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Assembly and Characterization of megaTALs for Hyperspecific Genome Engineering Applications

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Chromosomal Mutagenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1239))

Abstract

Rare-cleaving nucleases have emerged as valuable tools for creating targeted genomic modification for both therapeutic and research applications. MegaTALs are novel monomeric nucleases composed of a site-specific meganuclease cleavage head with additional affinity and specificity provided by a TAL effector DNA binding domain. This fusion product facilitates the transformation of meganucleases into hyperspecific and highly active genome engineering tools that are amenable to multiplexing and compatible with multiple cellular delivery methods. In this chapter, we describe the process of assembling a megaTAL from a meganuclease, as well as a method for characterization of nuclease cleavage activity in vivo using a fluorescence reporter assay.

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Author information

Authors and Affiliations

  1. Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, 98101, USA

    Sandrine Boissel & Andrew M. Scharenberg

  2. Department of Immunology, University of Washington, Seattle, WA, 98195, USA

    Andrew M. Scharenberg

  3. Department of Pediatrics, University of Washington, Box 359300, C9S-6, 1900 9th Avenue, Seattle, WA, 98101, USA

    Andrew M. Scharenberg

  4. Seattle Children’s Research Institute, University of Washington, Box 359300, C9S-6, 1900 9th Avenue, Seattle, WA, 98101, USA

    Andrew M. Scharenberg

Authors
  1. Sandrine Boissel
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  2. Andrew M. Scharenberg
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Corresponding author

Correspondence to Andrew M. Scharenberg .

Editor information

Editors and Affiliations

  1. Department of Genetics, Genome Engineering and iPSC Center, Washington University School of Medicine, St. Louis, Missouri, USA

    Shondra M. Pruett-Miller

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Boissel, S., Scharenberg, A.M. (2015). Assembly and Characterization of megaTALs for Hyperspecific Genome Engineering Applications. In: Pruett-Miller, S. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 1239. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_9

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  • DOI: https://doi.org/10.1007/978-1-4939-1862-1_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1861-4

  • Online ISBN: 978-1-4939-1862-1

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