In principle these technologies are capable of targeting virtually any site in the genome and editing the dna that is there. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. This new technology opens up revolutionary possibilities in agriculture, animal husbandry and molecular medicine. A dnabinding domain comprised of a chain of twofinger modules, each recognizing a unique hexamer 6 bp sequence of dna. Targeted genome editing in the eukaryotic systems using zinc.
The world now has two major technologies for targeted editing of the genome. The dsb is induced by a zinc finger nuclease zfn4,5, which is a designed, sequence specific endonuclease that can be customized to cleave a user chosen. A selection free, highly efficient targeted mutagenesis approach based on a novel zfn monomer arrangement for genome engineering in tomato reveals plant trait modifications. An individual zinc finger consists of approximately 30 amino acids in a conserved configuration figure 1a. One of the more advanced selectionbased design strategies currently in use is referred to as the open oligomerized pool engineering design 18, 19. Zinc finger nucleases zfns are synthetic restriction enzymes evolved by fusing zinc finger that facilitate genome editing by creating a doublestranded break in dna. For instance, four zinc finger units can be assembled into a zinc finger chain that recognizes a 12 base pair dna sequence.
A general method for improving the efficiency of gene targeting would be valuable in. Genome engineering with zincfinger nucleases genetics. A zinc finger is formed by repeated groupings of cysteine and histidine residues and recognize 3 nucleotides nt. Design, engineering, and characterization of zinc finger. Genome engineering with programmable nucleases depends on cellular. Faster method of engineering zincfinger nucleases developed.
Designer zinc finger proteins zfps are the sequence specific tools which are used to manipulate a gene of interest at genomic level. The first truly targetable reagents were the zinc finger nucleases zfns. Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. Twofinger modules are stitched together to form a zinc finger protein, each with specificity of. All members have committed to making all protocols, software, dna sequences, and reagents they publish available to the academic scientific community. Targeted genome engineering via zinc finger nucleases springerlink. Zinc finger nucleases as tools to understand and treat human. B revia enhancing gene targeting with designed zinc finger. To address this issue, zinc finger nuclease zfnbased. The generation of genetically modified animals or plants with genetargeted deletions or modifications is a powerful tool to analyze gene function, study disease and produce organisms of economical interest. Genome engineering of crops with designer nucleases. Pdf target zinc finger nucleases in genome engineering. Pdf genome targeting engineering is one of the most important advances in genetic. To assess the genome editing activities of the zfns, the dna.
Zinc finger nucleases zfnsproteins customdesigned to cut at specific dna sequenceswere originally developed in our lab for this purpose of delivering a targeted genomic dsb within plant and mammalian cells to enable such gene targeting experiments. Targeted genome editing using sitespecific nucleases. N2 recent advances in genome engineering provide newfound control over a plants genetic material. Thus, in addition to basic advances in dna manipulation, three specific research threads led to the production of the first zincfinger nucleases zfns. This approach is an outcome of the zinc finger consortium, a collaboration across multiple academic labs interested in promoting the application of zfps as a research tool. Genome editing uses engineered nucleases as powerful tools to target specific dna sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. Zinc finger nucleases zfns function as dimers and each monomer is a fusion protein of a zinc finger dna binding domain and a nonspecific foki nuclease domain 12. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. The emergence of sitespecific dna endonucleases and genome editing has. Improved cellpenetrating zincfinger nuclease proteins for. The zinc finger domain, which typically recognizes 3nucleotide motifs on dna, was the first to be exploited in this way. Numerous examples of successful genome editing now exist.
