The sequences of 3zincfinger arrays were then inserted into the sequences of zfn backbone, to design two pairs of zfns zfn1 and zfn2. Zinc finger nucleases and transcription activatorlike effector nucleases recognize the dna sequence based on specially designed protein motifs, which makes them more difficult to program and control. Zincfinger nucleases have proven to be successful as reagents for targeted genome manipulation in drosophila melanogaster and many. Each zinc finger nuclease zfn consists of twofunctional domains. In principle these technologies are capable of targeting virtually any site in the genome and editing the dna that is there. Starting from an archive of zinc finger modules known to function well together, effective multifinger arrays can be. By using engineered zinc finger nucleases zfns designed to target an integrated reporter and two endogenous rat genes, immunoglobulin m igm and rab38, we demonstrate that a single injection of dna or messenger rna encoding zfns into the one. It is one of three methods of changing the genome with engineered nucleases they have two parts. Twometaliondependent nucleases comprise the largest number of different tertiary folds and mediate the most diverse set of biological functions. Model experiments have demonstrated that a doublestrand break dsb. Diversifying the structure of zinc finger nucleases for high. Autologous tcells genetically modified at the ccr5 gene by.
A dnabinding domain comprised of a chain of twofinger modules, each. Engineered zinc finger nucleases zfns enable targeted genome modification. A common one is the cys2his2 finger, which utilizes two cysteine and two histidine residues to bind zinc. Aug 23, 2018 the binding specificity of the designed zinc finger domain points the zfn to a specific genomic site. Aug 01, 2011 zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Zinc finger motif 3d structural motif of the protein. Generally, zfns are made up of two modular domains, dna binding domain and dna cleavage domain that collectively mediate site directed mutagenesis porteus, 2008. A dnabinding domain comprised of a chain of two finger modules, each recognizing a unique hexamer 6 bp sequence of dna. Zinc finger nucleases are made with 36 zinc fingers. Custom zincfinger nucleases for use in human cells stephen alwin,1 maja b.
Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. One zinc finger can bind about 3 base pairs by itself. Genome engineering with zincfinger nucleases genetics. Zinc finger nucleases as tools to understand and treat human. Genome editing with compozr custom zinc finger nucleases. Zinc finger nuclease technology market study with market size, share, valuation, segmentwise analysis, competitive landscape analysis, regulatory framework analysis and impact of covid19 outbreak on zinc finger nuclease technology industry. Zinc finger engineering the zinc finger consortium. Zinc finger domains zinc finger is the general name given to a peptide, about 30 amino acids, which coordinates the metal zinc. Nuclear gene targeting in chlamydomonas using engineered.
By using engineered zincfinger nucleases zfns designed to target an integrated reporter and two endogenous rat genes, immunoglobulin m igm and rab38, we demonstrate that a single injection of dna or. Genome engineering with zinc finger nuclease and talens. Genome editing of human embryonic stem cells and induced pluripotent stem cells with zinc finger nucleases for cellular imaging. The fingers in the output from the same modular sources sgmo or barbas were selected to make the 3 zinc finger arrays of two pairs of zfns correspondingly table 1. A dsb is created in gene b, which is then processed to form free 3.
Zinc finger nuclease an overview sciencedirect topics. 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. Each zinc finger nuclease zfn consists of two functional domains. Nuclear gene targeting in chlamydomonas using engineered zinc. Zinc finger nucleases zfns are chimeric engineered nucleases designed to target userspecified sequences within the genome durai et al. Knockout rats via embryo microinjection of zincfinger. 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. Both local mutagenesis via nonhomologous end joining nhej and targeted gene replacement via homologous recombination hr have been achieved in up to 10% of all targets at a given locus. Autologous tcells genetically modified at the ccr5 gene by zinc finger nucleases sb728 for hiv zinc finger the safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Genome editing zinc finger nuclease part 1 lecture by vikas mangal vikas mangal. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would the extension of this genetic tool to a broader range of organisms.
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. Gene targetingthe process of gene replacement by homologous recombinationis a very useful but typically inefficient technique 11. Pdf selectionfree zincfingernuclease engineering by. Jun 16, 20 each zinc finger nuclease zfn consists of twofunctional domains. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. Zincfinger nucleases zfns are enzymes got by fusing a zinc finger to a dna cleavage enzyme called a. Zinc finger nucleases zfns show promise in improving the efficiency. Each zinc motif consist of the 30 amino acid and folds into. Comparing zinc finger nucleases and transcription activatorlike. Zinc finger nucleases zfns have been used to direct precise modifications of the genetic information in living cells at high efficiency. Nucleases definition of nucleases by medical dictionary. Zinc finger structure zinc finger is the name of many protein structures. Heritable targeted inactivation of myostatin gene in yellow.
