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Seattle, WA, United States

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. Source


Munoz N.M.,Fred Hutchinson Cancer Research Center | Beard B.C.,Fred Hutchinson Cancer Research Center | Ryu B.Y.,Northwest Genome Engineering Consortium | Ryu B.Y.,Seattle Childrens Research Institute | And 8 more authors.
Nucleic Acids Research | Year: 2012

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects. © The Author(s) 2011. Published by Oxford University Press. Source


Baxter S.K.,University of Washington | Baxter S.K.,Seattle Childrens Research Institute | Scharenberg A.M.,Northwest Genome Engineering Consortium | Scharenberg A.M.,University of Washington | And 3 more authors.
Methods in Molecular Biology | Year: 2014

LAGLIDADG homing endonucleases (LHEs) are valuable tools for genome engineering, and our ability to alter LHE target site specificity is rapidly evolving. However, widespread use of these enzymes is limited due to the small number of available engineering scaffolds, each requiring extensive redesign to target widely varying DNA sequences. Here, we describe a technique for the chimerization of homologous I-OnuI family LHEs. Chimerization greatly expands the pool of unique starting scaffolds, thereby enabling more effective and efficient LHE redesign. I-OnuI family enzymes are divided into N- and C-terminal halves based on sequence alignments, and then combinatorially rejoined with a hybrid linker. The resulting chimeric enzymes are expressed on the surface of yeast where stability, DNA binding affinity, and cleavage activity can be assayed by flow cytometry. © Springer Science+Business Media New York 2014. Source

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