Diversifying the structure of zinc finger nucleases for highprecision. Custom zinc finger nucleases for use in human cells stephen alwin,1 maja b. Donald kohn, director of the human gene medicine program. Our knowledge of the oglycoproteome nacetylgalactosamine galnac type is highly limited. Zinc finger nuclease zfn technology involves transfection of highly specific genetargeting reagents linked to dna cleavage enzymes, allowing exquisite specificity and total gene knockout ko in a. A dnabinding domain comprised of a chain of two finger. 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. The performance of our linkers in the nccn architecture suggested that we could combine two zfns containing an aminoterminal foki. High frequency modification of plant genes using engineered zinc finger nucleases article pdf available in nature 4597245. Pdf methods of modifying the human genome precisely and efficiently hold great. Aavmediated delivery of zinc finger nucleases targeting. Induction and repair of zinc finger nuclease targeted doublestrand breaks in caenorhabditis elegans somatic cells jason morton, m. By using an engineered chimeric protein containing a fusion of a foki nuclease with sitespecific zinc finger motifs, known collectively as zinc finger nucleases zfns, dna doublestrand breaks can be introduced at predetermined genomic loci. Zfninduced doublestrand breaks are subject to cellular dna repair.
Dec 11, 2009 this video provides a brief description of how zinc finger nucleases function and can be utilized to alter a dna of interest. A bottleneck in the application of zfn technology has been the generation of highly specific engineered zinc finger. Gene editing strategies, such as zinc finger nucleases zfns, transcription activatorlike effector nucleases talens, and clustered regularly interspaced short palindromic repeatcas9. One of the first breakthrough methods of gene targeting was the usage of chimeric proteins called zinc fingers nucleases zfn to create doublestrand breaks. Zinc finger engineering the zinc finger consortium. Dnabinding zinc finger proteins zfps and a nuclease.
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. Zfn dimers induce targeted dna dsbs that stimulate dna damage response pathways. 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. Knockout rats via embryo microinjection of zincfinger. Zfninduced doublestrand breaks are subject to cellular dna repair processes that. Zinc finger nucleases zfns drive efficient genome editing by introducing a doublestrand break into the targeted gene. The structure shown here on the left, from pdb entry 1znf, shows a classic zinc finger. Zinc finger nucleasebased doublestrand breaks attenuate. Use of zinc finger nuclease technology to knock out efflux. Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. Medical school, campus benjamin franklin, hindenburgdamm 27, 12203 berlin, germany 2skaggs institute for chemical biology and department of molecular biology, the. When you go to the pdb to explore zinc fingers, try not to be overwhelmed. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes.
Probing isoformspecific functions of polypeptide galnac. Custommade zinc finger nucleases zfns can induce targeted genome modifications with high efficiency in cell types including drosophila, c. Zinc finger nucleases zfns are synthetic restriction enzymes evolved by fusing. A dnabinding domain comprised of a chain of two finger modules, each recognizing a unique hexamer 6 bp sequence of dna. Here it is demonstrated that zincfinger nucleasesenzymes engineered to. Targeted gene knockout by direct delivery of zincfinger. Identity of zinc finger nucleases with specificity to herpes. An alternative repair pathway for dsbs, nonhomologous end joining, often joins. Pdf precise genome modification in the crop species zea. Zinc finger nuclease an overview sciencedirect topics.
