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      Programmable Sequence-Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors

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          Abstract

          The ability to direct functional domains to specific DNA sequences is a long sought-after goal for studying and engineering biological processes. Transcription activator like effectors (TALEs) from Xanthomonas sp. present a promising platform for designing sequence-specific DNA binding proteins. Here we describe a robust and rapid method for overcoming the difficulty of constructing TALE repeat domains. We synthesized 17 designer TALEs (dTALEs) that are customized to recognize specific DNA binding sites, and demonstrate that dTALEs can specifically modulate transcription of endogenous genes (Sox2 and Klf4) from the native genome in human cells. dTALEs provide a designable DNA targeting platform for the interrogation and engineering of biological systems.

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          A TALE nuclease architecture for efficient genome editing.

          Jeffrey Miller, Siyuan Tan, Guijuan Qiao … (2011)
          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.

            Michelle Christian, Tomas Cermak, Erin L. Doyle … (2010)
            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

              Carola Engler, Ramona Gruetzner, Romy Kandzia … (2009)
              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
                Journal ID (nlm-journal-id): 9604648
                Journal ID (pubmed-jr-id): 20305
                Journal ID (nlm-ta): Nat Biotechnol
                Title: Nature biotechnology
                ISSN (Print): 1087-0156
                ISSN (Electronic): 1546-1696
                Publication date Nihms-submitted: 8 February 2011
                Publication date (Electronic): 19 January 2011
                Publication date (Print): February 2011
                Publication date PMC-release: 1 August 2011
                Volume: 29
                Issue: 2
                Pages: 149-153
                Affiliations
                [1 ] Society of Fellows
                [2 ] Department of Genetics, Harvard Medical School
                [3 ] Wyss Institute for Biologically Inspired Engineering
                [4 ] Program in Biological and Biomedical Sciences, Harvard Medical School
                [5 ] Department of Stem Cell and Regenerative Biology Harvard University
                [6 ] Center for Regenerative Medicine and Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School
                Author notes
                [± ]To whom correspondence should be addressed: Feng Zhang, Ph.D., Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, zhang_f@ 123456mit.edu . George Church, Ph.D., Department of Genetics, Harvard Medical School, New Research Building, Rm 238, 77 Avenue Louis Pasteur, Boston, MA 02115, gmc@ 123456harvard.edu . Paola Arlotta, Ph.D., Department of Stem Cell and Regenerative Biology, Harvard University, Center for Regenerative Medicine, Massachusetts General Hospital, Simches Building 4238, 185 Cambridge Street, Boston, MA 02114, paola_arlotta@ 123456hms.harvard.edu
                [*]

                These authors contributed equally to this work.

                [#]

                These authors contributed equally to this work.

                [7]

                Present Address: Broad Institute of MIT and Harvard, Cambridge, MA, McGovern Institute for Brain Research, MIT, Cambridge, MA, Department of Brain and Cognitive Sciences, MIT Cambridge, MA

                Article
                Manuscript ID: nihpa264159
                DOI: 10.1038/nbt.1775
                PMC ID: 3084533
                PubMed ID: 21248753
                SO-VID: b4069536-27c6-42cc-92ba-0e3353d0ad37
                License:

                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

                History
                Funding
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R01 NS073124-01 ||NS
                Categories
                Subject: Article

                ScienceOpen disciplines: Biotechnology
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                ScienceOpen disciplines: Biotechnology

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