Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes
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....
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2020-01-01
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| Series: | Research in Pharmaceutical Sciences |
| Subjects: | |
| Online Access: | http://www.rpsjournal.net/article.asp?issn=1735-5362;year=2020;volume=15;issue=2;spage=182;epage=190;aulast=Hosseini |
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| author | Nafiseh Hosseini Hossein Khanahmad Bahram Nasr Esfahani Mojgan Bandehpour Laleh Shariati Nushin Zahedi Bahram Kazemi |
| author_facet | Nafiseh Hosseini Hossein Khanahmad Bahram Nasr Esfahani Mojgan Bandehpour Laleh Shariati Nushin Zahedi Bahram Kazemi |
| author_sort | Nafiseh Hosseini |
| collection | DOAJ |
| description | 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 andV. 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. |
| first_indexed | 2024-12-19T10:34:06Z |
| format | Article |
| id | doaj.art-3b75f8a3196a4df8b83d0f5bf9b0a76f |
| institution | Directory Open Access Journal |
| issn | 1735-5362 1735-9414 |
| language | English |
| last_indexed | 2024-12-19T10:34:06Z |
| publishDate | 2020-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
| record_format | Article |
| series | Research in Pharmaceutical Sciences |
| spelling | doaj.art-3b75f8a3196a4df8b83d0f5bf9b0a76f2022-12-21T20:25:39ZengWolters Kluwer Medknow PublicationsResearch in Pharmaceutical Sciences1735-53621735-94142020-01-0115218219010.4103/1735-5362.283818Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotesNafiseh HosseiniHossein KhanahmadBahram Nasr EsfahaniMojgan BandehpourLaleh ShariatiNushin ZahediBahram KazemiBackground 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 andV. 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.http://www.rpsjournal.net/article.asp?issn=1735-5362;year=2020;volume=15;issue=2;spage=182;epage=190;aulast=Hosseinictxa gene; gene editing tools; vibrio cholerae; zinc finger nuclease. |
| spellingShingle | Nafiseh Hosseini Hossein Khanahmad Bahram Nasr Esfahani Mojgan Bandehpour Laleh Shariati Nushin Zahedi Bahram Kazemi Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes Research in Pharmaceutical Sciences ctxa gene; gene editing tools; vibrio cholerae; zinc finger nuclease. |
| title | Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes |
| title_full | Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes |
| title_fullStr | Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes |
| title_full_unstemmed | Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes |
| title_short | Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes |
| title_sort | targeting of cholera toxin a ctxa gene by zinc finger nuclease pitfalls of using gene editing tools in prokaryotes |
| topic | ctxa gene; gene editing tools; vibrio cholerae; zinc finger nuclease. |
| url | http://www.rpsjournal.net/article.asp?issn=1735-5362;year=2020;volume=15;issue=2;spage=182;epage=190;aulast=Hosseini |
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