title: megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.
abstract: 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.
authors.affiliation: Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA 98101, USA, Pregenen, Inc., Seattle, WA 98103, USA, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA, Cellectis S.A., Paris, 75013, France, Division of Basic Sciences, Fred Hutch Cancer Research Center, Seattle, WA 98109, USA, Department of Biochemistry, University of Washington, Seattle, WA 98195, USA, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA and Department of Immunology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Boissel
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.forename: Alexander
authors.initials: A
authors.lastname: Astrakhan
authors.forename: Andrew
authors.initials: A
authors.lastname: Adey
authors.forename: Agnès
authors.initials: A
authors.lastname: Gouble
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.forename: Michael T
authors.initials: MT
authors.lastname: Certo
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
mesh_terms: D002478:Cells, Cultured; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D019673:Genes, T-Cell Receptor alpha; D005818:Genetic Engineering; D023281:Genomics / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases