Jeffrey C Miller

Publications (38)600.1 Total impact

• Source

  Available from: Laurence Cooper

  Article: Towards eliminating HLA class I expression to generate universal cells from allogeneic donors.

  Hiroki Torikai, Andreas Reik, Frank Soldner, Edus H Warren, Carrie Yuen, Yuanyue Zhou, Denise L Crossland, Helen Huls, Nicholas Littman, Ziying Zhang, Scott S Tykodi, Partow Kebriaei, Dean A Lee, Jeffrey C Miller, Edward J Rebar, Michael C Holmes, Rudolf Jaenisch, Richard E Champlin, Philip D Gregory, Laurence J N Cooper
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  ABSTRACT: Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as "non-self" by the recipient. To this end, we developed designer zinc finger nucleases (ZFNs) and employed a "hit-and-run" approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T-cells, including CD19-specific T-cells. The HLA-A(neg) T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by NK-cells of cells that had lost HLA expression was circumvented by enforced expression of non-classical HLA molecules. Furthermore, we demonstrate that ZFNs can eliminate HLA-A expression from embryonic stem cells which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically-appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from one donor can be administered to multiple recipients.
  Blood 06/2013; · 9.90 Impact Factor
• Source

  Available from: Laurence Cooper

  Article: A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR.

  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
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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
• Article: Transcriptional activation of Brassica napus β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor.

  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
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  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
• Article: Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme.

  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
• Article: An unbiased genome-wide analysis of zinc-finger nuclease specificity.

  Richard Gabriel, Angelo Lombardo, Anne Arens, Jeffrey C Miller, Pietro Genovese, Christine Kaeppel, Ali Nowrouzi, Cynthia C Bartholomae, Jianbin Wang, Geoffrey Friedman, Michael C Holmes, Philip D Gregory, Hanno Glimm, Manfred Schmidt, Luigi Naldini, Christof von Kalle
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  ABSTRACT: Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research.
  Nature Biotechnology 08/2011; 29(9):816-23. · 29.50 Impact Factor
• Source

  Available from: Te-Wen Lo

  Article: Targeted genome editing across species using ZFNs and TALENs.

  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
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  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
• Source

  Available from: Farhoud Faraji

  Article: Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.

  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
• Source

  Available from: Andrew A Lackner

  Article: Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

  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
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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
• Source

  Available from: um.ac.ir

  Article: A TALE nuclease architecture for efficient genome editing.

  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
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  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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  Available from:

  Article: Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.

  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
• Source

  Available from: berkeley.edu

  Article: Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.

  Yannick Doyon, Thuy D Vo, Matthew C Mendel, Shon G Greenberg, Jianbin Wang, Danny F Xia, Jeffrey C Miller, Fyodor D Urnov, Philip D Gregory, Michael C Holmes
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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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  Available from: Dirk Hockemeyer

  Article: Genetic engineering of human pluripotent cells using TALE nucleases.

  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
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  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
• Article: Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome.

  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
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  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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  Available from: PubMed Central

  Article: Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

  Salvatore J Orlando, Yolanda Santiago, Russell C DeKelver, Yevgeniy Freyvert, Elizabeth A Boydston, Erica A Moehle, Vivian M Choi, Sunita M Gopalan, Jacqueline F Lou, James Li, Jeffrey C Miller, Michael C Holmes, Philip D Gregory, Fyodor D Urnov, Gregory J Cost
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  ABSTRACT: We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.
  Nucleic Acids Research 08/2010; 38(15):e152. · 8.03 Impact Factor
• Article: Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.

  Laetitia Malphettes, Yevgeniy Freyvert, Jennifer Chang, Pei-Qi Liu, Edmond Chan, Jeffrey C Miller, Zhe Zhou, Thu Nguyen, Christina Tsai, Andrew W Snowden, Trevor N Collingwood, Philip D Gregory, Gregory J Cost
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  ABSTRACT: IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily alpha1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a alpha1,6-fucosyltransferase. FUT8(-/-) CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8(-/-) cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8(-/-) CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8(-/-) cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN-derived FUT8(-/-) cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8(-/-) cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8(-/-) cell lines was fourfold higher than in the best-producing clones of FUT8(-/-) cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8- phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies.
  Biotechnology and Bioengineering 08/2010; 106(5):774-83. · 3.95 Impact Factor
• Article: Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases.

  Pei-Qi Liu, Edmond M Chan, Gregory J Cost, Lin Zhang, Jianbin Wang, Jeffrey C Miller, Dmitry Y Guschin, Andreas Reik, Michael C Holmes, John E Mott, Trevor N Collingwood, Philip D Gregory
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  ABSTRACT: Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering.
  Biotechnology and Bioengineering 05/2010; 106(1):97-105. · 3.95 Impact Factor
• Article: Distinct factors control histone variant H3.3 localization at specific genomic regions.

  Aaron D Goldberg, Laura A Banaszynski, Kyung-Min Noh, Peter W Lewis, Simon J Elsaesser, Sonja Stadler, Scott Dewell, Martin Law, Xingyi Guo, Xuan Li, [......], Shahin Rafii, Chingwen Yang, Peter J Scambler, David Garrick, Richard J Gibbons, Douglas R Higgs, Ileana M Cristea, Fyodor D Urnov, Deyou Zheng, C David Allis
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  ABSTRACT: The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.
  Cell 03/2010; 140(5):678-91. · 32.40 Impact Factor
• Source

  Available from: Adam James Waite

  Article: A rapid and general assay for monitoring endogenous gene modification.

  Dmitry Y Guschin, Adam J Waite, George E Katibah, Jeffrey C Miller, Michael C Holmes, Edward J Rebar
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  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.
• Article: BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells.

  Gregory J Cost, Yevgeniy Freyvert, Annamaria Vafiadis, Yolanda Santiago, Jeffrey C Miller, Edward Rebar, Trevor N Collingwood, Andrew Snowden, Philip D Gregory
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  ABSTRACT: Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production.
  Biotechnology and Bioengineering 09/2009; 105(2):330-40. · 3.95 Impact Factor
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  Available from: Dirk Hockemeyer

  Article: Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.

  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
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  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