Zinc Finger Database (ZiFDB): a repository for information on C2H2 zinc fingers and engineered zinc-finger arrays

Zinc fingers are the most abundant DNA-binding motifs encoded by eukaryotic genomes and one of the best understood DNA-recognition domains. Each zinc finger typically binds a 3-nt target sequence, and it is possible to engineer zinc-finger arrays (ZFAs) that recognize extended DNA sequences by linking together individual zinc fingers. Engineered zinc-finger proteins have proven to be valuable tools for gene regulation and genome modification because they target specific sites in a genome. Here we describe ZiFDB (Zinc Finger Database; http://bindr.gdcb.iastate.edu/ZiFDB), a web-accessible resource that compiles information on individual zinc fingers and engineered ZFAs. To enhance its utility, ZiFDB is linked to the output from ZiFiT—a software package that assists biologists in finding sites within target genes for engineering zinc-finger proteins. For many molecular biologists, ZiFDB will be particularly valuable for determining if a given ZFA (or portion thereof) has previously been constructed and whether or not it has the requisite DNA-binding activity for their experiments. ZiFDB will also be a valuable resource for those scientists interested in better understanding how zinc-finger proteins recognize target DNA

Targeted Deletion and Inversion of Tandemly Arrayed Genes in Arabidopsis thaliana Using Zinc Finger Nucleases

Tandemly arrayed genes (TAGs) or gene clusters are prevalent in higher eukaryotic genomes. For example, approximately 17% of genes are organized in tandem in the model plant Arabidopsis thaliana. The genetic redundancy created by TAGs presents a challenge for reverse genetics. As molecular scissors, engineered zinc finger nucleases (ZFNs) make DNA double-strand breaks in a sequence-specific manner. ZFNs thus provide a means to delete TAGs by creating two double-strand breaks in the gene cluster. Using engineered ZFNs, we successfully targeted seven genes from three TAGs on two Arabidopsis chromosomes, including the well-known RPP4 gene cluster, which contains eight resistance (R) genes. The resulting gene cluster deletions ranged from a few kb to 55 kb with frequencies approximating 1% in somatic cells. We also obtained large chromosomal deletions of ~9 Mb at approximately one tenth the frequency, and gene cluster inversions and duplications also were achieved. This study demonstrates the ability to use sequence-specific nucleases in plants to make targeted chromosome rearrangements and create novel chimeric genes for reverse genetics and biotechnology

ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool

ZiFiT (Zinc Finger Targeter) is a simple and intuitive web-based tool that provides an interface to identify potential binding sites for engineered zinc finger proteins (ZFPs) in user-supplied DNA sequences. In this updated version, ZiFiT identifies potential sites for ZFPs made by both the modular assembly and OPEN engineering methods. In addition, ZiFiT now integrates additional tools and resources including scoring schemes for modular assembly, an interface with the Zinc Finger Database (ZiFDB) of engineered ZFPs, and direct querying of NCBI BLAST servers for identifying potential off-target sites within a host genome. Taken together, these features facilitate design of ZFPs using reagents made available to the academic research community by the Zinc Finger Consortium. ZiFiT is freely available on the web without registration at http://bindr.gdcb.iastate.edu/ZiFiT/

Efficient TALEN-mediated gene knockout in livestock

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications

TALEN-mediated editing of the mouse Y chromosome

The functional study of Y chromosome genes has been hindered by a lack of mouse models with specific Y chromosome mutations. We used transcription activator-like effector nuclease (TALEN)-mediated gene editing in mouse embryonic stem cells (mESCs) to produce mice with targeted gene disruptions and insertions in two Y-linked genes—Sry and Uty. TALEN-mediated gene editing is a useful tool for dissecting the biology of the Y chromosome.National Institutes of Health (U.S.) (US NIH grant R01-HG000257)National Institutes of Health (U.S.) (US NIH grant R01-CA084198)National Institutes of Health (U.S.) (US NIH grant R37-HD045022)Croucher Foundation (Scholarship)Howard Hughes Medical Institute (Investigator

