title: Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.
abstract: Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.
authors.affiliation: Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA.
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Maeder
authors.forename: Stacey
authors.initials: S
authors.lastname: Thibodeau-Beganny
authors.forename: Anna
authors.initials: A
authors.lastname: Osiak
authors.forename: David A
authors.initials: DA
authors.lastname: Wright
authors.forename: Reshma M
authors.initials: RM
authors.lastname: Anthony
authors.forename: Magdalena
authors.initials: M
authors.lastname: Eichtinger
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Foley
authors.forename: Ronnie J
authors.initials: RJ
authors.lastname: Winfrey
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Townsend
authors.forename: Erica
authors.initials: E
authors.lastname: Unger-Wallace
authors.forename: Jeffry D
authors.initials: JD
authors.lastname: Sander
authors.forename: Felix
authors.initials: F
authors.lastname: Müller-Lerch
authors.forename: Fengli
authors.initials: F
authors.lastname: Fu
authors.forename: Joseph
authors.initials: J
authors.lastname: Pearlberg
authors.forename: Carl
authors.initials: C
authors.lastname: Göbel
authors.forename: Justin P
authors.initials: JP
authors.lastname: Dassie
authors.forename: Shondra M
authors.initials: SM
authors.lastname: Pruett-Miller
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Porteus
authors.forename: Dennis C
authors.initials: DC
authors.lastname: Sgroi
authors.forename: A John
authors.initials: AJ
authors.lastname: Iafrate
authors.forename: Drena
authors.initials: D
authors.lastname: Dobbs
authors.forename: Paul B
authors.initials: PB
authors.lastname: McCray
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism* / Q000633:toxicity; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D016335:Zinc Fingers
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004720:Endonucleases