Feb 27, 2016 genome engineering with programmable nucleases depends on cellular responses to a targeted doublestrand break dsb. The spacer between the zincfinger binding sites, 6 bp in this case, is also indicated. Target zinc finger nucleases in genome engineering. Download citation genome engineering with zincfinger nucleases.
Genome editing with engineered nucleases allows us to precisely change the target genome of living cells and is a powerful way to control functional genes. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. Genome engineering with compozr custom zinc finger nucleases. Genome editing the new frontier of genome engineering with. Our zinc finger nuclease zfn technology is based on a naturally occurring class of proteins called zinc finger dnabinding proteins zfps, which recognize and bind to specific sequences of dna. Targeted genome editing using sitespecific nucleases. Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Here, we describe methods for the targeted modification of plant genomes using transcription activatorlike effector nucleases talens1.
Pdf target zinc finger nucleases in genome engineering. Genome engineering with targetable nucleases dana carroll department of biochemistry, university of utah school of medicine, salt lake city, utah 84112. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. Zinc finger nucleases to be useful for genome engineering, an endonuclease must exhibit an extraordinary combination of qualities. As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. Genome editing with engineered nucleases in plants plant. This genomic sequence information is critical for genome engineering of plant and.
Zinc finger nucleases zfns are a class of engineered dnabinding proteins that facilitate targeted editing of the genome by creating doublestrand breaks in dna at userspecified locations. Zinc finger nuclease an overview sciencedirect topics. The new frontier of genome engineering with crisprcas9. 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. Customdesigned zinc finger nucleases zfns, proteins designed to cut at specific dna sequences. Dna replicons for plant genome engineering plant cell. This video outlines these four basic genome editing. 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. Twofinger modules are stitched together to form azinc finger protein, each with specificity. This new technology opens up revolutionary possibilities in agriculture, animal husbandry and molecular medicine. Selectionfree zincfinger nuclease engineering by context. Gene editing using zincfinger nucleases zfns, transcription activator like effectors tales and crisprcas systems has been used to create changes in the genome in order to make it nonfunctional. The versatile toolbox for genetic engineering of the mouse embryo has been recently extended by genome editing approaches based on designer nucleases such as zinc finger nucleases zfn 46, transcription activatorlike effector nucleases talen 7,8, and the clustered regularly interspaced short palindromic. Genome editing with modularly assembled zincfinger nucleases.
T he ability to precisely and efficiently modify the human genome could lead to the development of novel and safer ways of treating human genetic and complex diseases. Recent advances in genome engineering provide newfound control over a plants genetic material. Here, we use geminivirusbased replicons for transient expression of sequencespecific nucleases zincfinger nucleases, transcription. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Genome engineering with zinc finger nuclease and talens. The zinc finger proteins could have effector or nuclease domains attached, allowing for gene regulation or gene replacement.
Over the past two decades, research groups in both academia and private industry have developed key technologies, including viral delivery vectors and engineered transposonbased or zinc finger proteinbased nucleases, towards achieving the longsought goal of therapeutic genome editing in humans. Genome engineering with zincfinger nucleases genetics. The new frontier of genome engineering with crisprcas9 science. Gene targeting is a process in which a dna molecule introduced into a cell replaces. Optimizing delivery and expression of designer nucleases for.
Genome engineering of crops with designer nucleases shaun j. Origins of programmable nucleases for genome engineering. Here we describe contextdependent assembly coda, a platform for engineering zfns using only standard cloning techniques or custom dna synthesis. 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. Keywords zincfinger nucleases, transcription activatorlike effector nucleases, talens, meganucleases, nonhomologous end joining, homologous recombination, gene targeting. Compozr custom zfn compozr zinc finger nucleases service. Further, genome engineering will enable plants biosynthetic capacity to be harnessed to produce the many agricultural products required by an expanding world population. We also consider some of the limitations that nucleasemediated crop improvement technologies may encounter. The technology has experienced remarkable development in the last few years with regard to both the target site specificity and the engineering platforms used to. Mice are by far the most popular platform for generating transgenic animal models.
