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      Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases

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          Abstract

          We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish ( Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg.

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          Most cited references 25

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          An improved zinc-finger nuclease architecture for highly specific genome editing.

          Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.
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            Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.

            A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.
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              Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression.

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                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nature biotechnology
                1087-0156
                1546-1696
                12 March 2009
                25 May 2008
                June 2008
                29 April 2009
                : 26
                : 6
                : 702-708
                Affiliations
                [1 ] Department of Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley, CA 94720-3200, USA
                [2 ] Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, CA
                Author notes
                [3 ]To whom correspondence should be addressed: amacher@ 123456berkeley.edu . Correspondence regarding, and requests for, zinc finger nuclease reagents described in the paper should be directed to furnov@ 123456sangamo.com
                [4]

                These authors contributed equally to this work.

                Article
                nihpa100553
                10.1038/nbt1409
                2674762
                18500334
                Funding
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-06A2S1 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-06A2 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-05 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-04 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-03 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-02 ||GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM061952-01A2 ||GM
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                Biotechnology

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