Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

Handle URI:
http://hdl.handle.net/10754/562063
Title:
Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification
Authors:
Li, Lixin; Piatek, Marek J.; Atef, Ahmed; Piatek, Agnieszka Anna ( 0000-0001-9587-6360 ) ; Wibowo, Anjar Tri; Fang, Xiaoyun; Sabir, Jamal Sabir M; Zhu, Jiankang; Mahfouz, Magdy M. ( 0000-0002-0616-6365 )
Abstract:
Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants. © 2012 Springer Science+Business Media B.V.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Center for Desert Agriculture; Bioscience Program
Publisher:
Springer Nature
Journal:
Plant Molecular Biology
Issue Date:
22-Jan-2012
DOI:
10.1007/s11103-012-9875-4
PubMed ID:
22271303
PubMed Central ID:
PMC3580834
Type:
Article
ISSN:
01674412
Additional Links:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580834
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.authorLi, Lixinen
dc.contributor.authorPiatek, Marek J.en
dc.contributor.authorAtef, Ahmeden
dc.contributor.authorPiatek, Agnieszka Annaen
dc.contributor.authorWibowo, Anjar Trien
dc.contributor.authorFang, Xiaoyunen
dc.contributor.authorSabir, Jamal Sabir Men
dc.contributor.authorZhu, Jiankangen
dc.contributor.authorMahfouz, Magdy M.en
dc.date.accessioned2015-08-03T09:43:53Zen
dc.date.available2015-08-03T09:43:53Zen
dc.date.issued2012-01-22en
dc.identifier.issn01674412en
dc.identifier.pmid22271303en
dc.identifier.doi10.1007/s11103-012-9875-4en
dc.identifier.urihttp://hdl.handle.net/10754/562063en
dc.description.abstractTranscription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants. © 2012 Springer Science+Business Media B.V.en
dc.publisherSpringer Natureen
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580834en
dc.subjectGenome engineeringen
dc.subjectGenome modificationsen
dc.subjectTALE nucleases (TALENs)en
dc.subjectTALE-based activators and repressorsen
dc.subjectTargeted activation and repressionen
dc.subjectTargeted mutagenesisen
dc.titleRapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modificationen
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentCenter for Desert Agricultureen
dc.contributor.departmentBioscience Programen
dc.identifier.journalPlant Molecular Biologyen
dc.identifier.pmcidPMC3580834en
dc.contributor.institutionDepartment of Biological Sciences, Faculty of Sciences, King Abdulaziz University, P.O. Box 80141, Jeddah 21589, Saudi Arabiaen
dc.contributor.institutionDepartment of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, United Statesen
kaust.authorLi, Lixinen
kaust.authorWibowo, Anjar Trien
kaust.authorMahfouz, Magdy M.en
kaust.authorPiatek, Marek J.en
kaust.authorPiatek, Agnieszka Annaen
kaust.authorFang, Xiaoyunen

Related articles on PubMed

All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.