Engineered zinc finger nucleases zfns enable targeted genome modification. The zinc atom binds the proteins and makes them more stable. Apr 28, 2014 zinc finger nucleases are reagents that induce dna doublestrand breaks at specific sites that can be repaired by nonhomologous end joining, inducing alterations in the genome. Gene targeting by homologous recombination in mouse. Unlimited genomic engineering possibilities in any species or cell line. The most useful of these was a set of cys 2 his 2 zinc fingers zfs in which each unit of. This modification of the cellular dna is permanent and can fully block gene function. Genome engineering with zincfinger nucleases dana carroll1 department of biochemistry, university of utah school of medicine, salt lake city, utah 841125650 abstract zinc. Zinc finger nucleases as tools to understand and treat human. We have been developing chimeric zinc finger nucleases zfns as potential stimulators of gene targeting 7, 8. Aug 23, 2018 one of the first breakthrough methods of gene targeting was the usage of chimeric proteins called zinc fingers nucleases zfn to create doublestrand breaks. Gt methods, an emerging technology involving engineered restriction enzymes known as zincfinger nucleases zfns has. The binding specificity of the designed zinc finger. Zinc finger nuclease zfn technology has recently been introduced to the zebrafish system as a complementary reverse genetic strategy to induce targeted mutations.
We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zincfinger nucleases zfns. Dnaworks software was used instead of manual design to obtain the. Here we describe contextdependent assembly coda, a platform for engineering zfns using only standard cloning. In principle these technologies are capable of targeting virtually any site in the genome and editing the dna that is there. Gene editing in human stem cells using zinc finger nucleases and integrasedefective lentiviral vector delivery. 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. Enhancing zincfingernuclease activity with improved. Gene disruption using zinc finger nuclease technology.
The nucleases used in our platform provide high specificity and optimized digestion conditions possible. Genome editing in plant cells by zinc finger nucleases. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. 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. Here, we have studied the dna cleavage by one such zfn. One zinc finger can bind about 3 base pairs by itself. The binding specificity of the designed zincfinger domain directs the zfn to a specific genomic site.
Induction and repair of zincfinger nucleasetargeted. Genome engineering with zincfinger nucleases genetics. After dimerization the nuclease is activated and cuts the dna in the spacer sequence. Zincfinger nuclease enhanced gene targeting in human. Pdf high frequency modification of plant genes using.
Zinc finger nucleases for precise editing of the cho cell. Your guide to investing in zincfinger zfn gene editing. Qnkfn, in order to gain insight into how zfns cleave dna and how two inverted sites promote doublestrand cleavage. A toolbox and procedural notes for characterizing novel. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. Genome editing with engineered zinc finger nucleases. Realizing the full potential of this technology requires the development of safe and effective methods. The oglycoproteome is differentially regulated in cells by dynamic expression of a subset of. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a zinc. In addition to this flexibility, gene therapy by zinc finger nucleases may enable.
The potential of gene targeting with zinc finger nucleases. Here we report that gene targeting is achieved in 1. 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. In this approach, human c2bbe1 cells a subclone of caco. 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.
Zincfinger nuclease dimers are more difficult to engineer than single. Zinc finger nuclease dimers are more difficult to engineer than single dnabinding domains, but the development of new methods could help. Weitzman,4 and toni cathomen1, 1institute of virology, charite. Zinc finger nucleases are made with 36 zinc fingers. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. The consortium is committed to developing resources, software, and other tools for engineering zinc fingers and for performing genome engineering that are robust, userfriendly, and publicly available. Based on our advanced techniques and rich experience, creative biolabs provides zinc finger nuclease service for our global clients, especially those who are currently working on drug target validation. A toolbox and procedural notes for characterizing novel zinc. Design, construction, and analysis of specific zinc finger nucleases. Apr 29, 2009 an efficient method for gene targeting in plants has been lacking until now, frustrating efforts to engineer crop plants. Targeted genome modification in mice using zincfinger nucleases. Each subunit encompasses three zincfingers orange, 123, which recognize 9 base pairs within the full target site, and the foki endonuclease domain green. 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. These chimeric nucleases are composed of programmable, sequencespecific.