Specific zfns targeting dicer like dcl genes and other. Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. The fingers in the output from the same modular sources sgmo or barbas were selected to make the 3zincfinger arrays of two pairs of zfns correspondingly table 1. 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. It seems timely to consider the adoption of terminology that more accurately reflects what is being done. Mar 01, 2015 zinc finger nucleases zfns are engineered restriction enzymes designed to target specific dna sequences within the genome. Here we describe contextdependent assembly coda, a platform for engineering zfns using only standard cloning techniques or custom dna synthesis. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. Thus, in principle, zincfinger domains can be designed to target a broad range of dna sequences. The zinc finger nuclease monopoly nature biotechnology. The binding specificity of the designed zinc finger domain directs the zfn to a specific genomic site.
Listing a study does not mean it has been evaluated by the u. Selectionfree zincfinger nuclease engineering by context. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate. Jul 24, 2009 the toolbox of rat genetics currently lacks the ability to introduce sitedirected, heritable mutations into the genome to create knockout animals. Zinc finger nucleases are chimeric proteins that consist of a specific dna. In addition, the impact on biomedical research of classical gene targeting. The zinc atom binds the proteins and makes them more stable.
Conversely, a single biological process can often be accomplished by multiple tertiary and quaternary folds and by more than one catalytic mechanism. 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. Contextdependent assembly coda of zinc finger nucleases is reported. Zinc finger nuclease zfn license agreement sigmaaldrich. Targeted mutagenesis using zincfinger nucleases in. Efficient gene targeting in drosophila with zincfinger. Lee, bruno huber, leszek lisowski, ping liang, mei huang, patricia e. We report very high gene targeting frequencies in drosophila by direct embryo injection of mrnas encoding specific zincfinger nucleases zfns. The toolbox of rat genetics currently lacks the ability to introduce sitedirected, heritable mutations into the genome to create knockout animals.
Zfns are engineered proteins comprised of a zinc finger dnabinding domain fused to the cleavage domain of the foki restriction endonuclease. Zinc finger nucleases zfns are synthetic restriction enzymes evolved by. A zfn designed to create a dna doublestrand break dsb in the target locus is composed of two. Despite the advantages of genome editing with zfn, there are several potential disadvantages. Diversifying the structure of zinc finger nucleases for. Twofinger modules are stitched together to form azinc finger protein, each with specificity. Zinc finger nuclease technology linkedin slideshare. Genome editing with engineered zinc finger nucleases. Zinc finger nucleases or zfn are a tool used to target genes and change dna. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. 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. Genome editing zinc finger nuclease part 1 lecture by. Dec 12, 2010 contextdependent assembly coda of zinc finger nucleases is reported. Genome editing of human embryonic stem cells and induced.
Originally coined to describe the finger like appearance of a hypothesized structure from xenopus laevis transcription factor iiia, the zinc finger name has now come to encompass a wide variety of differing protein structures. Each finger makes contact primarily with a separate dna triplet 2, 3. Zinc fingers are manmade molecules made of a protein and zinc. A suite of zfns were engineered by the recently described contextdependent assembly platforma rapid, opensource method for generating zincfinger arrays. Zincfinger nucleases zfns can be used to create targeted doublestranded breaks in the genome and have thus generated much excitement. Improved cellpenetrating zincfinger nuclease proteins. But all the buzz right now is about a new, comparatively untested but very promising alternative. Jan 19, 20 zinc finger nucleases are all grown up at this point, having already made it to a clinical trial. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a zinc atom to stabilize this protein motif. Induction and repair of zincfinger nucleasetargeted. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. In this video we discuss zinc finger nucleases, which are an important tool for cutting dna. Engineered zincfinger nucleases zfns can be used to introduce targeted alterations into genomes of model organisms, plants, and human cells.
