Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae

Handle URI:
http://hdl.handle.net/10754/575585
Title:
Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae
Authors:
Aouida, Mustapha ( 0000-0001-7945-5320 ) ; Piatek, Marek J.; Bangarusamy, Dhinoth Kumar; Mahfouz, Magdy M. ( 0000-0002-0616-6365 )
Abstract:
The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects. © 2013 Springer-Verlag.
KAUST Department:
Center for Desert Agriculture; Biosciences Core Lab; Bioscience Program
Publisher:
Springer Science + Business Media
Journal:
Current Genetics
Issue Date:
1-Oct-2013
DOI:
10.1007/s00294-013-0412-z
PubMed ID:
24081604
Type:
Article
ISSN:
01728083
Sponsors:
We thank the Bioscience Core Facility and Optical, Imaging Core Lab King Abdullah University of Science and Technology KAUST for technical assistance. We also thank Nina Fedoroff and the members of the genome engineering group at KAUST for their helpful discussions and technical assistance throughout the preparation of the manuscript. No conflict of interest declared. This research is funded from the Center for Desert Agriculture and genome engineering group baseline funding.
Appears in Collections:
Articles; Bioscience Program; Biosciences Core Lab; Center for Desert Agriculture

Full metadata record

DC FieldValue Language
dc.contributor.authorAouida, Mustaphaen
dc.contributor.authorPiatek, Marek J.en
dc.contributor.authorBangarusamy, Dhinoth Kumaren
dc.contributor.authorMahfouz, Magdy M.en
dc.date.accessioned2015-08-24T08:33:30Zen
dc.date.available2015-08-24T08:33:30Zen
dc.date.issued2013-10-01en
dc.identifier.issn01728083en
dc.identifier.pmid24081604en
dc.identifier.doi10.1007/s00294-013-0412-zen
dc.identifier.urihttp://hdl.handle.net/10754/575585en
dc.description.abstractThe development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects. © 2013 Springer-Verlag.en
dc.description.sponsorshipWe thank the Bioscience Core Facility and Optical, Imaging Core Lab King Abdullah University of Science and Technology KAUST for technical assistance. We also thank Nina Fedoroff and the members of the genome engineering group at KAUST for their helpful discussions and technical assistance throughout the preparation of the manuscript. No conflict of interest declared. This research is funded from the Center for Desert Agriculture and genome engineering group baseline funding.en
dc.publisherSpringer Science + Business Mediaen
dc.subjectGenome engineeringen
dc.subjectHomodimeric TALENsen
dc.subjectSaccharomyces cerevisiaeen
dc.subjectTALE-based nucleasesen
dc.subjectTALENsen
dc.subjectTargeted genome modificationen
dc.titleActivities and specificities of homodimeric TALENs in Saccharomyces cerevisiaeen
dc.typeArticleen
dc.contributor.departmentCenter for Desert Agricultureen
dc.contributor.departmentBiosciences Core Laben
dc.contributor.departmentBioscience Programen
dc.identifier.journalCurrent Geneticsen
kaust.authorAouida, Mustaphaen
kaust.authorPiatek, Marek J.en
kaust.authorMahfouz, Magdy M.en

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