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. Engineered zincfinger nucleases zfns are chimeras of a dnaspecific binding domain cys 2 his 2 zincfinger protein and a cutting domain endonuclease fok i 5. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Zinc finger nuclease an overview sciencedirect topics. Zinc finger nucleases zfns have been successfully used for rational genome engineering in a variety of cell types and organisms. The mrna or dna encoding a zincfinger nuclease zfn, a transcription activatorlike effector nuclease talen, or a clustered regularly interspaced short palindromic repeatcrisprassociated crisprcas system, and a repair template could potentially be introduced into cells using nonviral vectors to carry out gene editing. The most useful of these was a set of cys2his2 zinc fingers zfs in which each. In addition, the impact on biomedical research of classical gene targeting. Donald kohn, director of the human gene medicine program.
Only a few percent of the injected eggs giving rise totransgenic animals. Gene knockout technologies involving programmable nucleases have been used to create knockouts in several applications. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. The most common applications for engineered zinc finger arrays include zinc finger transcription factors and zinc finger nucleases, but other applications have also been described. Zinc finger arrays created using zfc reagents for either. Dnabinding zinc finger proteins zfps and a nuclease domain, such as the dna cleavage domain of a type. Genome engineering with zincfinger nucleases genetics. Pdf methods of modifying the human genome precisely and efficiently hold great promise for revolutionizing the gene therapy arena.
Targeted mutagenesis of betalactoglobulin gene in caprine. 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. Zincfinger nucleases are fusions of the nonspecific dna cleavage domain from the foki restriction endonuclease with zincfinger proteins. Transcription activatorlike effector nuclease wikipedia. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. Improved cellpenetrating zincfinger nuclease proteins. Targeted genome editing using engineered zinc finger nucleases. Zinc finger nuclease1 technique zfn1 zinc finger nucleases are protein chimeras composed of two covalently linked, functional domains. The binding specificity of the designed zincfinger domain directs the zfn. Zfn dimers induce targeted dna doublestrand breaks dsbs that stimulate dna damage response pathways. Highly specific genomic scissors more important was the discovery that the dnabinding properties of zinc fingers could be fused to proteins with dnacutting function.
Pdf high frequency modification of plant genes using. It seems timely to consider the adoption of terminology that more accurately reflects what is being done. Zinc finger nuclease technology zfn targeted genome. Targeted genome modification in mice using zincfinger nucleases. The zinc finger design can be used to construct dnabinding proteins for specific intervention in gene expression. Microinjection and homologous recombination inembryonic stem es cells, are robust but overallinefficient. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. High frequency modification of plant genes using engineered zinc finger nucleases article pdf available in nature 4597245. A potential answer comes from zinc finger nucleases zfns which have been demonstrated to efficiently block the activity of a gene by cleaving the human genome at a predetermined site and altering the genetic sequence via an errorprone dna repair process. By fusing selected zinc finger peptides to repression or activation.
Gene editing using zinc finger nucleases zfns, transcription. Zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Zinc finger simple english wikipedia, the free encyclopedia. Zfns are engineered nucleases that target and cut specific cellular dna sequences. 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. Recently, a new technology based on the use of gene targeted zincfinger nucleases zfns was developed and used for the generation of. Zinc finger nuclease 1 technique zfn1 zinc finger nucleases are protein chimeras composed of two covalently linked, functional domains. Zinc finger engineering the zinc finger consortium. Improved cellpenetrating zinc finger nuclease proteins for precision genome engineering jia liu1,2,3,4 thomas, gaj 1,2,3, mark c wallen and carlos f barbas iii1,2,3,5. In this video we discuss zinc finger nucleases, which are an important tool for cutting dna.
One of the most promising methods to achieve this is the use of zinc finger nucleases zfns. Targeted mutagenesis by zinc finger nucleases zfns can be used to generate knockout mammalian cell lines with high efficiency. Your guide to investing in zinc finger zfn gene editing if youre an investor whos heard of zinc finger proteins and gene editing but you arent sure what it all means, this can help answer. These models are especially valuable with the emerging need for validation of genes found in genomewide association studies for complex diseases. In addition, zinc fingers have become extremely useful in various therapeutic and research capacities. Zinc finger proteins znfs are one of the most abundant groups of proteins and have a wide range of molecular functions.
