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      Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

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          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 35 bp. 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 AddGene and a web-based version of our software is freely accessible online.

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          Most cited references21

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          A TALE nuclease architecture for efficient genome editing.

          Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
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            Targeting DNA double-strand breaks with TAL effector nucleases.

            Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
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              Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

              We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
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                Author and article information

                Journal
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                July 2011
                July 2011
                14 April 2011
                14 April 2011
                : 39
                : 12
                : e82
                Affiliations
                1Department of Genetics, Cell Biology & Development and Center for Genome Engineering, 321 Church Street SE, University of Minnesota, Minneapolis, MN 55455 2Department of Plant Pathology, 351 Bessey Hall, Iowa State University, Ames, IA 50011, USA, 3Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chendu 610054, China and 4Biomedical Informatics and Computational Biology Program, University of Minnesota Rochester, 111 South Broadway, Rochester, MN 55904, USA
                Author notes
                *To whom correspondence should be addressed. Tel: +1 612 626 4509; Fax: +1 612 626 2600; Email: voytas@ 123456umn.edu
                Correspondence may also be addressed to Adam J. Bogdanove. Tel: +1 515 294 3421; Fax: +1 515 294 9420; Email: ajbog@ 123456iastate.edu

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Article
                gkr218
                10.1093/nar/gkr218
                3130291
                21493687
                d9ec713f-8520-49ab-beec-11743010ea23
                © The Author(s) 2011. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 8 March 2011
                : 23 March 2011
                : 24 March 2011
                Page count
                Pages: 11
                Categories
                Methods Online

                Genetics
                Genetics

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