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      FLASH Assembly of TALENs Enables High-Throughput Genome Editing

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          Abstract

          Engineered transcription activator-like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) platform, a rapid and cost-effective method we developed to enable large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell-based EGFP reporter system and found that all 48 possessed efficient gene modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with engineered zinc-finger nucleases or meganucleases.

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          Most cited references 25

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          Cancer genes and the pathways they control.

          The revolution in cancer research can be summed up in a single sentence: cancer is, in essence, a genetic disease. In the last decade, many important genes responsible for the genesis of various cancers have been discovered, their mutations precisely identified, and the pathways through which they act characterized. The purposes of this review are to highlight examples of progress in these areas, indicate where knowledge is scarce and point out fertile grounds for future investigation.
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            Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

            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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              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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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat. Biotechnol.
                Nature biotechnology
                1087-0156
                1546-1696
                30 March 2012
                May 2012
                30 January 2013
                : 30
                : 5
                : 460-465
                Affiliations
                [1 ]Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129
                [2 ]Department of Genetics, Development & Cell Biology, Interdepartmental Graduate Program in Bioinformatics & Computational Biology, Iowa State University, Ames, IA 50011
                [3 ]Department of Pathology, Harvard Medical School, Boston, MA 02115
                Author notes
                [*]

                These authors contributed equally to this work

                Article
                NIHMS359665
                10.1038/nbt.2170
                3558947
                22484455

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Funding
                Funded by: National Cancer Institute : NCI
                Award ID: T32 CA009216-30 || CA
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: P50 HG005550-01 || HG
                Funded by: Office of the Director : NIH
                Award ID: DP1 OD006862-02 || OD
                Funded by: Office of the Director : NIH
                Award ID: DP1 OD006862-01 || OD
                Categories
                Article

                Biotechnology

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