The most useful of these was a set of cys 2 his 2 zinc fingers zfs in which each unit of. Genome engineering with zinc finger nuclease and talens. When you go to the pdb to explore zinc fingers, try not to be overwhelmed. The manipulation of the rat genome using engineered zinc finger nucleases now introduces a key technology for manipulating the rat genome, which is broadly applicable. Zincfinger nucleases zfns can be used to create targeted doublestranded breaks in the genome and have thus generated much excitement. Zincfinger nucleases make the cut in hiv to control hiv.
Figure 1zincfinger nuclease zfnmediated genome editing. In principle these technologies are capable of targeting virtually any site in the genome and editing the dna that is there. Zfninduced doublestrand breaks are subject to cellular dna. Members of the zinc finger consortium continue to work to develop robust, publicly available methods for engineering zinc finger nucleases that function well in various cellular environments. The zinc atom binds the proteins and makes them more stable. Genome editing zinc finger nuclease part 1 lecture by. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical. 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. Zinc finger nuclease zfn technology has recently been introduced to the zebrafish system as a complementary reverse genetic strategy to induce targeted mutations.
One zinc finger can bind about 3 base pairs by itself. Zinc finger nucleases zfns are synthetic restriction enzymes evolved by fusing zinc. Pdf diversifying the structure of zinc finger nucleases. Two finger modules are stitched together to form a zinc finger protein, each with specificity of. This page lists plasmid vectors designed to express nucleases in different cell types. Unlimited genomic engineering possibilities in any species or cell line. A dnacleaving domain comprised of the nuclease domain of fok i. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases. The potential of gene targeting with zinc finger nucleases. Knockoutratsviaembryomicroinjection of zincfinger nucleases. Genome editing in plant cells by zinc finger nucleases uf plant. 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 nuclear. Zinc finger nucelases genome editing nucleases ii zfn vs talens duration. Zinc finger nuclease zfn license agreement sigmaaldrich.
Engineering zinc fingers to have an affinity for a specific sequence is an area of active research, and zinc finger nucleases and zinc finger transcription factors are two of the most important applications of this to be realized to date. For instance, four zinc finger units can be assembled into a zinc finger chain that recognizes a 12 base pair dna sequence. The discovery of zinc fingers and their development for. Design, assembly, in vitro and in vivo validation of zinc finger nucleases the zfns were designed, assembled, and validated using strategies and procedures described elsewhere s1, s2. The structure shown here on the left, from pdb entry 1znf, shows a classic zinc finger.
Zinc finger nucleases zfns, transcription activatorlike effector nucleases talens, and clustered regularly interspaced short palindromic repeats crisprs are three commonly used biotechnologies in genome editing of mammalian animals, fish, and other species. The restriction enzymes can be introduced into cells, for use in gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement. Fusing the zinc finger module to the foki nuclease domain the zinc finger module normally the module is tripartite, that is, consisting of three zinc fingers is fused via its carboxylterminus to the foki nuclease domain.
Zincfinger nuclease dimers are more difficult to engineer than single dnabinding domains, but the development of new methods could help. Zinc finger nucleases zfns and transcription activatorlike effector nucleases talens comprise a powerful class of tools that are redefining the boundaries of biological research. Zfn dimers induce targeted dna doublestrand breaks. Crisprs clustered regularly interspaced short palindromic repeats, in particular, have been gaining popularity in recent years and are getting ready to make the leap to the clinic. In the present study, we have looked into the possibility of using six. Typical engineered zinc finger arrays have between 3 and 6 individual zinc finger motifs. Diversifying the structure of zinc finger nucleases for high. All members have committed to making all protocols, software, dna sequences, and reagents they publish available to the academic scientific community. Highly efficient modification of betalactoglobulin blg. 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 userchosen dna target.
The discovery of zinc fingers and their applications in gene. The finger is a selfcontained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. General position statement of the zkbs on the use of zinc. Doublestrand breaks are important for sitespecific mutagenesis in that they stimulate the cells natural dnarepair processes, namely homologous recombination and nonhomologous end joining nhej. Oct 19, 2014 in this video we discuss zinc finger nucleases, which are an important tool for cutting dna.
Zincfinger nuclease dimers are more difficult to engineer than single dna binding domains, but the development of new methods could help. 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. Genome editing with engineered zinc finger nucleases. The foki restriction enzyme is a dimer, with each of the two nuclease. These genemodified cells proliferated normally and were. A zfn designed to create a dna doublestrand break dsb in the target locus is composed of two.
