Seattle, WA, United States
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Baxter S.,University of Washington | Baxter S.,Seattle Childrens Research Institute | Lambert A.R.,Seattle Childrens Research Institute | Kuhar R.,Seattle Childrens Research Institute | And 7 more authors.
Nucleic Acids Research | Year: 2012

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N-and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70 amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70 of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications. © 2012 The Author(s).


Chan Y.-S.,University of Cambridge | Takeuchi R.,Fred Hutchinson Cancer Research Center | Jarjour J.,Pregenen Inc. | Huen D.S.,University of Cambridge | And 4 more authors.
PLoS ONE | Year: 2013

The homing endonuclease gene (HEG) drive system, a promising genetic approach for controlling arthropod populations, utilises engineered nucleases to spread deleterious mutations that inactivate individual genes throughout a target population. Previous work with a naturally occurring LAGLIDADG homing endonuclease (I-SceI) demonstrated its feasibility in both Drosophila and Anopheles. Here we report on the next stage of this strategy: the redesign of HEGs with customized specificity in order to drive HEG-induced 'homing' in vivo via break-induced homologous recombination. Variants targeting a sequence within the Anopheles AGAP004734 gene were created from the recently characterized I-OnuI endonuclease, and tested for cleavage activity and frequency of homing using a model Drosophila HEG drive system. We observed cleavage and homing at an integrated reporter for all endonuclease variants tested, demonstrating for the first time that engineered HEGs can cleave their target site in insect germline cells, promoting targeted mutagenesis and homing. However, in comparison to our previously reported work with I-SceI, the engineered I-OnuI variants mediated homing with a reduced frequency, suggesting that site-specific cleavage activity is insufficient by itself to ensure efficient homing. Taken together, our experiments take a further step towards the development of a viable HEG-based population control strategy for insects. © 2013 Chan et al.


Boissel S.,University of Washington | Boissel S.,Seattle Childrens Research Institute | Jarjour J.,Pregenen Inc. | Astrakhan A.,Pregenen Inc. | And 11 more authors.
Nucleic Acids Research | Year: 2014

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods. © 2013 The Author(s). Published by Oxford University Press.


Disclosed herein are compositions for inactivating the human TCR-alpha gene comprising engineered LAGLIDADG homing endonucleases (LHEs) and their derivatives, particularly derived from members of the \-Onul subfamily of LHEs. Polynucleotides encoding such endonucleases, vectors comprising said polynucleotides, cells comprising or having been treated with such endonucleases, and therapeutic compositions deriving therefore are also provided.


Disclosed herein are compositions for inactivating the human CCR5 gene comprising engineered LAGLIDADG homing endonucleases (LHEs) and their derivatives, particularly derived from members of the \-OnuI subfamily of LHE. Polynucleotides encoding such endonucleases, vectors comprising said polynucleotides, cells comprising or having been treated with such endonucleases, and therapeutic compositions deriving therefrom are also provided.


Osborn M.J.,University of Minnesota | Gabriel R.,National Center for Tumor Diseases | Gabriel R.,German Cancer Research Center | Webber B.R.,University of Minnesota | And 14 more authors.
Human Gene Therapy | Year: 2015

Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder. © Copyright 2015, Mary Ann Liebert, Inc. 2015.


Baxter S.K.,University of Washington | Baxter S.K.,Seattle Childrens Research Institute | Lambert A.R.,Seattle Childrens Research Institute | Scharenberg A.M.,University of Washington | And 4 more authors.
Methods in Molecular Biology | Year: 2013

A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNAbinding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry. © Springer Science+Business Media New York 2013.


Wang Y.,Seattle Childrens Research Institute | Khan I.F.,Seattle Childrens Research Institute | Boissel S.,Seattle Childrens Research Institute | Boissel S.,University of Washington | And 8 more authors.
Nucleic Acids Research | Year: 2014

LAGLIDADG homing endonucleases (LHEs) are compact endonucleases with 20-22 bp recognition sites, and thus are ideal scaffolds for engineering site-specific DNA cleavage enzymes for genome editing applications. Here, we describe a general approach to LHE engineering that combines rational design with directed evolution, using a yeast surface display high-throughput cleavage selection. This approach was employed to alter the binding and cleavage specificity of the I-Anil LHE to recognize a mutation in the mouse Bruton tyrosine kinase (Btk) gene causative for mouse X-linked immunodeficiency (XID) - a model of human X-linked agammaglobulinemia (XLA). The required re-targeting of I-AniI involved progressive resculpting of the DNA contact interface to accommodate nine base differences from the native cleavage sequence. The enzyme emerging from the progressive engineering process was specific for the XID mutant allele versus the wild-type (WT) allele, and exhibited activity equivalent to WT I-AniI in vitro and in cellulo reporter assays. Fusion of the enzyme to a site-specific DNA binding domain of transcription activator-like effector (TALE) resulted in a further enhancement of gene editing efficiency. These results illustrate the potential of LHE enzymes as specific and efficient tools for therapeutic genome engineering. © 2014 The Author(s) 2014.


Astrakhan A.,University of Washington | Astrakhan A.,Pregenen Inc. | Sather B.D.,Seattle Childrens Research Institute | Ryu B.Y.,Seattle Childrens Research Institute | And 9 more authors.
Blood | Year: 2012

The immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to lifethreatening hematopoietic cell dysfunction. We used WAS protein (WASp)- deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp + T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp + T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain + naive B cells, development of self-reactive CD11c +FAS + B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS. © 2012 by The American Society of Hematology.

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