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Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes.
Res Pharm Sci. 2020 Apr; 15(2):182-190.RP

Abstract

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.

Authors+Show Affiliations

Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, I.R. Iran.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.Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.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.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32582358

Citation

Hosseini, Nafiseh, et al. "Targeting of Cholera Toxin a (ctxA) Gene By Zinc Finger Nuclease: Pitfalls of Using Gene Editing Tools in Prokaryotes." Research in Pharmaceutical Sciences, vol. 15, no. 2, 2020, pp. 182-190.
Hosseini N, Khanahmad H, Esfahani BN, et al. Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes. Res Pharm Sci. 2020;15(2):182-190.
Hosseini, N., Khanahmad, H., Esfahani, B. N., Bandehpour, M., Shariati, L., Zahedi, N., & Kazemi, B. (2020). Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes. Research in Pharmaceutical Sciences, 15(2), 182-190. https://doi.org/10.4103/1735-5362.283818
Hosseini N, et al. Targeting of Cholera Toxin a (ctxA) Gene By Zinc Finger Nuclease: Pitfalls of Using Gene Editing Tools in Prokaryotes. Res Pharm Sci. 2020;15(2):182-190. PubMed PMID: 32582358.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes. AU - Hosseini,Nafiseh, AU - Khanahmad,Hossein, AU - Esfahani,Bahram Nasr, AU - Bandehpour,Mojgan, AU - Shariati,Laleh, AU - Zahedi,Nushin, AU - Kazemi,Bahram, Y1 - 2020/05/11/ PY - 2019/10/23/received PY - 2019/12/22/revised PY - 2020/04/29/accepted PY - 2020/6/26/entrez PY - 2020/6/26/pubmed PY - 2020/6/26/medline KW - Gene editing tools KW - Vibrio cholerae KW - Zinc finger nuclease KW - ctxA Gene SP - 182 EP - 190 JF - Research in pharmaceutical sciences JO - Res Pharm Sci VL - 15 IS - 2 N2 - 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. SN - 1735-5362 UR - https://www.unboundmedicine.com/medline/citation/32582358/Targeting_of_cholera_toxin_A_(ctxA)_gene_by_zinc_finger_nuclease:_pitfalls_of_using_gene_editing_tools_in_prokaryotes L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/32582358/ DB - PRIME DP - Unbound Medicine ER -
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