This combination of simplicity and flexibility has catapulted zinc finger nucleases zfns and transcription activatorlike effector nucleases talens to the forefront of genetic engineering. The second category is the zinc finger nucleases zfns, which are made from a series of. Zinc finger nuclease technology linkedin slideshare. Zfns are chimeric dna restriction enzymes that consist of a customdesigned zinc finger zf dnabinding domain and the nonspecific nuclease. Please view the pdf by using the full text pdf link under view to the left. This combination of simplicity and flexibility has catapulted zincfinger nucleases zfns and transcription activatorlike effector nucleases talens to the forefront of genetic engineering. From meganucleases, to zinc fingers, to talens, and now crisprcas, the use of engineered nucleases for genome editing has been gathering pace. Much like zfns, talens are chimeric proteins made by fusing an engineered dnabinding domain with the catalytic domain of foki endonuclease christian et al. Targeted genome engineering via zinc finger nucleases.
Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. Zincfinger nucleases zfns consist of dnabinding modules derived from natural transcription factors tfs that are linked to the nuclease domain of the type iis restriction enzyme, foki 18, 19. By taking advantage of endogenous dna repair machinery, these reagents can be. Two finger modules are stitched together to form azinc finger protein, each with specificity. To date, zinc finger nucleases zfns have been the most promising reagents for potential. In the past few years, transcription activatorlike effector nucleases have emerged as the reagent of choice for many genome engineering applications bogdanove and voytas, 2011. To recognize a specific sequence of dna, the zinc fingers could be attached to each other, with six zinc fingers recognizing a unique 18base pair sequence in an organisms genome. Aug 24, 2015 from mega nucleases, to zinc fingers, to talens, and now crisprcas, the use of engineered nucleases for genome editing has been gathering pace. The dnabinding domains of zinc finger zf proteins were the first to be used as genome editing tools, in the form of designed zf nucleases zfns. Zfn, talen, and crisprcasbased methods for genome engineering.
Zinc finger nucleases zfns are artificial restriction enzymes made by fusing reprogrammable zinc finger dnabinding units to the foki nuclease domain, which efficiently induce, sitespecific. The plant genome abstract original research genome. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. More recently, transcription activatorlike effector nucleases talens, as well as the clustered regularly interspaced short palindromic repeatscas9 crisprcas9 system, which utilizes rnadna. As the archetypal platform for programmable dna cleavage 1, zincfinger nucleases zfns have had a central role in the development and application of genome engineering technologies. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. Thus, in addition to basic advances in dna manipulation, three specific research threads led to the production of the first zincfinger nucleases zfns.
Studies within this dissertation seek to develop novel methods for delivering genome engineering reagents to whole plants. Using genome engineering to understand huntingtons disease. Genome engineering with programmable nucleases depends on cellular responses to a targeted doublestrand break dsb. The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Transcription activatorlike effector nucleases enable. 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. Dec 28, 2010 kim s, lee mj, kim h, kang m, kim js 2010b preassembled zinc finger arrays for rapid construction of zinc finger nucleases. Dec 11, 20 t he ability to precisely and efficiently modify the human genome could lead to the development of novel and safer ways of treating human genetic and complex diseases. In a correspondence in nature methods, some members of the zinc finger consortium reported discouragingly high failure rates. Genome editing with engineered zinc finger nucleases. Genome engineering of crops with designer nucleases. Optimizing delivery and expression of designer nucleases. Zfn, the firstgeneration genome engineering effector, was constructed by combining a zinc finger motif, which recognizes dna sequences, with the foki nuclease domain kim and kim, 2011 fig. Pdf targeted genome engineering via zinc finger nucleases.
