Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays

被引：134

作者：

Maeder, Morgan L. ^{[1
,2
,3
]}

Thibodeau-Beganny, Stacey ^{[1
,2
]}

Sander, Jeffry D. ^{[4
]}

Voytas, Daniel F. ^{[4
,6
,7
]}

Joung, J. Keith ^{[1
,2
,3
,5
]}

机构：

[1] Massachusetts Gen Hosp, Ctr Canc Res, Mol Pathol Unit, Charlestown, MA USA

[2] Massachusetts Gen Hosp, Ctr Computat & Integrat Biol, Charlestown, MA USA

[3] Harvard Univ, Sch Med, Biol & Biomed Sci Program, Boston, MA USA

[4] Iowa State Univ, Dept Genet Dev & Cell Biol, Roy J Carver Co Lab, Ames, IA USA

[5] Harvard Univ, Sch Med, Dept Pathol, Boston, MA 02115 USA

[6] Univ Minnesota, Dept Genet Cell Biol & Dev, Minneapolis, MN USA

[7] Univ Minnesota, Ctr Genome Engn, Minneapolis, MN USA

来源：

NATURE PROTOCOLS | 2009年 / 4卷 / 10期

基金：

美国国家卫生研究院; 美国国家科学基金会;

关键词：

CONTROLLING GENE-EXPRESSION; DNA-BINDING PROTEINS; HOMOLOGOUS RECOMBINATION; TRANSCRIPTION FACTORS; RESTRICTION ENZYMES; MAJOR DETERMINANT; HUMAN GENOME; NUCLEASES; CLEAVAGE; CELLS;

D O I：

10.1038/nprot.2009.98

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN selections are carried out in Escherichia coli using a bacterial two-hybrid system and do not require specialized equipment. Here we provide a detailed protocol for carrying out OPEN to engineer zinc-finger arrays that have a high probability of functioning as ZFNs. Using OPEN, researchers can generate multiple, customized ZFNs in similar to 8 weeks.

引用

页码：1471 / 1501

页数：31

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