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: 8.36). 09/2009; 4(10):1471-501. DOI: 10.1038/nprot.2009.98
Source: PubMed

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.

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Available from: Daniel F Voytas, Apr 10, 2014
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    • "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). "
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    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 · 5.68 Impact Factor
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    • "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). "
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    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 · 2.51 Impact Factor
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    • "Using the Zinc-Finger Targeter (ZiFiT) online program (Sander et al., 2007), the most promising ZFN recognition site was found at position 4699–4722 in exon 13. For the right half-site, ZiFiT identified a distinct 3-finger ZFA (database entry OZ179) as being capable of activating the transcription of a reporter gene by more than 14-fold in a bacterial two-hybrid assay (Wright et al., 2005; Maeder et al., 2009). This ZFN was named ChR1-R. "
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    ABSTRACT: The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide toolset for genetic manipulation has been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes 1) design of gene-specific ZFNs using available online tools; 2) evaluation of the designed ZFNs in a Chlamydomonas in situ model system; 3) optimization of ZFN activity by modification of the nuclease domain; and 4) application of the most suitable enzymes for the mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs for targeting the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin resistant (Pm-R) clones with active nucleases. Of these Pm-R clones, 1% contained a modified COP3 locus as well. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified accordingly by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
    The Plant Journal 11/2012; 73(5). DOI:10.1111/tpj.12066 · 6.82 Impact Factor
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