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

Nat Protoc. 2009;4(10):1471-501. doi: 10.1038/nprot.2009.98. Epub 2009 Sep 17.

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 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 approximately 8 weeks.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacteriological Techniques
  • Bacteriophages / genetics
  • Binding Sites
  • Escherichia coli / genetics
  • Gene Library
  • Genomics / methods
  • Polymerase Chain Reaction
  • Protein Engineering / methods*
  • Recombination, Genetic
  • Software
  • Zinc Fingers*