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Judith Straimer,
Marcus C S Lee,
Andrew H Lee,
Bryan Zeitler,
April E Williams,
Jocelynn R Pearl,
Lei Zhang, Edward J Rebar,
Philip D Gregory,
Manuel Llinás,
Fyodor D Urnov,
David A Fidock
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ABSTRACT: Malaria afflicts over 200 million people worldwide, and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum-induced pathogenesis, including drug-resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene-deletion parasites with unprecedented speed (2 weeks), both with and without direct selection. ZFNs engineered against the parasite gene pfcrt, responsible for escape under chloroquine treatment, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. This method will enable a diverse array of genome-editing approaches to interrogate this human pathogen.
Nature Methods 08/2012; 9(10):993-8. · 19.28 Impact Factor
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H Steve Zhang,
Dingang Liu,
Yan Huang,
Stefan Schmidt,
Reed Hickey,
Dmitry Guschin,
Haili Su,
Ion S Jovin,
Mike Kunis,
Sarah Hinkley,
Yuxin Liang,
Linda Hinh,
S Kaye Spratt,
Casey C Case, Edward J Rebar,
Barbara Ehrlich,
Philip D Gregory,
Frank J Giordano
[show abstract]
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ABSTRACT: Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.
Molecular Therapy 07/2012; 20(8):1508-15. · 6.87 Impact Factor
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Hiroki Torikai,
Andreas Reik,
Pei-Qi Liu,
Yuanyue Zhou,
Ling Zhang,
Sourindra Maiti,
Helen Huls,
Jeffrey C Miller,
Partow Kebriaei,
Brian Rabinovitch,
Dean A Lee,
Richard E Champlin,
Chiara Bonini,
Luigi Naldini, Edward J Rebar,
Philip D Gregory,
Michael C Holmes,
Laurence J N Cooper
[show abstract]
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ABSTRACT: Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR(+) T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR(+)TCR(neg) T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.
Blood 04/2012; 119(24):5697-705. · 9.90 Impact Factor
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Manju Gupta,
Russell C DeKelver,
Asha Palta,
Carla Clifford,
Sunita Gopalan,
Jeffrey C Miller,
Stephen Novak,
Daniel Desloover,
Daniel Gachotte,
James Connell,
Josh Flook,
Thomas Patterson,
Kelly Robbins, Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Joseph F Petolino
[show abstract]
[hide abstract]
ABSTRACT: Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species.
Plant Biotechnology Journal 04/2012; 10(7):783-91. · 5.44 Impact Factor
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Frank Soldner,
Josée Laganière,
Albert W Cheng,
Dirk Hockemeyer,
Qing Gao,
Raaji Alagappan,
Vikram Khurana,
Lawrence I Golbe,
Richard H Myers,
Susan Lindquist,
Lei Zhang,
Dmitry Guschin,
Lauren K Fong,
B Joseph Vu,
Xiangdong Meng,
Fyodor D Urnov, Edward J Rebar,
Philip D Gregory,
H Steve Zhang,
Rudolf Jaenisch
[show abstract]
[hide abstract]
ABSTRACT: Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinson's disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.
Cell 07/2011; 146(2):318-31. · 32.40 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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Andrew J Wood,
Te-Wen Lo,
Bryan Zeitler,
Catherine S Pickle,
Edward J Ralston,
Andrew H Lee,
Rainier Amora,
Jeffrey C Miller,
Elo Leung,
Xiangdong Meng,
Lei Zhang, Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Barbara J Meyer
[show abstract]
[hide abstract]
ABSTRACT: Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.
Science 06/2011; 333(6040):307. · 31.20 Impact Factor
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Deniz Simsek,
Erika Brunet,
Sunnie Yan-Wai Wong,
Sachin Katyal,
Yankun Gao,
Peter J McKinnon,
Jacqueline Lou,
Lei Zhang,
James Li, Edward J Rebar,
Philip D Gregory,
Michael C Holmes,
Maria Jasin
[show abstract]
[hide abstract]
ABSTRACT: Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.
PLoS Genetics 06/2011; 7(6):e1002080. · 8.69 Impact Factor
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Craig B Wilen,
Jianbin Wang,
John C Tilton,
Jeffrey C Miller,
Kenneth A Kim, Edward J Rebar,
Scott A Sherrill-Mix,
Sean C Patro,
Anthony J Secreto,
Andrea P O Jordan, [......],
Bruce A Bunnell,
Andrew A Lackner,
James A Hoxie,
Gwenn A Danet-Desnoyers,
Frederic D Bushman,
James L Riley,
Philip D Gregory,
Carl H June,
Michael C Holmes,
Robert W Doms
[show abstract]
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ABSTRACT: HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.
