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. A dnabinding domain comprised of a chain of two finger modules, each recognizing a unique hexamer 6 bp sequence of dna. Your guide to investing in zincfinger zfn gene editing. The zfas are fused to a foki nuclease domain, and a 57 bp spacer separates the target.
Zinc finger nucleases zfns can actively introduce a doublestrand break dsb at target loci in eukaryotic genomes and this can improve the efficiency of gene knockout or knockin by several. Genome editing in plant cells by zinc finger nucleases uf plant. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical. An alternative technology used in genetic engineering is based on targeted nucleases. May 21, 2009 zinc finger nucleases zfns enzymes engineered to create dna double strand breaks at specific loci are potent stimulators of gene targeting,1, 2 including at engineered reporter genes in plants. A novel arrangement of zinc finger nuclease system for in. Each zinc finger nuclease zfn consists of twofunctional domains. Gene targeting is a process in which a dna molecule introduced into a cell replaces.
Zfns have been shown to be capable of efficiently modifying engineered loci both in human cells6 and in model plants7,8. Zincfinger nucleases are synthetic designer proteins combining a zincfinger domain, engineered to bind a particular dna sequence, with an enzyme that breaks both dna strands at. Presentations of the thesis these topics have been presented at national or international conferences as poster or oral presentations. Download citation genome engineering with zincfinger nucleases.
However, the ability to modify dna, particularly in plant cells, does not solely depend on the activity of the sequence specific nuclease. 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. 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. More recently, transcription activatorlike effector nucleases talens, as well as the clustered regularly interspaced short palindromic repeatscas9 crisprcas9 system, which utilizes rnadna. Genome editing with engineered nucleases in plants plant. Faster method of engineering zincfinger nucleases developed. The expression of zinc finger nucleases zfns can induce genomic doublestrand breaks. Zincfinger nucleases foki cleavage domain the familiar type ii restriction endonucleaseslongtime workhorses of molecular biologyrecognize a short dna sequence and cut within it.
Recently, a new technology based on the use of genetargeted zincfinger nucleases zfns was developed and used for the generation of organisms with genetargeted deletions andor modifications when combined with hr. 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. 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. The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. Zinc finger nuclease zfnmediated gene targeting is rapidly becoming a powerful tool for gene editing and directed mutagenesis of plant and mammalian genomes including the. Jun 16, 20 each zinc finger nuclease zfn consists of twofunctional domains. Twofinger modules are stitched together to form azinc finger protein, each with specificity. 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. Each zinc finger nuclease zfn consists of two functional domains. Part of thebioinformatics commons,computational biology commons, and thegenomics commons. Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins. Genome engineering of crops with designer nucleases shaun j.
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. Two landmark papers demonstrated the utility of zfns for making targeted modifications to the drosophila melanogaster and human homo sapiens sapiens genomes bibikova et al. All members have committed to making all protocols, software, dna sequences, and reagents they publish available to the academic scientific community. 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. 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.
Genome editing driven by zincfinger nucleases zfns yields high genemodification efficiencies 10% by introducing a recombinogenic. Genome engineering with targetable nucleases dana carroll. Zinc finger nucleases for precise editing of the cho cell genome. 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. Thus, in addition to basic advances in dna manipulation, three specific research threads led to the production of the first zincfinger nucleases zfns. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a. Zinc finger nucleases to be useful for genome engineering, an endonuclease must exhibit an extraordinary combination of qualities. The hybrid enzymes proved effective and were called zinc finger nucleases zfns. Zinc finger nucleases for precise editing of the cho cell. Zincfinger nuclease mediated genome modification in maize. Targeted genome engineering via zinc finger nucleases springerlink. 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.
To be useful for genome engineering, an endonuclease. A general method for improving the efficiency of gene targeting would be valuable in. The structurebased design of zinc finger nucleases can. Genome engineering of crops with designer nucleases. Rapid opensource engineering of customized zincfinger nucleases for highly efficient gene modification. Recent advances in genome engineering provide newfound control over a plants genetic material. 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 corresponding author. Dec 17, 2010 zinc finger nucleases are synthetic designer proteins combining a zinc finger domain, engineered to bind a particular dna sequence, with an enzyme that breaks both dna strands at the targeted site. Plant genome engineering with sequencespecific 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.
