Sumitra , Shankar and Ahalya, Sreekumar and Deepti, Prasad and Ani V. , Das and Pillai, M. R (2018) Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design. Cancer cell international, 18 (169). ISSN 1475-2867

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Abstract

Gene 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. Methods HPV 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. Results Although 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. Conclusions Our 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 material The online version of this article (10.1186/s12935-018-0666-0) contains supplementary material, which is available to authorized users.

Item Type:	Article
Uncontrolled Keywords:	Cervical cancer; Gene editing; HPV; TALEN; Zinc-finger nucleases
Subjects:	Cancer Research
Depositing User:	Central Library RGCB
Date Deposited:	10 Dec 2018 12:03
Last Modified:	10 Dec 2018 12:03
URI:	http://rgcb.sciencecentral.in/id/eprint/710

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