Simultaneous zincfinger nuclease editing of the hiv. Zinc finger nucleases as tools to understand and treat human. Zincfinger nucleases zfns are synthetic enzymes that can be designed to generate targeted doublestranded breaks in dna, giving the scientific community a way to effectively stimulate genomic changes. Sangamos zinc finger nucleases do not, however, have the field of gene editing to themselves. This modification of the cellular dna is permanent and can fully block gene function. 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. Zinc finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a doublestrand break induced by the resulting zinc finger nuclease can create. Given the wide variety of zinc finger domains, znfs are able to. Zfn design made use of an archive of prevalidated 2 finger and 1 finger modules s1, s35. Your guide to investing in zincfinger zfn gene editing. A lightactivated zfn will competence the conditional generation of gene. 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. Zinc finger nucleases are made with 36 zinc fingers.
Knockout rats via embryo microinjection of zincfinger nucleases article pdf available in science 3255939. One of the first breakthrough methods of gene targeting was the usage of chimeric proteins called zinc fingers nucleases zfn to create doublestrand breaks. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. Gene targeting by homologous recombination in mouse. Nov 30, 2017 i would say that zinc finger nucleases, talen, and meganucleases all work well and can be as active and specific as crispr, said dr. Highly efficient endogenous human gene correction using. Zfninduced doublestrand breaks are subject to cellular dna repair processes that. The crystal structure of a set of three fingers bound to dna showed that each finger contacts primarily 3 bp of dna in remarkably modular fashion pavletich and pabo 1991. Gregory says they should be ready to file an investigational new drug ind application for this programme by the middle of this year. Apr 29, 2009 the scope for improvement of yield and disease resistance of crop plants by genetic engineering has been limited by the lack of an efficient method for targeted gene modification.
The binding specificity of the designed zincfinger domain directs the zfn to a specific genomic site. Generation of esr1knockout rats using zinc finger nuclease. An account is given of the discovery of the classical cyshis zinc finger, arising from the interpretation of biochemical studies on the interaction of the protein transcription factor iiia with 5s rna, and of structural studies on its structure and its interaction with dna. Zfns consist of a nonspecific foki endonuclease domain and a specific zinc finger dnabinding domain. However, recent advances in generating customized zinc finger nucleases zfns that can create a dna doublestrand break dsb at preselected sites in the human genome have paved the way for hr. Zincfinger proteins in health and disease cell death.
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. 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. Targeted genome editing using sitespecific nucleases. Dual zincfinger nucleases block hiv infection blood. Diversifying the structure of zinc finger nucleases for highprecision genome editing article pdf available in nature communications 101 december 2019 with 95 reads how we measure reads.
Singlestranded oligonucleotides are used as donor templates for zincfinger nucleases to create targeted genomic deletions and mutations in mammalian cell lines. Zinc finger structure zinc finger is the name of many protein structures. These genemodified cells proliferated normally and. Tools that are mentioned include zinc finger nucleases zfns, transcription activatorlike effector nucleases talens, and crisprcas9, all of which have received much attention in recent years as.
Zfn design made use of an archive of prevalidated 2 finger and 1 finger. Zfns are engineered zinc finger proteins containing nucleotide sequencespecific dna binding with the endonuclease activity of the restriction enzyme foki. A number of different methods have been developed for the design and assembly of genespecific zfns, making them easily accessible to researchers. Zfn dimers induce targeted dna dsbs that stimulate dna damage response pathways. Precise genome modification in the crop species zea mays. These chimeric nucleases are composed of programmable, sequencespecific. To date, zinc finger nucleases zfns have been the most promising reagents for potential.
Knockout rats via embryo microinjection of zincfinger. A binding sites underlined of the zincfinger nucleases produced. Diversifying the structure of zinc finger nucleases for. Zinc fingers comprise a class of phylogenetically highly conserved protein structures which are especially important for the regulation of dna transcription. Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. The mrna or dna encoding a zincfinger nuclease zfn, a transcription activatorlike effector nuclease talen, or a clustered regularly interspaced short palindromic repeatcrisprassociated. When the dnabinding and dnacleaving domains are fused together, a highlyspecific pair of genomic scissors are created. Zinc finger arrays created using zfc reagents for either open or modular assembly can be easily transferred inframe to any of these vectors using unique cloning sites.
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