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1.  Rapid Mutation of Endogenous Zebrafish Genes Using Zinc Finger Nucleases Made by Oligomerized Pool ENgineering (OPEN) 
PLoS ONE  2009;4(2):e4348.
Background
Customized 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 Findings
Here 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 Significance
In 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.
doi:10.1371/journal.pone.0004348
PMCID: PMC2634973  PMID: 19198653
2.  Targeted gene inactivation in zebrafish using engineered zinc finger nucleases 
Nature biotechnology  2008;26(6):695-701.
In most vertebrate model systems direct genomic manipulation at a specific locus is still not feasible. Zinc finger nucleases (ZFNs) provide a system to introduce genomic lesions at a specific site in vertebrate cell lines1-3. Here, we adapt this technology to create targeted mutations in the zebrafish germline in vivo. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdra. Co-injection of mRNAs encoding these ZFNs into 1-cell stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germline with high frequency. Thus, engineered ZFNs can introduce heritable mutations into a vertebrate genome. Importantly, this in vivo gene inactivation approach obviates the need for embryonic stem cell lines and will be applicable to most animal species, especially in cases where early stage embryos are easily accessible.
doi:10.1038/nbt1398
PMCID: PMC2502069  PMID: 18500337
3.  Directed evolution of an enhanced and highly efficient FokI cleavage domain for Zinc Finger Nucleases 
Journal of molecular biology  2010;400(1):96-107.
Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The high specificity and affinity of these chimeric enzymes are based on custom-designed zinc finger proteins (ZFPs). In order to improve the performance of existing ZFN technology, we developed an in vivo evolution-based approach to improve the efficacy of the FokI cleavage domain (FCD). After multiple rounds of cycling mutagenesis and DNA shuffling, a more efficient nuclease variant (Sharkey) was generated. In vivo analyses indicated that Sharkey is >15-fold more active than wild-type FCD on a diverse panel of cleavage sites. Further, a mammalian cell-based assay showed a 3 to 6-fold improvement in targeted mutagenesis for ZFNs containing derivatives of the Sharkey cleavage domain. We also identified mutations that impart sequence specificity to the FCD that might be utilized in future studies to further refine ZFNs through cooperative specificity. In addition, Sharkey was observed to enhance the cleavage profiles of previously published and newly selected heterodimer ZFN architectures. This enhanced and highly efficient cleavage domain will aid in a variety of ZFN applications in medicine and biology.
doi:10.1016/j.jmb.2010.04.060
PMCID: PMC2885538  PMID: 20447404
zinc finger nuclease; directed evolution; gene targeting
4.  Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases 
Nature biotechnology  2008;26(6):702-708.
We describe here the use of zinc finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), where targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury genes. In both cases, injection of ZFN-encoding mRNA into 1-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Disrupted ntl alleles were transmitted from ZFN mRNA-injected founder animals in over half the adults tested at frequencies averaging 20%. The frequency and precision of gene disruption events observed, in combination with the ability to design ZFNs against any locus, open fundamentally novel avenues of experimentation, and suggest that ZFN technology may be widely applied to many organisms that allow mRNA delivery into the fertilized egg.
doi:10.1038/nbt1409
PMCID: PMC2674762  PMID: 18500334
5.  Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases 
Nature protocols  2009;4(12):1855.
Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months.
doi:10.1038/nprot.2009.209
PMCID: PMC2814337  PMID: 20010934
6.  Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells 
Nucleic Acids Research  2005;33(18):5978-5990.
Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.
doi:10.1093/nar/gki912
PMCID: PMC1270952  PMID: 16251401
7.  Creating Designed Zinc Finger Nucleases with Minimal Cytotoxicity 
Journal of molecular biology  2010;405(3):630-641.