An affinity-based scoring scheme for predicting DNA-binding activities of modularly assembled zinc-finger proteins

Zinc-finger proteins (ZFPs) have long been recognized for their potential to manipulate genetic information because they can be engineered to bind novel DNA targets. Individual zinc-finger domains (ZFDs) bind specific DNA triplet sequences; their apparent modularity has led some groups to propose methods that allow virtually any desired DNA motif to be targeted in vitro. In practice, however, ZFPs engineered using this ‘modular assembly’ approach do not always function well in vivo. Here we report a modular assembly scoring strategy that both identifies combinations of modules least likely to function efficiently in vivo and provides accurate estimates of their relative binding affinities in vitro. Predicted binding affinities for 53 ‘three-finger’ ZFPs, computed based on energy contributions of the constituent modules, were highly correlated (r = 0.80) with activity levels measured in bacterial two-hybrid assays. Moreover, Kd values for seven modularly assembled ZFPs and their intended targets, measured using fluorescence anisotropy, were also highly correlated with predictions (r = 0.91). We propose that success rates for ZFP modular assembly can be significantly improved by exploiting the score-based strategy described here

Selection-Free Zinc-Finger Nuclease Engineering by Context-Dependent Assembly (CoDA)

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe Context-Dependent Assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations

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

<p>Abstract</p> <p>Background</p> <p>Zinc 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.</p> <p>Description</p> <p>ZFNGenome 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; <it>S. cerevisiae, C. reinhardtii, A. thaliana</it>, <it>D. melanogaster, D. rerio, C. elegans</it>, and <it>H. sapiens </it>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 <it>in vivo</it>. 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.</p> <p>Conclusions</p> <p>ZFNGenome 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.</p> <p>ZFNGenome is freely available at: <url>http://bindr.gdcb.iastate.edu/ZFNGenome</url> or at the Zinc Finger Consortium website: <url>http://www.zincfingers.org/</url>.</p

Evolution of Genome Size and Complexity in Pinus

BACKGROUND: Genome evolution in the gymnosperm lineage of seed plants has given rise to many of the most complex and largest plant genomes, however the elements involved are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Gymny is a previously undescribed retrotransposon family in Pinus that is related to Athila elements in Arabidopsis. Gymny elements are dispersed throughout the modern Pinus genome and occupy a physical space at least the size of the Arabidopsis thaliana genome. In contrast to previously described retroelements in Pinus, the Gymny family was amplified or introduced after the divergence of pine and spruce (Picea). If retrotransposon expansions are responsible for genome size differences within the Pinaceae, as they are in angiosperms, then they have yet to be identified. In contrast, molecular divergence of Gymny retrotransposons together with other families of retrotransposons can account for the large genome complexity of pines along with protein-coding genic DNA, as revealed by massively parallel DNA sequence analysis of Cot fractionated genomic DNA. CONCLUSIONS/SIGNIFICANCE: Most of the enormous genome complexity of pines can be explained by divergence of retrotransposons, however the elements responsible for genome size variation are yet to be identified. Genomic resources for Pinus including those reported here should assist in further defining whether and how the roles of retrotransposons differ in the evolution of angiosperm and gymnosperm genomes

De novo domestication of wild tomato using genome editing

Breeding of crops over millennia for yield and productivity1 has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost2. We devised a CRISPR–Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines. We report that editing of six loci that are important for yield and productivity in present-day tomato crop lines enabled de novo domestication of wild Solanum pimpinellifolium. Engineered S. pimpinellifolium morphology was altered, together with the size, number and nutritional value of the fruits. Compared with the wild parent, our engineered lines have a threefold increase in fruit size and a tenfold increase in fruit number. Notably, fruit lycopene accumulation is improved by 500% compared with the widely cultivated S. lycopersicum. Our results pave the way for molecular breeding programs to exploit the genetic diversity present in wild plants