Targeting of cholera toxin A ( ctxA ) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes.

Autor: Hosseini N; Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran., Khanahmad H; Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran., Esfahani BN; Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran., Bandehpour M; Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran., Shariati L; Biosensor Research Center, Department of Biomaterials, Nanotechnology and Tissue Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran., Zahedi N; Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran., Kazemi B; Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.; Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.
Jazyk: angličtina
Zdroj: Research in pharmaceutical sciences [Res Pharm Sci] 2020 May 11; Vol. 15 (2), pp. 182-190. Date of Electronic Publication: 2020 May 11 (Print Publication: 2020).
DOI: 10.4103/1735-5362.283818
Abstrakt: Background and Purpose: The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene ( ctxA ) for inhibiting CT toxin production in Vibrio cholera (V. cholera) .
Experimental Approach: An engineered ZFN was designed to target the catalytic site of the ctxA gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and E2-crimson plasmid and transformed to Escherichia coli (E. coli) Top10 and V. cholera . The efficiency of ZFN was evaluated by colony counting.
Findings/results: No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed E. coli . The ctxA gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of E. coli Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of V. cholera with E2-crimson vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay.
Conclusions and Implications: ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.
Competing Interests: The authors declare no conflict of interest for this study.
(Copyright: © 2020 Research in Pharmaceutical Sciences.)
Databáze: MEDLINE
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