PLoS Pathogens 04/2011; 7(4):e1002020. · 9.13 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
[show abstract]
[hide abstract]
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
[show abstract]
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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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Jeffrey C Miller,
Siyuan Tan,
Guijuan Qiao,
Kyle A Barlow,
Jianbin Wang,
Danny F Xia,
Xiangdong Meng,
David E Paschon,
Elo Leung,
Sarah J Hinkley,
Gladys P Dulay,
Kevin L Hua,
Irina Ankoudinova,
Gregory J Cost,
Fyodor D Urnov,
H Steve Zhang,
Michael C Holmes,
Lei Zhang,
Philip D Gregory, Edward J Rebar
[show abstract]
[hide abstract]
ABSTRACT: 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.
Nature Biotechnology 02/2011; 29(2):143-8. · 29.50 Impact Factor
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Dirk Hockemeyer,
Haoyi Wang,
Samira Kiani,
Christine S Lai,
Qing Gao,
John P Cassady,
Gregory J Cost,
Lei Zhang,
Yolanda Santiago,
Jeffrey C Miller,
Bryan Zeitler,
Jennifer M Cherone,
Xiangdong Meng,
Sarah J Hinkley, Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Rudolf Jaenisch
[show abstract]
[hide abstract]
ABSTRACT: Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).
Nature Biotechnology 01/2011; 29(8):731-4. · 29.50 Impact Factor
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Josee Laganiere,
Adrian P Kells,
Jeffrey T Lai,
Dmitry Guschin,
David E Paschon,
Xiangdong Meng,
Lauren K Fong,
Qi Yu, Edward J Rebar,
Philip D Gregory,
Krystof S Bankiewicz,
John Forsayeth,
H Steve Zhang
[show abstract]
[hide abstract]
ABSTRACT: Loss of dopaminergic neurons is primarily responsible for the onset and progression of Parkinson's disease (PD); thus, neuroprotective and/or neuroregenerative strategies remain critical to the treatment of this increasingly prevalent disease. Here we explore a novel approach to neurotrophic factor-based therapy by engineering zinc finger protein transcription factors (ZFP TFs) that activate the expression of the endogenous glial cell line-derived neurotrophic factor (GDNF) gene. We show that GDNF activation can be achieved with exquisite genome-wide specificity. Furthermore, in a rat model of PD, striatal delivery of an adeno-associated viral vector serotype 2 encoding the GDNF activator resulted in improvements in forelimb akinesia, sensorimotor neglect, and amphetamine-induced rotations caused by 6-hydroxydopamine (6-OHDA) lesion. Our results suggest that an engineered ZFP TF can drive sufficient GDNF expression in the brain to provide functional neuroprotection against 6-OHDA; therefore, targeted activation of the endogenous gene may provide a method for delivering appropriate levels of GDNF to PD patients.
Journal of Neuroscience 12/2010; 30(49):16469-74. · 7.11 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.
Nature Reviews Genetics 09/2010; 11(9):636-46. · 38.08 Impact Factor
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Russell C DeKelver,
Vivian M Choi,
Erica A Moehle,
David E Paschon,
Dirk Hockemeyer,
Sebastiaan H Meijsing,
Yasemin Sancak,
Xiaoxia Cui,
Eveline J Steine,
Jeffrey C Miller, [......],
Jeremy M Rock,
Lei Zhang,
Gregory D Davis, Edward J Rebar,
Iain M Cheeseman,
Keith R Yamamoto,
David M Sabatini,
Rudolf Jaenisch,
Philip D Gregory,
Fyodor D Urnov
[show abstract]
[hide abstract]
ABSTRACT: Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.
Genome Research 08/2010; 20(8):1133-42. · 13.61 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: The development of zinc finger nucleases for targeted gene modification can benefit from rapid functional assays that directly quantify activity at the endogenous target. Here we describe a simple procedure for quantifying mutations that result from DNA double-strand break repair via non-homologous end joining. The assay is based on the ability of the Surveyor nuclease to selectively cleave distorted duplex DNA formed via cross-annealing of mutated and wild-type sequence.
Methods in molecular biology (Clifton, N.J.) 01/2010; 649:247-56.
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Dirk Hockemeyer,
Frank Soldner,
Caroline Beard,
Qing Gao,
Maisam Mitalipova,
Russell C DeKelver,
George E Katibah,
Ranier Amora,
Elizabeth A Boydston,
Bryan Zeitler,
Xiangdong Meng,
Jeffrey C Miller,
Lei Zhang, Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Rudolf Jaenisch
[show abstract]
[hide abstract]
ABSTRACT: Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.
Nature Biotechnology 09/2009; 27(9):851-7. · 29.50 Impact Factor
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Aron M Geurts,
Gregory J Cost,
Yevgeniy Freyvert,
Bryan Zeitler,
Jeffrey C Miller,
Vivian M Choi,
Shirin S Jenkins,
Adam Wood,
Xiaoxia Cui,
Xiangdong Meng, [......],
Hartmut Weiler,
Séverine Ménoret,
Ignacio Anegon,
Gregory D Davis,
Lei Zhang, Edward J Rebar,
Philip D Gregory,
Fyodor D Urnov,
Howard J Jacob,
Roland Buelow
[show abstract]
[hide abstract]
ABSTRACT: The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.
Science 08/2009; 325(5939):433. · 31.20 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
[show abstract]
[hide abstract]
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