Starting from an archive of zinc finger modules known to function well together, effective multifinger arrays can be. Instead, it also depends on the efficiency with which the genome engineering reagents are delivered, the cells they are delivered to, and the effectiveness of selecting or screening for cells with the desired modification. Trautman, danacarroll gene targetingthe process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 1. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger. Selection free zincfingernuclease engineering by contextdependent assembly coda. Genome engineering with compozr custom zinc finger. For instance, four zinc finger units can be assembled into a zinc finger chain. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zinc finger nucleases to target unique sequences within complex genomes. Microinjection and homologous recombination inembryonic stem es cells, are robust but overallinefficient. Genome editing is an exciting technology that allows for specific manipulation of complex genomes. Zinc finger engineering the zinc finger consortium. Three types of engineered nucleases have been used to date for this purpose. T1 plant genome engineering with sequencespecific nucleases.
Targeted genome editing in the eukaryotic systems using. Genome engineering with compozr custom zinc finger nucleases. Solisleal a and berges bk department of microbiology and molecular biology, brigham young university, usa. It is now possible for most bench scientists to alter dna in living plant cells in a variety of ways, including introducing specific nucleotide substitutions in a gene that change a proteins amino acid sequence, deleting genes or chromosomal segments, and inserting foreign dna at precise genomic. Part of thebioinformatics commons,computational biology commons, and thegenomics commons. Genome editing with modularly assembled zincfinger nucleases. Genome editing with engineered zinc finger nucleases nature. Us2055793a1 sitespecific modification of the human. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. In a correspondence in nature methods, some members of the zinc finger consortium reported discouragingly high failure rates. Geen is an effective genetic engineering method that uses molecular scissors, or artificially engineered nucleases, to target and digest dna at specific locations in the genome.
Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement. Genome engineering with zinc finger nuclease and talens. Mock indicates cells treated with serumfree medium. Mar 06, 20 rote 20 presentation targeted genome editing in the eukaryotic systems using zinc finger nucleases joe frangipane, sigmaaldrich. Zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. References 117 and 119 demonstrate two different methods to identify offtarget sites. Genome engineering with targetable nucleases dana carroll. Buy targeted genome editing using sitespecific nucleases. Genome engineering with targetable nucleases annual.
Recent advances in genome engineering provide newfound control over a plants genetic material. The cys 2his 2 zinc finger domain is among the most common types of dnabinding motifs found in eukaryotes and represents the second most frequently encoded protein domain in the human genome. Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins. Twofinger modules are stitched together to form a zinc. How to achieve precise gene targeting in plants and especially in crops remains a longsought goal for elucidating gene function and advancing molecular breeding. Targeted genetic modification of the porcine genome using. Genome editing with engineered nucleases in plants yuriko osakabe. The first truly targetable reagents were the zinc finger nucleases zfns showing that arbitrary dna sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. Genome engineering of crops with designer nucleases shaun j. Zinc finger nucleases zfns are versatile tools for making precise modifications to genomes, and their use is now established in a range of model. Jul 29, 2016 a selection free, highly efficient targeted mutagenesis approach based on a novel zfn monomer arrangement for genome engineering in tomato reveals plant trait modifications. Rote 20 presentation targeted genome editing in the eukaryotic systems using zinc finger nucleasesjoe frangipane, sigmaaldrich. Us2055793a1 us11989,417 us98941706a us2010055793a1 us 2055793 a1 us2055793 a1 us 2055793a1 us 98941706 a us98941706 a us 98941706a us 2010055793 a1 us2010055793 a1 us 2010055793a1 authority us united states prior art keywords gene method zinc finger binding domain finger binding prior art date 20050725 legal status the legal status is an.
Customdesigned zinc finger nucleases zfns, proteins designed to. Targeted genome editing in human cells with zinc finger nucleases. Two studies show signs that the introduced dna is functioning, but its too early to know if patients actually benefit. Zinc finger nuclease an overview sciencedirect topics. Zfns, talens, and the crisprcas9 system on free shipping on qualified orders. Targeted genetic modification of the porcine genome using zincfinger nucleases thesis submitted in partial fulfilment of the requirements for the degree doctor of philosophy phd at the university of veterinary medicine hannover by janet hauschildquintern bremen hannover 2012. Discover more publications, questions and projects in zinc. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a zinc atom to stabilize this protein motif. Programmable nucleases including zfns, talens and rgens derived from the prokaryotic crisprcas system enable targeted highprecision genome engineering.