Customdesigned zinc finger nucleases zfns, proteins designed to cut at specific dna sequences, are becoming powerful tools in gene targetingthe process of replacing a gene within a genome by homologous recombination hr. Jun 16, 20 each zinc finger nuclease zfn consists of twofunctional domains. It is feasible in almost all organisms ranging from bacteria to plants and animals, as well as in cultured cells such as es and ips cells. Genome editing the new frontier of genome engineering. However, difficulties of protein design, synthesis, and validation remained a barrier to the new frontier of. 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. Keywords zinc finger nucleases, transcription activatorlike effector nucleases, talens, meganucleases, nonhomologous end joining, homologous recombination, gene targeting. Oct 21, 2014 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. Here, we use geminivirusbased replicons for transient expression of sequencespecific nucleases zincfinger nucleases, transcription activatorlike.
Sep 15, 2017 to recognize a specific sequence of dna, the zinc fingers could be attached to each other, with six zinc fingers recognizing a unique 18base pair sequence in an organisms genome. Engineered zinc finger nucleases zfns enable targeted genome modification. Gene editing using zinc finger nucleases zfns, transcription activator like effectors tales and crisprcas systems has been used to create changes in the genome in order to make it nonfunctional. Zinc finger nuclease zfn technology has recently been introduced to the zebrafish system as a complementary reverse genetic strategy to induce targeted mutations. Each zinc finger nuclease zfn consists of two functional domains. Using coda zfns, we rapidly altered 20 genes in zebrafish, arabidopsis, and soybean. Zincfinger nucleases zfns are a powerful tool that can be used to edit the human genome ad libitum.
Compozr zinc finger nucleases zfns are a class of engineered dnabinding proteins that facilitate highly specific targeted editing of any genome by creating doublestranded breaks in dna at user specified locations. In the present study, we have looked into the possibility of using six. Unlimited genomic engineering possibilities in any species or cell line. Aug 01, 2011 zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Diversifying the structure of zinc finger nucleases for high. Plant genome engineering with sequencespecific nucleases. Because the nuclease domain must dimerize to cut dna, two zfn molecules are required to target a single site. Kim s, lee mj, kim h, kang m, kim js 2010b preassembled zinc finger arrays for rapid construction of zinc finger nucleases. The zinc finger proteins could have effector or nuclease domains.
Sequencespecific nucleases enable facile editing of higher eukaryotic genomic dna. Diversifying the structure of zinc finger nucleases for. Genome engineering with compozr custom zinc finger. 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. As the archetypal platform for programmable dna cleavage 1, zinc finger nucleases zfns have had a central role in the development and application of genome engineering technologies. The technology has experienced remarkable development in the last few years with regard to both the target site specificity and the engineering platforms used to generate zinc finger proteins. Genome engineering with targetable nucleases annual. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. By taking advantage of endogenous dna repair machinery zfns can be designed to precisely alter the genomes of higher organisms, allowing almost any region of the genome to be targeted fig. A dnabinding domain comprised of a chain of twofinger modules, each.
Zinc finger nucleases zfns are artificial restriction enzymes made by fusing reprogrammable zincfinger dnabinding units to the foki nuclease domain. Here, we summarize the role of three classes of nucleases. Methods were optimized using tobacco nicotiana tabacum protoplasts and talens1 targeting the acetolactate synthase. However, difficulties of protein design, synthesis, and validation remained a barrier to the new frontier of genome engineering with crisprcas9 genome editing. Zincfinger nucleases zfns and transcription activatorlike effector nucleases talens. Nat methods in press klug a 2010 the discovery of zinc fingers and their applications in gene regulation and genome manipulation. Targeted genome engineering via zinc finger nucleases springerlink. Genome engineering with zincfinger nucleases researchgate. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical. 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. A dnabinding domain comprised of a chain of two finger modules, each recognizing a unique hexamer 6 bp sequence of dna. Engineered zincfinger nucleases zfns can be used to introduce targeted alterations into genomes of model organisms, plants, and human cells.
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