Targeting of photoreceptor genes in chlamydomonas reinhardtii via zinc finger nucleases and crisprcas9 andre greiner,a,1,2 simon kelterborn,a,1 heide evers,a georg kreimer,b irina sizova,a,2,3. It seems timely to consider the adoption of terminology that more accurately reflects what is being done. Pdf gene editing in human stem cells using zinc finger. Zinc finger nuclease zfn has become a new tool for gene knockout in other. Zinc finger nuclease mediated gene knockout results in. 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. Binding of two zinc finger nuclease monomers to two specific. Highly efficient homologydriven genome editing in human t. The discovery of zinc fingers and their applications in gene regulation and genome manipulation aaron klug mrc laboratory of molecular biology, cambridge cb2 0qh, united kingdom. 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. The discovery of zinc fingers and their applications in. Compozr custom zfn compozr zinc finger nucleases service. Zinc finger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical.
The finger is a selfcontained domain stabilized by a zinc. Enhancing gene targeting with designed zinc finger nucleases. Each zinc finger nuclease zfn consists of two functional domains. Gene targeting is a process in which a dna molecule introduced into a cell replaces.
Other recent approaches to targeted genome modification zinc finger nucleases zfns, 7 and transcriptionactivator like effector nucleases talens 8 enable researchers to generate permanent mutations by introducing doublestranded breaks to activate repair pathways. 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. By taking advantage of endogenous dna repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement. Since other nucleases with a similar function to zfn are considered in this opinion the term sitedirected nuclease. Customdesigned zinc finger nucleases zfns are becoming powerful tools in gene targetingthe process of replacing a gene within a genome by homologous recombination. With the advent of zinc finger nucleases zfns to the world of genetic research, the theoretical ideal became a reality. 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. Weber1,2, daniel stone1, ruth hall sedlak2, harshana s. Safc has created a series of zinc finger nuclease zfn modified cho cell lines with characteristics that are desirable in a platform cell line. Targeted mutagenesis of duplicated genes in soybean with. Scientific opinion addressing the safety assessment of. Microinjection and homologous recombination inembryonic stem es cells, are robust but overallinefficient.
Zinc finger nuclease technology market report offers accurate regionwise market projections and forecasts, market share, size, segmentwise analysis, regulatory framework assessment, opportunities and challenges for stakeholders, and impact of key industry trends. This strategy has enabled highly efficient gene disruption in numerous cell types and model organisms opening a door for new therapeutic applications. Zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. In addition, the impact on biomedical research of classical gene targeting. Zinc finger nucleases zfns are versatile reagents that have redefined genome engineering. Highfrequency modification of plant genes using engineered. Jun 24, 2011 identification of hsv2 genome specific zinc finger array zfas and zinc finger nucleases zfn u sing the fasta format of the nucleotide sequences of the whole genome of hsv2 strain hg52 dolan et al. Zinc finger structure zinc finger is the name of many protein structures. A zfn designed to create a dna doublestrand break dsb in the target locus is composed of two. A suite of zfns were engineered by the recently described contextdependent assembly platforma rapid, opensource method for generating zinc finger. When a dna vector with homology to both sides of the dna double strand break is added, the genomic.
Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins. The zinc finger zf consortium was established to ensure and to promote continued research and development of engineered zinc finger technology. In addition, zinc fingers have become extremely useful in various therapeutic and research capacities. The zfns have a dna recognition domain composed of three cys 2 his 2 zinc. Engineering zinc fingers to have an affinity for a specific sequence is an area of active research, and zinc finger nucleases and zinc finger. 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. Oct 19, 2014 in this video we discuss zinc finger nucleases, which are an important tool for cutting dna. 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. Improved cellpenetrating zincfinger nuclease proteins. Zinc finger simple english wikipedia, the free encyclopedia. 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. Zinc finger nucleases are useful to manipulate the genomes of many plants and animals. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools.
The world now has two major technologies for targeted editing of the genome. Pdf selectionfree zincfingernuclease engineering by. Knockoutratsviaembryomicroinjection of zincfinger nucleases. 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. Aavmediated delivery of zinc finger nucleases targeting hepatitis b virus inhibits active replication nicholas d. Zfn design made use of an archive of prevalidated 2 finger and 1 finger. Zinc finger nucleases zfns and transcription activatorlike effector nucleases talens comprise a powerful class of tools that are redefining the boundaries of biological research.
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