Only a few percent of the injected eggs giving rise totransgenic animals. Origins of programmable nucleases for genome engineering. Zinc finger nucleases zfns are artificial dna restriction enzymes. Zinc finger nucleases zfns are versatile tools for making precise modifications to genomes, and their use is now established in a range of model systems.
Zinc finger nuclease the new technology for targeted genome. The plant genome original research genome engineering of crops with designer nucleases view my binders. Several methods are being used to modify the human genome precisely and efficiently. We previously reported that zincfinger nuclease zfn proteins have the innate. Zinc finger nuclease zfn modify human genome precisely. Genome editing with engineered nucleases in plants plant.
In most nucleases, dna binding and nuclease functions are structurally integrated, making them difficult to. Zfn, talen and crisprcasbased methods for genome engineering. Zinc finger nuclease the new technology for targeted genome modification in animals. A guide to genome engineering with programmable nucleases. An unbiased genome wide analysis of zinc finger nuclease specificity. Selectionfree zincfingernuclease engineering by context. Studies within this dissertation seek to develop novel methods for delivering genome engineering reagents to whole plants. Zinc finger nuclease news, articles the scientist magazine.
Targeted genome editing using engineered zinc finger nucleases. These methods hold great promise to revolutionize the gene therapy arena. It is possible to target the dna sequences probing gene function. Until recently, the generation of animals with gene targeted manipulations has been accomplished by homologous recombination hr in embryonic stem es cells or cloning through. Target zinc finger nucleases in genome engineering. As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. In principle these technologies are capable of targeting virtually any site in. Zinc finger nuclease zfn technology has recently been introduced to the zebrafish system as a complementary reverse genetic strategy to induce targeted mutations. Genome editing with engineered nucleases in plants yuriko osakabe1 and keishi osakabe2, 1riken center for sustainable resource science, 311 koyadai, tsukuba, ibaraki, 3050074 japan 2center for collaboration among agriculture, industry and commerce, the university of tokushima, 31815 kuramotocho, tokushima, 7708503 japan. Selection free zincfingernuclease engineering by contextdependent assembly. Plant genome engineering with sequencespecific nucleases. The plant genome original research genome engineering of. The second category is the zinc finger nucleases zfns, which are made from a series of.
Highly specific genomic scissors more important was the discovery that the dnabinding properties of zinc fingers could be fused to proteins with dnacutting function. Zincfinger nucleases foki cleavage domain the familiar type ii restriction endonucleaseslongtime workhorses of molecular biologyrecognize a short dna sequence and cut within it. Genome engineering with zinc finger nuclease and talens with the advances in largescale genome sequencing, it is now possible to sequence whole genomes quite readily. Genomeengineering tools with improved design and efficiency will become widely used. Targeted genome engineering via zinc finger nucleases. The donor design service can be used in conjunction with functionally validated compozr zfns to perform precise genome modifications in a variety of organisms or cell lines. This site includes information about the zinc finger consortium, including. Zinc finger nucleases to be useful for genome engineering, an endonuclease must exhibit an extraordinary combination of qualities. The zincfinger domain, which typically recognizes 3nucleotide motifs on dna, was the first to be exploited in this way. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Genome engineering with programmable nucleases depends on cellular responses to a targeted doublestrand break dsb. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a. The spacer between the zincfinger binding sites, 6 bp in this case, is also indicated. Contextdependent assembly coda of zinc finger nucleases is reported.
Each zinc finger nuclease zfn consists of two functional domains. Sep 26, 2009 the generation of genetically modified animals or plants with genetargeted deletions or modifications is a powerful tool to analyze gene function, study disease and produce organisms of economical interest. T1 design, engineering, and characterization of zinc finger nucleases. This makes targeted gene correction or genome editing a viable option in human cells. Zinc finger nuclease the new technology for targeted.