For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Here, we demonstrate a rapid singlestep approach to targeted gene knockout in mammalian cells, using engineered zinc finger nucleases zfns. Carbery, diana ji, anne harrington, victoria brown, edward j. Apr 29, 2009 an efficient method for gene targeting in plants has been lacking until now, frustrating efforts to engineer crop plants. Weinstein, lucy liaw and xiaoxia cui,1 sigma advanced genetic engineering labs, sigmaaldrich biotechnology, st. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005.
Both studies provide insights into the precision of this tool. Enhancing gene targeting with designed zinc finger nucleases marinabibikova,kellybeumer,jonathank. To address this issue, zinc finger nuclease zfnbased. 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. It is one of three methods of changing the genome with engineered nucleases. We report here an efficient method for making targeted mutations in arabidopsis thaliana genes. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Zfnmediated genome editing takes place in the nucleus when a zfn pair targeting. Letters precise genome modification in the crop species zea mays using zincfinger nucleases vipula k. Genome editing with engineered zinc finger nucleases.
Zinc fingers arent yet being used in any approved therapies, but choo notes that the opportunities are there for it to happen soon. Targeted genome editing using engineered zinc finger. In principle these technologies are capable of targeting virtually any site in the genome and editing the dna that is there. How to achieve precise gene targeting in plants and especially in crops remains a longsought goal for elucidating gene function and advancing molecular breeding. The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. Female and male galnegative piglets were produced by using zfnmediated gene targeting and scnt. Zincfinger nucleases allow targeted genetic modification at loci chosen by the investigator, but the extent of their offtarget activitywhich.
This evidence, along with ongoing improvements in the design and engineering of lentiviral 33,34 and parvovirus 35 vectors. We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zincfinger nucleases zfns. Zincfinger nucleases allow targeted genetic modification at loci chosen by the investigator, but the extent of their offtarget activitywhich could be toxic to cellshas not been evaluated. Targeted gene knockout in mammalian cells by using. Analysis of illegitimate genomic integration mediated by zinc. Zinc finger nucleases zfns show promise in improving the efficiency of gene targeting by introducing dna doublestrand breaks in target genes, which then stimulate the cells endogenous. In this study, we applied an approach using gene repair with zinc finger nucleases zfns.
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 genetic modification of the porcine genome using zincfinger nucleases. Previous generations of geneediting tools are still very much in play particularly zinc finger nucleases, talen, and meganucleases. Targeted gene knockout in mammalian cells by using engineered. The approach uses zincfinger nucleases zfns enzymes engineered to create dna doublestrand breaks at specific target loci. The world now has two major technologies for targeted editing of the genome. We have been developing chimeric zinc finger nucleases zfns as potential stimulators of gene targeting 7, 8. This method was much more efficient than conventional gene targeting. A selectionfree, highly efficient targeted mutagenesis approach based on a novel zfn monomer arrangement for genome engineering in tomato reveals plant trait modifications.
Zinc finger nuclease zfn technology has recently been introduced to the zebrafish system as a complementary reverse genetic strategy to induce targeted mutations. The top panel schematic illustrates the native hpitx3 locus before targeting. The offtarget effects of a genomeediting technology called zincfinger nucleases zfns are reported online this week in nature biotechnology and nature methods. Targeted mutagenesis of duplicated genes in soybean with.
Zinc finger nuclease zfn has become a new tool for gene knockout in other. Gene targeting in nod mouse embryos using zincfinger. An unbiased genomewide analysis of zincfinger nuclease. We have previously shown that the zifzfn stimulates gene targeting as a heterodimer when the heterodimer target binding sites are oriented as a zfn full site. 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. A suite of zfns were engineered by the recently described contextdependent assembly platforma rapid, opensource method for generating zincfinger arrays. Zinc finger nuclease the new technology for targeted genome modification in animals. Enhancing gene targeting with designed zinc finger nucleases. In most nucleases, dna binding and nuclease functions are structurally integrated, making them difficult to. To further explore novel methods of genetic manipulation in this mouse model, we tested the feasibility of using zinc finger nucleases zfns to knock out a gene directly in a pure nod background. Recent work has shown that it is possible to target regulatory elements to dna sequences of an investigators choosing, increasing the armamentarium for probing gene function.
