Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification.
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.
Maeder
Morgan L
ML
Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA.
Thibodeau-Beganny
Stacey
S
Osiak
Anna
A
Wright
David A
DA
Anthony
Reshma M
RM
Eichtinger
Magdalena
M
Jiang
Tao
T
Foley
Jonathan E
JE
Winfrey
Ronnie J
RJ
Townsend
Jeffrey A
JA
Unger-Wallace
Erica
E
Sander
Jeffry D
JD
Müller-Lerch
Felix
F
Fu
Fengli
F
Pearlberg
Joseph
J
Göbel
Carl
C
Dassie
Justin P
JP
Pruett-Miller
Shondra M
SM
Porteus
Matthew H
MH
Sgroi
Dennis C
DC
Iafrate
A John
AJ
Dobbs
Drena
D
McCray
Paul B
PB
Cathomen
Toni
T
Voytas
Daniel F
DF
Joung
J Keith
JK
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
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004720:Endonucleases