Protocol abstract

Nature Protocols 4, 1471 - 1501 (2009)
Published online: 17 September 2009 | doi:10.1038/nprot.2009.98

Subject Categories: Biochemistry and protein analysis | Nucleic acid based molecular biology

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

Morgan L Maeder1,2, Stacey Thibodeau-Beganny1, Jeffry D Sander3, Daniel F Voytas3,5 & J Keith Joung1,2,4

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 approx8 weeks.

  1. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
  2. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.
  3. Department of Genetics, Development & Cell Biology, Roy J. Carver Co-Laboratory, Iowa State University, Ames, Iowa, USA.
  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
  5. Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.

Correspondence to: J Keith Joung1,2,4 e-mail: JJOUNG@PARTNERS.ORG


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