Zinc finger nucleases (ZFNs) have emerged as powerful tools for delivering a targeted genomic double-strand break (DSB) to either stimulate local homologous recombination (HR) with investigator-provided donor DNA or induce gene mutations at the site of cleavage in absence of a donor by non-homologous end joining (NHEJ), both in plant and mammalian cells, including human cells. ZFNs are formed by fusing zinc finger proteins (ZFPs) to the non-specific cleavage domain of FokI restriction enzyme. ZFN-mediated gene targeting yields high gene modification efficiencies (>10%), in a variety of cells and cell types by delivering a recombinogenic DSB to the targeted chromosomal locus, using two designed ZFNs. Mechanism of DSB by ZFNs requires that two ZFN monomers bind to their adjacent cognate sites on DNA and the FokI nuclease domains dimerize to form the active catalytic center for the induction of the DSB. In the case of ZFNs fused to wild-type FokI cleavage domains, homodimers may also form, which could limit the efficacy and safety of the ZFNs by inducing off-target cleavage. In this article, we report further refinements to obligate heterodimer variants of FokI cleavage domain for creating custom ZFNs with minimal cellular toxicity. The efficacy and efficiency of the re-engineered obligate heterodimer variants of FokI cleavage domain were tested using the GFP gene targeting reporter system. The 3- and 4-finger ZFP fusions to REL_DKK pair among the newly generated FokI nuclease domain variants appear to eliminate or greatly reduce the toxicity of designer ZFNs to human cells.
doi:10.1016/j.jmb.2010.10.043
PMCID: PMC3017627  PMID: 21094162
8.  Engineered Zinc Finger Nuclease–Mediated Homologous Recombination of the Human Rhodopsin Gene 
Zinc finger nuclease–mediated homologous recombination is examined as a permanent genetic approach to treat retinitis pigmentosa.
Purpose.
Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment.
Methods.
Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination.
Results.
Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene.
Conclusions.
ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination.
doi:10.1167/iovs.10-5781
PMCID: PMC3055761  PMID: 20671268
9.  Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132 
PLoS ONE  2013;8(1):e54282.
Background
Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The characterization of ZFN protein stability and the development of simple methods to improve ZFN function would facilitate the application of this promising technology. However, the factors that affect ZFN protein stability and function are not yet clear. Here, we determined the stability and half-life of two ZFN proteins and examined the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-Hl), a proteasome inhibitor, on ZFN-mediated gene modifications.
Methodology/Principal Findings
ZFN proteins were expressed in 293T cells after transfection of ZFN-encoding plasmids. We studied two ZFN pairs: Z-224, which targets the CCR5 gene, and K-230, which targets a region 230 kbp upstream of CCR5. Western blotting after treatment with cycloheximide showed that the half-life of these ZFN proteins was around two hours. An immunoprecipitation assay revealed that the ZFN interacts with ubiquitin molecules and undergoes polyubiquitination in vivo. Western blotting showed that the addition of MG132, a proteasomal inhibitor, increased ZFN protein levels. Finally, a surrogate reporter assay and a T7E1 assay revealed that MG132 treatment enhanced ZFN-directed gene editing.
Conclusions
To our knowledge, this is the first study to investigate ZFN protein stability and to show that a small molecule can increase ZFN activity. Our protein stability study should lay the foundation for further improvement of ZFN technology; as a first step, the use of the small molecule MG132 can enhance the efficiency of ZFN-mediated gene editing.
doi:10.1371/journal.pone.0054282
PMCID: PMC3554775  PMID: 23365657
10.  Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs) 
PLoS ONE  2012;7(5):e37877.
Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%–76.8% compared to 1.1%–3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.
doi:10.1371/journal.pone.0037877
PMCID: PMC3360007  PMID: 22655075
11.  Zinc Finger–Based Knockout Punches for Zebrafish Genes 
Zebrafish  2008;5(2):121-123.
The ability to manipulate the genome is critical to develop and test hypotheses based on genetics. Knockdown strategies focused on RNAi and/or morpholinos are excellent genetic tools, but they come with substantial technical limitations. A new gene targeting approach employing synthetic zinc finger nuclease (ZFN) technology is a powerful and complementary approach to directly modify genetic loci for many diverse applications, notably enhancing Danio rerio (the zebrafish) as an experimental organism for understanding human disease. This ZFN-based technology to generate targeted knockouts in this aquatic animal opens the door to an array of new biological models of human disease and genetic testing.
doi:10.1089/zeb.2008.9988
PMCID: PMC2849655  PMID: 18554175
12.  Zinc Finger–Based Knockout Punches for Zebrafish Genes 
Zebrafish  2008;5(2):121-123.
