Oligomerized Pool ENgineering Research Articles

Simultaneous screening and validation of effective zinc finger nucleases in yeast.

Zinc finger nucleases (ZFNs) have been successfully used for genome modification in various cell types and species. However, construction of an effective ZFN remained challenging. Previous studies all focused on obtaining specific zinc finger proteins (ZFPs) first via bacterial 2-hybrid approach, and then fusing selected ZFPs to FokI nuclease domain. These assembled ZFNs have high rate of failing to cleave target sites in vivo. In this study, we developed a simultaneous screening and validation system to obtain effective ZFNs directly in yeast AH109. This system is based on Gal4 reporter system carrying a unique intermediate reporter plasmid with two 30-bp Gal4 homology arms and a ZFN target site. DNA double strand breaks introduced on target sequence by ZFNs were repaired by single strand annealing (SSA) mechanism, and the restored Gal4 drove reporter genes expression. Taking the advantage of OPEN (Oligomerized Pool ENgineering) selection, we constructed 3 randomized ZFNs libraries and 9 reporter strains for each target gene. We tested this system by taking goat α s1-casein as target gene following three-step selection. Consequently, 3 efficient pairs of ZFNs were obtained from positive colonies on selective medium. The ZFNs achieved a 15.9% disruption frequency in goat mammary epithelial cells. In conclusion, we created a novel system to obtain effective ZFNs directly with simultaneous screening and validation.

Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification.

Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid “linker” and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the “spacer” separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5–7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.

Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos

Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.

A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks.

Profile of Keith Joung

Profile of Keith Joung

Targeting DNA With Fingers and TALENs.

Researchers have long recognized that the random introduction of new DNA into the genome could result in unpredictable genetic effects due to bilateral influences between transgenic and endogenous sequences. Hence, precise editing or replacement of mutant genes has been a major goal of gene therapy since its inception. That goal looked possible with the publication of studies demonstrating that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and separate, designer DNA-recognition domains.1,2 Zinc-finger nucleases (ZFNs) are artificial endonucleases that consist of a FokI cleavage domain tethered to engineered Cys2His2 zinc-finger, DNA-binding polypeptides.3,4,5 ZFNs have gained considerable momentum and are widely considered the most mature nuclease technology in the gene therapy field.6 However, after 15 years in the spotlight, a new chimeric nuclease has emerged, Transcription Activator-Like (TAL) Effector Nucleases (TALENs).7,8,9,10 Here, we describe functional and design characteristics of ZFNs and TALENs and discuss their expanding role as tools for research and gene therapy.

Engineering Zinc Finger Proteins using Accessory Binding Modules

We report on the engineering of zinc finger proteins (ZFPs) as synthetic transcription factors by appending accessory modules to enhance protein binding affinity to target DNA. Over the last few years, transcription factor engineering has provided a valuable approach to genetically engineer a wide variety of cell types. ZFP technology is now being used to directly edit chromosomal DNA by creating synthetic nucleases. Recently, this approach has been used to produce genetically enhanced crops and improved biophama products and to cure genetic disease. The central feature of this technology is generation of synthetic transcription factors that bind to “target” DNA sequences with high specificity. In this way, modular assembly provides a convenient approach to synthesize target-specific ZFPs by generating modular proteins consisting of tandem repeats of well-characterized zinc fingers through standard molecular cloning. Oligomerized pool engineering (OPEN) and context-dependent assembly (CoDA) are alternative, albeit more complex protocols for creating ZFPs. In this work, we describe the direct engineering of two- and three-finger ZFPs to improve DNA binding site recognition specificity and affinity. We generated chimeric ZFPs containing the accessory module PAR from the yeast transcriptional activator ADR1, a zinc finger transcription factor from Saccharomyces cerevisiae. We observed that addition of the PAR binding module increased the binding affinity for chimeric ZFPs to target DNA sequences without affecting the selectivity for two-finger ZFPs. Two-finger ZFPs are generally not used for targeted DNA binding, because the functionality of ZFPs containing only two modules results in a loss in binding affinity and selectivity. Here, we show that ZFPs consisting of only two-finger modules can be successfully engineered to target specific DNA sequences by appending accessory binding modules. Based on this work, we anticipate that these chimeric ZFPs will enable new applications involving genetic modification.

