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

Morgan L. Maeder, Stacey Thibodeau-Beganny, Jeffry D. Sander, Daniel F. Voytas, J. Keith Joung

Research output: Contribution to journalArticlepeer-review

144 Scopus citations

Abstract

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 selectionbased 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 ∼8 weeks.

Original languageEnglish (US)
Pages (from-to)1471-1501
Number of pages31
JournalNature Protocols
Volume4
Issue number10
DOIs
StatePublished - Sep 21 2009

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