De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

Handle URI:
http://hdl.handle.net/10754/561706
Title:
De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks
Authors:
Mahfouz, Magdy M. ( 0000-0002-0616-6365 ) ; Li, Lixin; Shamimuzzaman, Md.; Wibowo, Anjar Tri; Fang, Xiaoyun; Zhu, Jian-Kang
Abstract:
Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.
KAUST Department:
Center for Desert Agriculture; Bioscience Program; Biological and Environmental Sciences and Engineering (BESE) Division
Publisher:
National Academy of Sciences
Journal:
Proceedings of the National Academy of Sciences of the United States of America
Issue Date:
24-Jan-2011
DOI:
10.1073/pnas.1019533108
PubMed ID:
21262818
PubMed Central ID:
PMC3038751
Type:
Article
ISSN:
00278424
Additional Links:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038751
Appears in Collections:
Articles; Bioscience Program; Center for Desert Agriculture; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorMahfouz, Magdy M.en
dc.contributor.authorLi, Lixinen
dc.contributor.authorShamimuzzaman, Md.en
dc.contributor.authorWibowo, Anjar Trien
dc.contributor.authorFang, Xiaoyunen
dc.contributor.authorZhu, Jian-Kangen
dc.date.accessioned2015-08-03T09:02:46Zen
dc.date.available2015-08-03T09:02:46Zen
dc.date.issued2011-01-24en
dc.identifier.issn00278424en
dc.identifier.pmid21262818en
dc.identifier.doi10.1073/pnas.1019533108en
dc.identifier.urihttp://hdl.handle.net/10754/561706en
dc.description.abstractSite-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.en
dc.publisherNational Academy of Sciencesen
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038751en
dc.subjectArtificial activatorsen
dc.subjectMolecular scissorsen
dc.subjectNontransgenic mutagenesisen
dc.titleDe novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaksen
dc.typeArticleen
dc.contributor.departmentCenter for Desert Agricultureen
dc.contributor.departmentBioscience Programen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalProceedings of the National Academy of Sciences of the United States of Americaen
dc.identifier.pmcidPMC3038751en
kaust.authorMahfouz, Magdy M.en
kaust.authorLi, Lixinen
kaust.authorWibowo, Anjar Trien
kaust.authorZhu, Jian-Kangen
kaust.authorShamimuzzaman, Md.en
kaust.authorFang, Xiaoyunen

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