Potential application of FoldX force field based protein modeling in zinc finger nucleases design

Engineered sequence-specific zinc finger nucleases (ZFNs) make the highly efficient modification of eukaryotic genomes possible. However, most current strategies for developing zinc finger nucleases with customized sequence specificities require the construction of numerous tandem arrays of zinc finger proteins (ZFPs), and subsequent largescale in vitro validation of their DNA binding affinities and specificities via bacterial selection. The labor and expertise required in this complex process limits the broad adoption of ZFN technology. An effective computational assisted design strategy will lower the complexity of the production of a pair of functional ZFNs. Here we used the FoldX force field to build 3D models of 420 ZFP-DNA complexes based on zinc finger arrays developed by the Zinc Finger Consortium using OPEN (oligomerized pool engineering). Using nonlinear and linear regression analysis, we found that the calculated protein-DNA binding energy in a modeled ZFP-DNA complex strongly correlates to the failure rate of the zinc finger array to show significant ZFN activity in human cells. In our models, less than 5% of the three-finger arrays with calculated protein-DNA binding energies lower than -13.132 kcal mol(-1) fail to form active ZFNs in human cells. By contrast, for arrays with calculated protein-DNA binding energies higher than -5 kcal mol(-1), as many as 40% lacked ZFN activity in human cells. Therefore, we suggest that the FoldX force field can be useful in reducing the failure rate and increasing efficiency in the design of ZFNs.

P22 Age and sex related differences in satellite cell number, proliferation and self renewal

knockdown of FKRP expression by anti-sense morpholino (MO) injection into zebrafish embryos. This system gives a range of phenotypes but the knockdown has limited duration. A stable mutant line would negate the problems of a transient knockdown system which include decrease of knockdown efficiency due to cell division and consequent dilution of MO with time. Zinc Finger Nucleases (ZFNs) which are artificial nucleases specific to 9 base pairs (bp) target regions either side of a 5–7bp spacer. ZFNs are capable of causing a range of genomic mutations at the target site by inducing double-strand break repair through non-homologous end joining. There are two strategies being adopted, these are Oligomerized Pool Engineering (OPEN) and Context Dependant Assembly (CODA). The sequences generated in each approach for the zinc fingers are cloned into a vector containing the Fok-I nuclease domain. RNA from the expression vectors is injected into the embryo at the single cell stage. Mutant embryos will be screened by genotype against database sequences for mutations. Immunostaining will be used to investigate the phenotypes of the mutants focussing on eyes and muscle.

ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

BackgroundZinc Finger Nucleases (ZFNs) have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN) method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms.DescriptionZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; S. cerevisiae, C. reinhardtii, A. thaliana, D. melanogaster, D. rerio, C. elegans, and H. sapiens and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s). Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence). Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter) "confidence" scores that estimate the likelihood that a chosen ZFN target site will function in vivo. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the effectiveness of a given ZFN in creating double-stranded breaks.ConclusionsZFNGenome provides a user-friendly interface that allows researchers to access resources and information regarding genomic target sites for engineered ZFNs in seven model organisms. This genome-wide database of potential ZFN target sites should greatly facilitate the utilization of ZFNs in both basic and clinical research.ZFNGenome is freely available at: http://bindr.gdcb.iastate.edu/ZFNGenome or at the Zinc Finger Consortium website: http://www.zincfingers.org/.

Predicting success of oligomerized pool engineering (OPEN) for zinc finger target site sequences.

BackgroundPrecise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN) is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs) designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable.ResultsAnalysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity+ of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88%) and specificity+ (92%), but with reduced ROC AUC (0.77). Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT.ConclusionZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function successfully in vivo, substantially reducing the experimental effort required. ZiFOpT is freely available and incorporated in the Zinc Finger Targeter web server (http://bindr.gdcb.iastate.edu/ZiFiT).

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

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 double-strand 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, approximately 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 ZFN-induced 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.