Abstract
The ability to manipulate the genome is critical to develop and test hypotheses based on genetics. Knockdown strategies focused on RNAi and/or morpholinos are excellent genetic tools, but they come with substantial technical limitations. A new gene targeting approach employing synthetic zinc finger nuclease (ZFN) technology is a powerful and complementary approach to directly modify genetic loci for many diverse applications, notably enhancing Danio rerio (the zebrafish) as an experimental organism for understanding human disease. This ZFN-based technology to generate targeted knockouts in this aquatic animal opens the door to an array of new biological models of human disease and genetic testing.
doi:10.1089/zeb.2008.9988
PMCID: PMC2849655  PMID: 18554175
13.  Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA 
Nucleic Acids Research  2008;36(12):3926-3938.
The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases.
doi:10.1093/nar/gkn313
PMCID: PMC2475635  PMID: 18511461
14.  Custom-designed zinc finger nucleases: What is next? 
Custom-designed zinc finger nucleases (ZFNs) – proteins designed to cut at specific DNA sequences – combine the non-specific cleavage domain (N) of Fok I restriction endonuclease with zinc finger proteins (ZFPs). Because the recognition specificities of the ZFPs can be easily manipulated experimentally, ZFNs offer a general way to deliver a targeted site-specific double-strand break (DSB) to the genome. They have become powerful tools for enhancing gene targeting – the process of replacing a gene within a genome of cells via homologous recombination (HR) – by several orders of magnitude. ZFN-mediated gene targeting thus confers molecular biologists with the ability to site-specifically and permanently alter not only plant and mammalian genomes but also many other organisms by stimulating HR via a targeted genomic DSB. Site-specific engineering of the plant and mammalian genome in cells so far has been hindered by the low frequency of HR. In ZFN-mediated gene targeting, this is circumvented by using designer ZFNs to cut at the desired chromosomal locus inside the cells. The DNA break is then patched up using the new investigator-provided genetic information and the cells’ own repair machinery. The accuracy and high efficiency of the HR process combined with the ability to design ZFNs that target most DNA sequences (if not all) makes ZFN technology not only a powerful research tool for site-specific manipulation of the plant and mammalian genomes, but also potentially for human therapeutics in the future, in particular for targeted engineering of the human genome of clinically transplantable stem cells.
doi:10.1007/s00018-007-7206-8
PMCID: PMC2921987  PMID: 17763826
Zinc finger nucleases; gene targeting; genome engineering; site-specific modification; targeted mutagenesis; gene correction; homologous recombination; non-homologous end-joining
15.  Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes 
Summary
Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.
doi:10.1007/978-1-59745-483-4_40
PMCID: PMC2921164  PMID: 19488728
Gene therapy; Nonviral vectors; Zinc finger nucleases; Gene targeting; Genome engineering; Site-specific modification; Targeted mutagenesis; Gene correction; Homologous recombination; Nonhomologous end joining
16.  Zinc-finger Nucleases as Gene Therapy Agents 
Gene therapy  2008;15(22):1463-1468.
Zinc-finger nucleases (ZFNs) are powerful tools for experimental gene manipulation. A number of recent papers have shown how this technology can be applied effectively to models of human gene therapy. Significant target genes and useful methods of ZFN delivery have been reported. Important strides have been made in minimizing toxic side effects observed with some ZFNs, which bodes well for their ultimate safety. New tools are available for the design and testing of ZFNs for new target genes. Applications of ZFNs to stem cells have been described, and genuine gene therapy trials appear to be on the immediate horizon.
doi:10.1038/gt.2008.145
PMCID: PMC2747807  PMID: 18784746
Zinc-finger nucleases; gene targeting; non-homologous end joining; homologous recombination
17.  The CRISPR System—Keeping Zebrafish Gene Targeting Fresh 
Zebrafish  2013;10(1):116-118.
Abstract
We are entering a new era in our ability to modify and edit the genomes of model organisms. Zinc finger nucleases (ZFNs) opened the door to the first custom nuclease-targeted genome engineering in the late 1990s. However, ZFNs remained out of reach for most research labs because of the difficulty of production, high costs, and modest efficacy in many applications. Transcription activator-like effector nucleases (TALENs) were built upon a DNA binding system discovered in a group of plant bacterial pathogens and broadened custom nuclease technology, showing significant improvements in both targeting flexibility and efficiency. Perhaps most importantly, TALENs are open source and easy to produce, providing zebrafish laboratories around the world with affordable tools that can be made in-house rapidly, at low cost, and with reliably high activity. Now a new system for targeted genome engineering derived from the CRISPR/Cas system in eubacteria and archaea promises to simplify this process further. Together, these tools will help overcome many of the bottlenecks that have constrained gene targeting in zebrafish, paving the way for advanced genome engineering applications in this model teleost.
doi:10.1089/zeb.2013.9999
PMCID: PMC3629780  PMID: 23536990
18.  Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification 
Molecular cell  2008;31(2):294-301.
