apoptosis, but instead features of cell cycle arrest and necrosis. Collectively, these studies show that a pleiotropic route to tumor inhibition occurs upon oncogene inactivation in HPV-related cancers.…”