Reply to “Genome editing with modularly assembled zinc-finger nucleases”

To the Editor: The publications cited by Kim et al. describing successful construction of ZFNs by modular assembly only further support our original conclusion that this method has a high failure rate for engineering functional zinc finger arrays.1 Two of the three reports cited provide data that enable calculation of failure rates for modular assembly.2, 3 Although it is true that modular assembly yielded ZFNs for ~25% of the DNA sites targeted, failure rates measured instead by the number of zinc finger proteins tested are remarkably consistent with those reported in our original Correspondence.1 For example, at the human CCR5 gene, Kim et al. screened 315 pairs of ZFNs for activity;3 this large-scale effort yielded only a small number of functional ZFN pairs (93.3% failure rate for ZFN pairs tested). Similarly, for the tobacco SuRB gene,2 we tested 32 zinc finger arrays in vitro but identified only three with functional activity (91.6% failure rate for zinc finger arrays tested). These data are consistent with our original predicted failure rates of ~94% and ~76% for modularly assembled ZFN pairs and zinc finger arrays, respectively.1 We believe that failure rates measured by numbers of zinc finger arrays or ZFN pairs tested rather than by numbers of DNA sites targeted are more relevant statistics for potential ZFN users because these influence how many proteins must be modularly assembled and tested for each potential site. We also respectfully disagree with various statements Kim et al. make regarding Oligomerized Pool ENgineering (OPEN), a selection-based method for engineering zinc finger arrays.4 Kim et al. contend that our recent report2 shows that “modularly assembled ZFNs…outperformed OPEN ZFNs in terms of mutation frequencies” and that “each method [modular assembly and OPEN] gave rise to successful genome modification at one out of four target sites.” However, the modularly assembled and OPEN ZFNs in our study were designed to recognize different DNA target sites and therefore Kim et al.’s conclusion is based on an indirect comparison. For the single site where direct comparison was possible, only the OPEN approach yielded functional ZFNs. In addition, we found in a different direct comparison of OPEN and modular assembly at five different target sites in the EGFP gene that OPEN ZFNs were active at four sites whereas modularly assembled ZFNs showed activity at only one site.4 Furthermore, the OPEN ZFNs outperformed the modularly assembled ZFNs at this one site. We believe that the higher activity and success rate of OPEN ZFNs is most likely due to the method’s explicit consideration of the well-established context-dependent behavior of zinc fingers,1, 4 a parameter that is largely ignored by modular assembly. Kim et al assert that “OPEN ZFNs thus far are largely limited to target GNN repeat sequences (i.e. 5’-GNN GNN GNN-3’)”. However, ~40% (11 out of 28) of the half-sites successfully targeted in endogenous genes by OPEN ZFNs to date actually contain one or more non-GNN subsites (Supplementary Table 1). Furthermore, potential users of the ZFN technology should be aware that with both modular assembly and OPEN it is easiest to target sequences composed entirely of GNN subsites. Kim et al. further suggest “that careful choice and use of reliable modules could improve success rates [of modular assembly].” However, given that our original Correspondence showed that modular assembly has considerably higher failure rates for target sites harboring one or more non-GNN subsites than for those composed solely of GNN subsites,1 taking this approach could also substantially restrict the targeting range of the method. In summary, we advise potential users to carefully weigh the effort required both to engineer and to validate ZFNs. Although modular assembly is simpler to perform than the selection-based OPEN method, our direct comparisons suggest that OPEN is more efficient than modular assembly for engineering functional ZFNs. As researchers who have practiced both methods,2, 4–6 we have concluded that modular assembly requires as much (if not more) time and effort to use than OPEN when one considers the requirement to screen hundreds of largely non-functional modularly assembled ZFNs for cellular activity. Nonetheless, the Zinc Finger Consortium continues to make reagents and software for both modular assembly and OPEN available to academic scientists (http://www.addgene.org/zfc; www.zincfingers.org/software-tools.htm). We believe that rather than attempting to improve the success rate of modular assembly, future efforts should instead focus on further simplification of selection-based techniques or development of more effective design-based methods that account for the context-dependent behavior of zinc finger domains.

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

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.

415. Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering): A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases

415. Further Improvements and Applications of OPEN (Oligomerized Pool ENgineering): A Rapid, Robust, and Publicly Available Method for Engineering Customized Zinc Finger Nucleases

Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN)

BackgroundCustomized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells.Methodology/Principal FindingsHere we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts.Conclusions and SignificanceIn summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.

Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.