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Jianbin Wang,
Geoffrey Friedman, Yannick Doyon,
Nathaniel S Wang,
Carrie Jiaxin Li,
Jeffrey C Miller,
Kevin L Hua,
Jenny Jiacheng Yan,
Joshua E Babiarz,
Philip D Gregory,
Michael C Holmes
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ABSTRACT: Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%-8%. Interestingly, ZFNickases targeting the AAVS1 "safe harbor" locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition-suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.
Genome Research 03/2012; 22(7):1316-26. · 13.61 Impact Factor
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Hojun Li,
Virginia Haurigot, Yannick Doyon,
Tianjian Li,
Sunnie Y Wong,
Anand S Bhagwat,
Nirav Malani,
Xavier M Anguela,
Rajiv Sharma,
Lacramiora Ivanciu,
Samuel L Murphy,
Jonathan D Finn,
Fayaz R Khazi,
Shangzhen Zhou,
David E Paschon,
Edward J Rebar,
Frederic D Bushman,
Philip D Gregory,
Michael C Holmes,
Katherine A High
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ABSTRACT: Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.
Nature 06/2011; 475(7355):217-21. · 36.28 Impact Factor
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John J Young,
Jennifer M Cherone, Yannick Doyon,
Irina Ankoudinova,
Farhoud M Faraji,
Andrew H Lee,
Catherine Ngo,
Dmitry Y Guschin,
David E Paschon,
Jeffrey C Miller,
Lei Zhang,
Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Richard M Harland,
Bryan Zeitler
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ABSTRACT: The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.
Proceedings of the National Academy of Sciences 04/2011; 108(17):7052-7. · 9.68 Impact Factor
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Jeffrey B Doyon,
Bryan Zeitler,
Jackie Cheng,
Aaron T Cheng,
Jennifer M Cherone,
Yolanda Santiago,
Andrew H Lee,
Thuy D Vo, Yannick Doyon,
Jeffrey C Miller,
David E Paschon,
Lei Zhang,
Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
David G Drubin
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ABSTRACT: Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.
Nature Cell Biology 02/2011; 13(3):331-7. · 19.49 Impact Factor
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ABSTRACT: Animal models, including the zebrafish, without a reliable embryonic stem cell system are not easily amenable to targeted mutagenesis for studying gene function. Three recent publications have shown that zinc finger nucleases (ZFNs) have circumvented this shortcoming in zebrafish. Similar to restriction enzymes, ZFNs can introduce site-specific double-strand breaks (DSBs); moreover, they can be designed to recognize virtually any target sequence. Because the preferred DSB repair pathway in zebrafish embryos, non-homologous end joining, is error-prone, ZFNs can be used to create mutations in a gene of interest. Here we review the protocols for a yeast-based assay to detect effective ZFNs. Additionally, we detail the procedures for synthesis and injection of ZFN-encoding mRNA into zebrafish embryos, screening of injected embryos for induced mutations in the soma, and recovery of germline mutations.
Methods in molecular biology (Clifton, N.J.) 01/2011; 770:505-27.
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ABSTRACT: Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means for obtaining highly efficient and specific genome modification.
Nature Methods 01/2011; 8(1):74-9. · 19.28 Impact Factor
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ABSTRACT: Zinc-finger nucleases (ZFNs) are powerful tools for editing the genomes of cell lines and model organisms. Given the breadth of their potential application, simple methods that increase ZFN activity, thus ensuring genome modification, are highly attractive. Here we show that transient hypothermia generally and robustly increased the level of stable, ZFN-induced gene disruption, thereby providing a simple technique to enhance the experimental efficacy of ZFNs.
Nature Methods 06/2010; 7(6):459-60. · 19.28 Impact Factor
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Vipula K Shukla, Yannick Doyon,
Jeffrey C Miller,
Russell C DeKelver,
Erica A Moehle,
Sarah E Worden,
Jon C Mitchell,
Nicole L Arnold,
Sunita Gopalan,
Xiangdong Meng, [......],
David McCaskill,
Matthew A Simpson,
Beth Blakeslee,
Scott A Greenwalt,
Holly J Butler,
Sarah J Hinkley,
Lei Zhang,
Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov
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ABSTRACT: Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis. Because targeted genome modification in plants has been intractable, plant trait engineering remains a laborious, time-consuming and unpredictable undertaking. Here we report a broadly applicable, versatile solution to this problem: the use of designed zinc-finger nucleases (ZFNs) that induce a double-stranded break at their target locus. We describe the use of ZFNs to modify endogenous loci in plants of the crop species Zea mays. We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide-tolerance gene at the intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes to the next generation. Insertional disruption of one target locus, IPK1, results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds. ZFNs can be used in any plant species amenable to DNA delivery; our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications.
Nature 05/2009; 459(7245):437-41. · 36.28 Impact Factor
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Charles Q Cai, Yannick Doyon,
W Michael Ainley,
Jeffrey C Miller,
Russell C Dekelver,
Erica A Moehle,
Jeremy M Rock,
Ya-Li Lee,
Robbi Garrison,
Lisa Schulenberg, [......],
Lisa Baker,
Farhoud Faraji,
Lei Zhang,
Michael C Holmes,
Edward J Rebar,
Trevor N Collingwood,
Beth Rubin-Wilson,
Philip D Gregory,
Fyodor D Urnov,
Joseph F Petolino
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ABSTRACT: Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.
Plant Molecular Biology 01/2009; 69(6):699-709. · 4.15 Impact Factor
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Yannick Doyon,
Jasmine M McCammon,
Jeffrey C Miller,
Farhoud Faraji,
Catherine Ngo,
George E Katibah,
Rainier Amora,
Toby D Hocking,
Lei Zhang,
Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Sharon L Amacher
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ABSTRACT: We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which 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 (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.
Nature Biotechnology 07/2008; 26(6):702-8. · 29.50 Impact Factor
