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      The I-TevI Nuclease and Linker Domains Contribute to the Specificity of Monomeric TALENs

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          Abstract

          Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5′-CNNNG-3′. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints.

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          Most cited references 28

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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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              FLASH Assembly of TALENs Enables High-Throughput Genome Editing

              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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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                16 April 2014
                June 2014
                : 4
                : 6
                : 1155-1165
                Affiliations
                [* ]Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1
                []Department of Plant Pathology and Plant-Microbe Biology, Cornell University, 334 Plant Science, Ithaca, New York 14853
                []Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5B7
                Author notes

                This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Supporting information is available online at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.114.011445/-/DC1

                [1]

                These authors contributed equally to the work.

                [2]

                Present address: Molecular Pathology Unit, Massachusetts General Hospital, 149 13 th Street, Charlestown, MA 02129.

                [3 ]Corresponding authors: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada N6A 3B1. E-mail: dedgell@ 123456uwo.ca ; and Department of Plant Pathology and Plant-Microbe Biology, Cornell University, 334 Plant Science, Ithaca, NY 14853. E-mail: ajb7@ 123456cornell.edu
                Article
                GGG_011445
                10.1534/g3.114.011445
                4065259
                24739648
                Copyright © 2014 B. P. Kleinstiver et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Pages: 11
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                Categories
                Investigations
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                v1

                Genetics

                genome editing, tal effector, i-tevi, giy-yig nuclease, monomeric talen

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