Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting
Tomas Cermak,Erin L. Doyle,Michelle Christian,Li-Li Wang,Yong Zhang,Clarice Schmidt,Joshua A. Baller,Nikunj V. Somia,Adam J. Bogdanove,Daniel F. Voytas +9 more
Reads0
Chats0
TLDR
A method and reagents for efficiently assembling TALEN constructs with custom repeat arrays are presented and design guidelines based on naturally occurring TAL effectors and their binding sites are described.Abstract:
TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGeneread more
Citations
More filters
Journal ArticleDOI
Genome engineering using the CRISPR-Cas9 system
F. Ann Ran,Patrick D. Hsu,Jason Wright,Vineeta Agarwala,Vineeta Agarwala,David A. Scott,Feng Zhang +6 more
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Journal ArticleDOI
ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
TL;DR: A review of achievements made possible by site-specific nuclease technologies and applications of these reagents for genetic analysis and manipulation, including the therapeutic potential of ZFNs and TALENs, and future prospects for the field are discussed.
Journal ArticleDOI
Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew
TL;DR: It is shown that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew, and provides a methodological framework to improve polyploid crops.
Journal ArticleDOI
TALENs: a widely applicable technology for targeted genome editing
J. Keith Joung,Jeffry D. Sander +1 more
TL;DR: The newly-developed transcription activator-like effector nucleases (TALENs) comprise a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain that can be easily engineered so that TALens can target essentially any sequence.
Conflict of interest statement. None declared.
TL;DR: It is found that women over 50 are more likely to have a family history of diabetes, especially if they are obese, than women under the age of 50.
References
More filters
Book
Molecular Cloning: A Laboratory Manual
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Journal ArticleDOI
Genome sequence of the human malaria parasite Plasmodium falciparum
Malcolm J. Gardner,Neil Hall,Eula Fung,Owen White,Matthew Berriman,Richard W. Hyman,Jane M. Carlton,Arnab Pain,Karen E. Nelson,Sharen Bowman,Ian T. Paulsen,Keith D. James,Jonathan A. Eisen,Kim Rutherford,Steven L. Salzberg,Alister Craig,Sue Kyes,Man Suen Chan,Vishvanath Nene,Shamira J. Shallom,Bernard B. Suh,Jeremy Peterson,Samuel V. Angiuoli,Mihaela Pertea,Jonathan E. Allen,Jeremy D. Selengut,Daniel H. Haft,Michael W. Mather,Akhil B. Vaidya,David M. A. Martin,Alan H. Fairlamb,Martin Fraunholz,David S. Roos,Stuart A. Ralph,Geoffrey I. McFadden,Leda M. Cummings,G. Mani Subramanian,Christopher J. Mungall,J. Craig Venter,Daniel J. Carucci,Stephen L. Hoffman,Chris I. Newbold,Ronald W. Davis,Claire M. Fraser,Bart Barrell +44 more
TL;DR: The genome sequence of P. falciparum clone 3D7 is reported, which is the most (A + T)-rich genome sequenced to date and is being exploited in the search for new drugs and vaccines to fight malaria.
Journal ArticleDOI
Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors
Jens Boch,Heidi Scholze,Sebastian Schornack,Angelika Landgraf,Simone Hahn,Sabine Kay,Thomas Lahaye,Anja Nickstadt,Ulla Bonas +8 more
TL;DR: The functionality of a distinct type of DNA binding domain is described and allows the design ofDNA binding domains for biotechnology.
Journal ArticleDOI
A TALE nuclease architecture for efficient genome editing
Jeffrey C. Miller,Siyuan Tan,Guijuan Qiao,Kyle A. Barlow,Jianbin Wang,Danny F Xia,Xiangdong Meng,David Paschon,Elo Leung,Sarah J. Hinkley,Gladys P Dulay,Kevin Hua,Irina Ankoudinova,Gregory J. Cost,Fyodor D. Urnov,H. Steve Zhang,Michael C. Holmes,Lei Zhang,Philip D. Gregory,Edward J. Rebar +19 more
TL;DR: This study identifies TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and uses them to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%.
Journal ArticleDOI
Genome editing with engineered zinc finger nucleases
TL;DR: A broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair.