Maeder, ML, Thibodeau-Beganny, S, Sander, JD, Voytas, DF and Joung, JK. Oligomerized pool engineering (OPEN): an ‘open-source’ protocol for making customized zinc-finger arrays. Nat Protoc 4: 1471-1501

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.
Nature Protocol (Impact Factor: 9.67). 09/2009; 4(10):1471-501. DOI: 10.1038/nprot.2009.98
Source: PubMed


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.


Available from: Daniel F Voytas, Apr 10, 2014
  • Source
    • "Each zinc finger motif is composed of approximately 30 amino acids and binds to three nucleotides. Several approaches have been developed for the assembly of zinc finger arrays to user-defined target sequences including the modular assembly and oligomerized pool engineering (OPEN) (Maeder et al., 2009). Assembled zinc finger arrays customized to bind to a user-selected DNA sequence were fused to the nonspecific catalytic domain of FokI endonuclease to create a chimeric zinc finger nuclease (ZFN) capable of generating site-specific DSBs (Morton et al., 2006) (Figure 2a). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.
    Plant Biotechnology Journal 10/2014; 12(8). DOI:10.1111/pbi.12256 · 5.75 Impact Factor
  • Source
    • "Thus, combinatorial assembly of pre-existing fingers has met with only modest success (Sander et al. 2010; Thibodeau-Beganny et al. 2010). In addition, target-driven selection procedures for new finger combinations typically are laborious and uncertain of success (Thibodeau-Beganny and Joung 2007; Maeder et al. 2009). TALENs use DNA-recognition modules that recognize single base pairs, linked to the same FokI-derived cleavage domain that is used in ZFNs (Gaj et al. 2013; Joung and Sander 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Zinc-finger nucleases (ZFNs) have proved successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially higher than for ZFNs, and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.
    G3-Genes Genomes Genetics 08/2013; 3(10). DOI:10.1534/g3.113.007260 · 3.20 Impact Factor
    • "Despite the advantages offered by ZFN technology, ZFNs have proven difficult for non-specialist investigators to engineer from scratch because it has not been straightforward to successfully assemble zinc finger domains to bind an extended stretch of nucleotides (Ramirez et al., 2008). Although a library of zinc finger components and protocols to perform screens to identify optimized ZFNs has been made freely available to the academic community (Maeder et al., 2008; Maeder et al., 2009), it can take months for non-specialists to engineer ZFNs that target a genomic site with high efficiency. Furthermore, target-site selection is limited – these freely available ZFN components can only be used for binding sites every few hundred bp throughout the genome. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Disease modeling with human pluripotent stem cells has come into the public spotlight with the awarding of the Nobel Prize in Physiology or Medicine for 2012 to Drs John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent. This discovery has opened the door for the generation of pluripotent stem cells from individuals with disease and the differentiation of these cells into somatic cell types for the study of disease pathophysiology. The emergence of genome-editing technology over the past few years has made it feasible to generate and investigate human cellular disease models with even greater speed and efficiency. Here, recent technological advances in genome editing, and its utility in human biology and disease studies, are reviewed.
    Disease Models and Mechanisms 06/2013; 6(4). DOI:10.1242/dmm.012054 · 4.97 Impact Factor
Show more