Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

Handle URI:
http://hdl.handle.net/10754/209404
Title:
Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application
Authors:
Wibowo, Anjar
Abstract:
Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.
Advisors:
Zhu, Jian-Kang ( 0000-0001-5134-731X )
Committee Member:
Gehring, Christoph A ( 0000-0003-4355-4591 ) ; Xiong, Liming ( 0000-0001-8099-0806 )
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
6-Jun-2011
Type:
Thesis
Appears in Collections:
Bioscience Program; Theses; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.advisorZhu, Jian-Kangen
dc.contributor.authorWibowo, Anjaren
dc.date.accessioned2012-02-04T08:39:30Z-
dc.date.available2012-02-04T08:39:30Z-
dc.date.issued2011-06-06en
dc.identifier.urihttp://hdl.handle.net/10754/209404en
dc.description.abstractGene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.en
dc.language.isoenen
dc.titleTranscription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Applicationen
dc.typeThesisen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberGehring, Christoph Aen
dc.contributor.committeememberXiong, Limingen
thesis.degree.disciplineBioscienceen
thesis.degree.nameMaster of Scienceen
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