Furthermore, the ideal target will be unique in the genome, with few closely related sequenc es that could potentially draw offtarget binding targets and off. Targeted genetic modification of the porcine genome using zinc finger 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. This video provides a brief description of how zincfinger nucleases function and can be utilized to alter a dna of interest. Compared with endogenous homologous recombination, the authors. Zfns can be used to advance both basic research and clinical applications, including gene therapy.
Precise genome modification in the crop species zea mays. An efficient method for gene targeting in plants has been lacking until now, frustrating efforts to engineer crop plants. A dnabinding domain comprised of a chain of twofinger modules, each recognizing a unique hexamer 6 bp sequence of dna. Highfrequency modification of plant genes using engineered. Engineered zinc finger nucleasemediated homologous. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wildtype wt p53 function have been investigated.
Gene targeting is a process in which a dna molecule introduced into a cell replaces. The target site and zfps were selected and designed with zinc finger tools, while the. The first generation zfp design entailed the use of a modular assembly, in which individual zinc fingers were optimized against target triplet. Research article open access zinc finger nucleases for. Zincfinger nucleases zfns are enzymes got by fusing a zinc finger to a dna cleavage enzyme called a fokl. Oct 19, 2014 in this video we discuss zinc finger nucleases, which are an important tool for cutting dna. In principle these technologies are capable of targeting virtually any site in. In the case of genome editing using donor constructs, drug selection is often also not required, and the mutations created can be as small as a singlebasepair change a process referred to as gene correction. D in recent years investigators have successfully developed superefficient systems using alternative technologies to generate genetically engineered mice much faster and more economically compared to traditional targeted mutation methods. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain.
Please use one of the following formats to cite this article in your essay, paper or report. Gene editing in human stem cells using zinc finger nucleases and integrasedefective lentiviral vector delivery. Targeted mutagenesis in arabidopsis using zincfinger. Efficient gene targeting by tal effector nucleases. Recently, the ability to engineer zfns that target any desired genomic dna sequence with high fidelity has improved. Zfns consist of a nonspecific foki endonuclease domain and a specific zincfinger dna. Recently, the ability to engineer zfns that target any desired genomic dna sequence with high fidelity. For example, sangamo is currently using zinc finger nucleases. Jul 29, 2016 a selectionfree, highly efficient targeted mutagenesis approach based on a novel zfn monomer arrangement for genome engineering in tomato reveals plant trait modifications. Testing zinc finger nucleases using the gfp gene targeting system. B revia enhancing gene targeting with designed zinc finger. Design, construction, and analysis of specific zinc finger nucleases.
Novel zinc finger nucleases zfns were designed to target the human rhodopsin gene. Zinc finger nuclease the new technology for targeted. Zinc finger engineering the zinc finger consortium. A comprehensive resource for locating zinc finger nuclease target sites in model organisms deepak reyon1,6, jessica r kirkpatrick1, jeffry d sander2,3, feng zhang4,5, daniel f voytas4, j keith joung2,3, drena dobbs1,6, clark r coffman1 abstract background. Dnaworks software was used instead of manual design to obtain the. Only a few percent of the injected eggs giving rise totransgenic animals. Zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as gene targeting tools. Existing methods for gene disruption are limited by their efficiency, time to completion, andor the potential for confounding offtarget effects. A suite of zfns were engineered by the recently described contextdependent assembly platforma rapid, opensource method for generating zinc finger arrays. Genetic correction using engineered nucleases for gene. 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. A general method for improving the efficiency of gene targeting would be valuable in.
Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. Here it is demonstrated that zincfinger nucleasesenzymes engineered to. Zinc finger nucleases zfns have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. May 10, 2010 formation of site specific genomic double strand breaks dsbs, induced by the expression of a pair of engineered zinc finger nucleases zfns, dramatically increases the rates of homologous recombination hr between a specific genomic target and a donor plasmid. Pdf donor dna utilization during gene targeting with. Genome editing using zincfinger nucleases zfns and. Gene targeting using zinc finger nucleases amazon s3. The zfns have a dna recognition domain composed of three cys 2 his 2 zinc fingers linked to a nonspecific dna cleavage domain. Targeted genetic modification of the porcine genome using zinc finger nucleases. In situations in which zinc finger nucleases are used to drive targeted integration, selection is. Targeted genetic modification of the porcine genome using. Pros and cons of znfs, talens, and crisprcas jim yeadon, ph.
Pdf donor dna utilization during gene targeting with zinc. Aug 23, 2018 please use one of the following formats to cite this article in your essay, paper or report. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as gene targeting tools. Targeting genes with zinc fingers december 20, 2010 issue. Zinc finger nucleases or zfn are a tool used to target genes and change dna.
Microinjection and homologous recombination inembryonic stem es cells, are robust but overallinefficient. Targeted mutagenesis of duplicated genes in soybean with zinc. Abstract zincfinger nucleases zfns have been successfully used for rational genome engineering in a variety of cell types and organisms. Zincfinger nucleases zfns are versatile reagents that have redefined genome engineering. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical. Twofinger modules are stitched together to form a zinc finger protein, each with specificity of. Zinc finger nucleases zfns have been successfully used for rational genome engineering in a variety of cell types and organisms. Letters precise genome modification in the crop species zea mays using zinc finger nucleases vipula k. Highly specific genomic scissors more important was the discovery that the dnabinding properties of zinc fingers could be fused to proteins with dnacutting function. It is one of three methods of changing the genome with engineered nucleases they have two parts. Zinc finger nucleases zfns have tremendous potential as tools to facilitate. Each zinc finger nuclease zfn consists of two functional domains. Mammalian gene targeting with designed zinc finger nucleases. Targeting genes with zinc fingers december 20, 2010.
The bottom panel schematic depicts the hpitx3 locus following correct insertion of the egfp transgene. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. A schematic of the southern blot strategy employed to validate targeting. Zinc fingers are manmade molecules made of a protein and zinc. Here, we demonstrate a rapid singlestep approach to targeted gene knockout in mammalian cells, using engineered zincfinger nucleases zfns. Validation of egfp targeting to the hpitx3 locus using zinc finger nucleases in hescs. We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zinc finger nucleases zfns. Zinc finger nuclease an overview sciencedirect topics. All members have committed to making all protocols, software, dna sequences, and reagents they publish available to the academic scientific community. However, for the safe use of zfn induced hr in practical applications, possible adverse effects of the technology. However, for the safe use of zfn induced hr in practical applications, possible adverse effects of the technology such as.
Zfns consist of a nonspecific foki endonuclease domain and a specific zinc finger dnabinding domain. Abstract zinc finger nucleases zfns have been successfully used for rational genome engineering in a variety of cell types and organisms. For instance, four zinc finger units can be assembled into a zinc finger chain. Highly efficient gene targeting of expressed and silent genes in human escs and ipscs using zinc finger nucleases.
Zinc finger nuclease the new technology for targeted genome. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement. Formation of site specific genomic double strand breaks dsbs, induced by the expression of a pair of engineered zincfinger nucleases zfns, dramatically increases the rates of homologous recombination hr between a specific genomic target and a donor plasmid. Zinc finger nucleases as tools to understand and treat human. In this video we discuss zinc finger nucleases, which are an important tool for cutting dna. Zfnmediated gene targeting thus confers molecular biologists with the ability to sitespecifically and permanently alter not only plant and mammalian genomes.
Donor dna utilization during gene targeting with zincfinger nucleases. Louis, missouri 63146 and maine medical center research institute, scarborough. Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. The ability to modify a complex genome with precision transformed biology in the late 1980s and early 1990s. Pdf gene targeting is the term commonly applied to experimental gene replacement by homologous recombination hr. Trautman, danacarroll gene targetingthe process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 1. For instance, four zinc finger units can be assembled into a zinc finger chain that recognizes a 12 base pair dna sequence.
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