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

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

doi:10.1038/nprot.2009.98

Cited by 184 publications

(164 citation statements)

References 79 publications

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“…We used OPEN (17) to generate ZFAs for potential target sites in two Arabidopsis genes: ALCOHOL DEHYDROGENASE1 (ADH1; AT1G77120) and TRANSPARENT TESTA4 (TT4; AT5G13930) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…ZFNs that recognize the Arabidopsis ADH1 and TT4 genes were generated by using a publicly available platform (Oligomerized Pool ENgineering, OPEN) that is sufficiently robust to target most Arabidopsis genes (16,17). To make our approach more accessible, we developed a web-based genome browser that identifies potential OPEN ZFN target sites in the Arabidopsis genome.…”

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confidence: 99%

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High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Zhang

Maeder

Unger-Wallace

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

336

227

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We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA doublestrand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, ≈20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFNinduced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.nonhomologous end-joining | gene knockout | alcohol dehydrogenase | chalcone synthase

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“…We used OPEN (17) to generate ZFAs for potential target sites in two Arabidopsis genes: ALCOHOL DEHYDROGENASE1 (ADH1; AT1G77120) and TRANSPARENT TESTA4 (TT4; AT5G13930) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Zhang

Maeder

Unger-Wallace

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

336

227

View full text Add to dashboard Cite

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“…[10][11][12][13][14] Approaches other than modular assembly also provide access to polydactyl ZF proteins. 15 Artificial transcription factors that use ZFs for DNA recognition typically use six ZFs to recognize 18 bases of DNA; this length of sequence is theoretically unique in the genome. 16 Nuclear receptors are ligand-activated transcription factors that regulate growth, development and homeostasis.…”

Section: Introductionmentioning

confidence: 99%

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

2011

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Targeted zinc-finger (ZF) DNA-binding domains in conjunction with nuclear receptor ligand-binding domains (LBDs) produce chemically inducible gene switches that have applications in gene therapy and proteomic and genomic research. The benzoate X receptor-b (BXRb) LBD was used to construct homodimer and single-chain ZF transcription factors (ZF TF s). These ZF TF s specifically regulated the transcription of target genes in response to two ligands, ethyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, in a dose-dependent manner. The ZF TF s also regulated the expression of endogenous intercellular adhesion molecule-1 in response to either ligand. The advantage of BXRb-based ZF TF s is that the ligands are inexpensive and easily synthetically modified, making the system a base for creation of orthogonal ligand-receptor pairs and expanding the gene-switch toolbox.

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“…Many tools have been developed for this purpose, including zinc finger nucleases (ZFNs) (2), transcription activator-like effector nucleases (TALENs) (3), CRISPR/Cas9 endonucleases (CRISPRs) (4,5), and meganucleases (6). ZFNs can be purchased or (with effort) engineered using publicly available resources (7,8) but often display measurable off-target activity (9,10). In contrast, TALENs and CRISPRs can be more easily reprogrammed to a wider range of DNA sequences (11), but their lengthy reading frames may complicate packaging and delivery in certain contexts and applications (12).…”

mentioning

confidence: 99%

“…Meganucleases and zinc finger arrays for ZFNs have been generally redesigned using cell-based selection systems, where cell culturing is time-consuming and the efficiency of transformation limits the number of mutants to be screened (6,7,22). In contrast, selection systems such as in vitro compartmentalization (IVC) and mRNA display (in which protein-coding libraries are readily generated by PCR-based approaches) allow for screening much more complex libraries (10 10 to 10 12 of variant genes) (23,24).…”

mentioning

confidence: 99%

Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization

Takeuchi

Choi

Stoddard

2014

Proc. Natl. Acad. Sci. U.S.A.

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LAGLIDADG homing endonucleases (meganucleases) are sequencespecific DNA cleavage enzymes used for genome engineering. Recently, meganucleases fused to transcription activator-like effectors have been demonstrated to efficiently introduce targeted genome modifications. However, retargeting meganucleases to genomic sequences of interest remains challenging because it usually requires extensive alteration of a large number of amino acid residues that are situated in and near the DNA interface. Here we describe an effective strategy to extensively redesign such an extensive biomolecular interface. Well-characterized meganucleases are computationally screened to identify the best candidate enzyme to target a genomic region; that protein is then redesigned using iterative rounds of in vitro selections within compartmentalized aqueous droplets, which enable screening of extremely large numbers of protein variants at each step. The utility of this approach is illustrated by engineering three different meganucleases to cleave three human genomic sites (found in two exons and one flanking intron in two clinically relevant genes) and a fourth endonuclease that discriminates between single-nucleotide polymorphism variants of one of those targets. Fusion with transcription activator-like effector DNA binding domains significantly enhances targeted modification induced by meganucleases engineered in this study. Simultaneous expression of two such fusion endonucleases results in efficient excision of a defined genomic region.

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

Cited by 184 publications

References 79 publications

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription

Redesign of extensive protein–DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization

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