Zincfinger nuclease dimers are more difficult to engineer than single. Efficient gene targeting in drosophila by direct embryo. Originally coined to describe the fingerlike appearance of a hypothesized structure from xenopus laevis transcription factor iiia, the zinc finger name has now come to encompass a wide variety of differing protein structures. Assembly of zinc finger dnabinding domain to a dnacleavage domain enables the enzyme machinery to target unique locus in the genome and invoke endogenous dna repair mechanisms. Starting from an archive of zinc finger modules known to function well. Knockout of myostatin by zinc finger nuclease in sheep fibroblasts and embryos there are four major classes of gene editing technologies, including meganuclease and their derivatives, zinc finger nucleases zfns, transcription activatorlike effector nucleases talens, and clustered regularly interspaced short palindromic crisprassociated. Knockout rats via embryo microinjection of zincfinger nucleases. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. When bound as a heterodimer, zfns create a doublestranded break at a user specified dna sequence. Selectionfree zincfingernuclease engineering by context. We designed a test system for zfns in chlamydomonas that is based on repair of an inactivated marker gene that was stably integrated into the genome. Targeted mutagenesis of duplicated genes in soybean with zinc.
This product and its use are the subject of one or more of the following patents controlled by sangamo biosciences, inc u. With the advent of zinc finger nucleases zfns to the world of genetic research, the theoretical ideal became a reality. Engineered zinc finger nucleases zfns can be used to introduce targeted alterations into genomes of model organisms, plants, and human cells. Autologous tcells genetically modified at the ccr5 gene by zinc finger nucleases sb728 for hiv zincfinger the safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Targeted mutagenesis of duplicated genes in soybean with. We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zincfinger nucleases zfns. Oct 19, 2014 in this video we discuss zinc finger nucleases, which are an important tool for cutting dna. The benefit of building a nuclease using zinc fingers is that this. 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. Autologous tcells genetically modified at the ccr5 gene. Specific zfns targeting dicer like dcl genes and other genes involved in rna silencing were. Natural and artificial zinc fingers have been characterized that bind to all. Heritable targeted inactivation of myostatin gene in. But that could change thanks to advances in genome editing approaches, including talen, crisprcas9, and the oldest, and most researched approach.
Thus, in principle, zinc finger domains can be designed to target a broad range of dna sequences. 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. The sequences of 3 zinc finger arrays were then inserted into the sequences of zfn backbone, to design two pairs of zfns zfn1 and zfn2. These problems have been solved by the introduction of a different genomicediting system, where the recognition of the target dna region is. Each zinc finger contacts primarily 3 bp of dna through interactions in the major groove, and fingers that recognize many of the 64 triplets have been isolated s egal et al. For nucleases not listed here, see the specific term. Apr 01, 2006 each zinc finger contacts primarily 3 bp of dna through interactions in the major groove, and fingers that recognize many of the 64 triplets have been isolated s egal et al. Figure 1zincfinger nuclease zfnmediated genome editing. Zinc finger nucleases are useful to manipulate the genomes of many plants and animals. Efficient gene targeting in drosophila with zincfinger nucleases. In tissue culture systems, a chromosomal integrated reporter inactivated gfp gene that harbors the zfn target site is commonly used to test designed. The world now has two major technologies for targeted editing of the genome. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical.
Zinc finger nuclease technology market study with market size, share, valuation, segmentwise analysis, competitive landscape analysis, regulatory framework analysis and impact of covid19 outbreak on. Zfns are chimeric proteins composed of dnabinding cys 2his 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme foki 1. Carroll d genome engineering with zincfinger nucleases. Two finger modules are stitched together to form azinc finger protein, each with specificity. The binding specificity of the designed zincfinger domain points the zfn to a specific genomic site. Pdf methods of modifying the human genome precisely and efficiently hold. This modification of the cellular dna is permanent and can fully block gene function. 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. 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.
Gene targeting using zinc finger nucleases nature biotechnology. Characterized by the coordination of one or more zn ions. Engineering a zinc finger nuclease zfn to introduce a doublestrand break at a specific single chromosomal locus and induce homologydirected repair with an exogenously added donor dna sequence. Zfn dimers induce targeted dna dsbs that stimulate dna damage response pathways. Your guide to investing in zincfinger zfn gene editing.
Enhancing gene targeting with designed zinc finger nucleases. Zinc ion is held in place by two cysteine and two histidine r group. Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins. Zinc finger simple english wikipedia, the free encyclopedia.
1217 763 1562 1466 112 34 309 180 1278 857 304 139 1462 941 391 1314 886 816 228 914 698 225 434 1010 530 895 449 1008 108 1109 1112 724 323 1412 140 1511 39 489 878 1369 1393 617 1255 647 851 285 1008 764