Safc has created a series of zinc finger nuclease zfn modified cho. Genome engineering with compozr custom zinc finger. Zinc finger nuclease the new technology for targeted. Zfns have been used to generate modified organisms such as plants, drosophila, zebra fish and rats with genetargeted mutations. Your guide to investing in zincfinger zfn gene editing if youre an investor whos heard of zincfinger proteins and gene editing but you arent sure what it all means, this can help answer. From meganucleases, to zinc fingers, to talens, and now crisprcas, the use of engineered nucleases for genome editing has been gathering pace. Zfns are obtained by fusing dnabinding domains of eukaryotic transcription factors with the cleavage domain of foki restriction. Origins of programmable nucleases for genome engineering. Carbery, diana ji, anne harrington, victoria brown, edward j. Design, engineering, and characterization of zinc finger. Indeed, zfns have been used to drive the repla cement of native dna. T1 design, engineering, and characterization of zinc finger nucleases. The expression of zinc finger nucleases zfns can induce genomic double strand breaks.
To address this issue, zinc finger nuclease zfnbased. Genome engineering with zinc finger nuclease and talens. Because the zinc finger domain9 can be engineered to recognize novel dna sequences 10,11, zfns have been widely exploited for rational genome engineering at endogenous loci in many eukaryotic. One of the more advanced selectionbased design strategies currently in use is referred to as the open oligomerized pool engineering design 18, 19. By taking advantage of endogenous dna repair machinery, these reagents can be. Genome engineering with compozr custom zinc finger nucleases. The plant genome original research genome engineering of. Zinc finger nuclease zfn modify human genome precisely.
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 dnabinding do mains of zinc finger zf proteins were the first to be used as genome editing tools, in the form of designed zf nucleases. A dnabinding domain comprised of a chain of twofinger modules, each. These recombinant proteins can be designed to recognize virtually any dna sequence in the human genome and to stimulate homologous recombination by creating a targeted doublestrand break dsb. 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. This new technology opens up revolutionary possibilities in agriculture, animal husbandry and molecular medicine. Two finger modules are stitched together to form azinc finger protein, each with specificity. Targeted genetic modification of the porcine genome using. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. Genome editing with engineered zinc finger nucleases nature.
Louis, missouri 63146 and maine medical center research institute, scarborough. A selectionfree, highly efficient targeted mutagenesis approach based on a novel zfn monomer arrangement for genome engineering in tomato reveals plant trait modifications. Enhancing gene targeting with designed zinc finger nucleases marinabibikova,kellybeumer,jonathank. Sep 26, 2009 recently, a new technology based on the use of genetargeted zinc finger nucleases zfns was developed and used for the generation of organisms with genetargeted deletions andor modifications when combined with hr. Zinc finger nucleases as tools to understand and treat human. High frequency modification of plant genes using engineered. These enzymes sequencespecific nucleases have proven to be powerful reagents for editing dna in highereukaryotic cells. Weinstein, lucy liaw and xiaoxia cui,1 sigma advanced genetic engineering labs, sigmaaldrich biotechnology, st. Trautman, danacarroll gene targetingthe process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 1. 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. 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. Introduction genome editing using engineered nucleases geen is an effective genetic engineering method that uses molecular scissors, or arti.
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 nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Zinc finger nuclease knockout mouse zfn assembly time. In this study, we applied an approach using gene repair with zinc finger nucleases zfns. Zfns were engineered that recognize sur loci using publicly available resources provided by the zinc finger consortium.
Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Zinc finger nuclease technology linkedin slideshare. Zinc finger nucleases zfns are versatile tools for making precise modifications to genomes, and their use is now established in a range of model. Zinc finger nucleases zfns are chimeric, engineered transcription factors fused to the nonspecific endonuclease domain of restriction enzymes such as foki. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wildtype wt p53 function have been investigated. Engineered zinc finger nucleasemediated homologous. Zinc finger nuclease an overview sciencedirect topics. Sangamo therapeutics will use zinc finger nucleases to introduce the gene for a missing clotting factor into the livers of men with hemophilia b. Zinc finger nucleases are useful to manipulate the genomes of many plants and animals. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a zinc atom to stabilize this protein motif.
B revia enhancing gene targeting with designed zinc finger. Genome engineering with programmable nucleases depends on cellular responses to a targeted doublestrand break dsb. Genome editing with engineered nucleases in plants oxford. Genome engineering with zincfinger nucleases researchgate. Genome editing with engineered zinc finger nucleases.
1249 194 274 1190 96 525 1472 656 10 284 1592 975 1331 1259 820 1289 1426 357 833 1415 783 1631 593 1526 285 174 1035 298 1524 680 1093 703 1358 600 140 1606 1650 891 1056 167 211 139 1103 1372 1055 1200 1077