Summary
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 multi-finger 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 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, 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.
doi:10.1016/j.molcel.2008.06.016
PMCID: PMC2535758  PMID: 18657511
19.  Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases 
PLoS ONE  2010;5(1):e8870.
Background
Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc.
Methodology/Principal Findings
We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype.
Conclusions and Significance
The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.
doi:10.1371/journal.pone.0008870
PMCID: PMC2810328  PMID: 20111598
20.  Efficient Methods for Targeted Mutagenesis in Zebrafish Using Zinc-Finger Nucleases: Data from Targeting of Nine Genes Using CompoZr or CoDA ZFNs 
PLoS ONE  2013;8(2):e57239.
Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich) or the CoDA (context-dependent assembly) platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1) evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2) a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5%) when compared to CopmoZr ZFNs (9–98%). This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.
doi:10.1371/journal.pone.0057239
PMCID: PMC3579846  PMID: 23451191
21.  ZFN-Site searches genomes for zinc finger nuclease target sites and off-target sites 
BMC Bioinformatics  2011;12:152.
Background
Zinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence.
Results
ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases.
Conclusions
ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.
doi:10.1186/1471-2105-12-152
PMCID: PMC3113941  PMID: 21569489
22.  Simultaneous Screening and Validation of Effective Zinc Finger Nucleases in Yeast 
PLoS ONE  2013;8(5):e64687.
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.
doi:10.1371/journal.pone.0064687
PMCID: PMC3669427  PMID: 23741369
23.  An Over Expression APP Model for Anti-Alzheimer Disease Drug Screening Created by Zinc Finger Nuclease Technology 
PLoS ONE  2013;8(11):e75493.
Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer’s disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.
doi:10.1371/journal.pone.0075493
PMCID: PMC3819351  PMID: 24223114
24.  Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion 
Nucleic Acids Research  2010;38(22):8269-8276.
Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.
doi:10.1093/nar/gkq720
PMCID: PMC3001086  PMID: 20716517
25.  Synthetic Zinc Finger Nuclease Design and Rapid Assembly 
Human Gene Therapy  2011;22(9):1155-1165.
Abstract
Engineered zinc finger nucleases (ZFNs) are a tool for genome manipulation that are of great interest to scientists in many fields. To meet the needs of researchers wishing to employ ZFNs, an inexpensive, rapid assembly procedure would be beneficial to laboratories that do not have access to the proprietary reagents often required for ZFN production. Using freely available sequence data derived from the Zinc Finger Targeter database, we developed a protocol for synthesis and directed insertion of user-defined ZFNs into a versatile plasmid expression system. This oligonucleotide-based isothermal DNA assembly protocol was used to determine whether we could generate functional nucleases capable of endogenous gene editing. We targeted the human α-l-iduronidase (IDUA) gene on chromosome 4, mutations of which result in the severe lysosomal storage disease mucopolysaccharidosis type I. In approximately 1 week we were able to design, assemble, and test six IDUA-specific ZFNs. In a single-stranded annealing assay five of the six candidates we tested performed at a level comparable to or surpassing previously reported ZFNs. One of the five subsequently showed nuclease activity at the endogenous genomic IDUA locus. To our knowledge, this is the first demonstration of in silico-designed, oligonucleotide-assembled, synthetic ZFNs, requiring no specialized templates or reagents that are capable of endogenous human gene target site activity. This method, termed CoDA-syn (context-dependent assembly-synthetic), should facilitate a more widespread use of ZFNs in the research community.
Osborn and colleagues report development of a protocol for synthesis and direct insertion of user-defined zinc finger nucleases (ZFN) into versatile expression systems. ZFNs targeting the human α-L-iduronidase (IDUA) gene designed in this manner performed comparably to previously reported ZFNs. This method, termed CoDA-syn (Context Dependent Assembly-synthetic), should facilitate a more widespread utilization of ZFNs in the research community.
doi:10.1089/hum.2011.072
PMCID: PMC4076977  PMID: 21663559

Results 1-25 (756865)