Gene disruption using zinc finger nuclease technology. Safc has created a series of zinc finger nuclease zfn modified cho cell lines with characteristics that are desirable in a platform cell line. Zfninduced doublestrand breaks are subject to cellular dna repair processes that. High frequency modification of plant genes using engineered zinc finger nucleases article pdf available in nature 4597245. Zinc finger nuclease mediated gene knockout results in. 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. Our knowledge of the oglycoproteome nacetylgalactosamine galnac type is highly limited. Zincfinger nucleases zfns are targetable dna cleavage reagents that have. Zinc finger nucleases zfns and transcription activatorlike effector nucleases talens comprise a powerful class of tools that are redefining the boundaries of biological research. The most useful of these was a set of cys 2 his 2 zinc fingers zfs in which each unit of.
The performance of our linkers in the nccn architecture suggested that we could combine two zfns containing an aminoterminal foki. 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. Unlimited genomic engineering possibilities in any species or cell line. Here it is demonstrated that zincfinger nucleasesenzymes engineered to. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. A zfn designed to create a dna doublestrand break dsb in the target locus is composed of two. Realizing the full potential of this technology requires the development of safe and effective methods. 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. Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. Targeted gene knockout by direct delivery of zincfinger. 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.
An alternative repair pathway for dsbs, nonhomologous end joining, often joins. Zinc finger nucleases zfns are synthetic restriction enzymes evolved by fusing. Zinc finger nucleases as tools to understand and treat human. Pdf precise genome modification in the crop species zea. Gene editing in human stem cells using zinc finger nucleases and integrasedefective lentiviral vector delivery. When you go to the pdb to explore zinc fingers, try not to be overwhelmed. One of the first breakthrough methods of gene targeting was the usage of chimeric proteins called zinc fingers nucleases zfn to create doublestrand breaks. Zincfinger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools.
Zinc finger structure zinc finger is the name of many protein structures. Zinc finger domains can be engineered to target specific desired dna sequences and this enables zincfinger nucleases to target unique sequences within complex genomes. Pdf high frequency modification of plant genes using. 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. 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. Gt methods, an emerging technology involving engineered restriction enzymes known as zincfinger nucleases zfns has. A dnabinding domain comprised of a chain of two finger modules, each recognizing a unique hexamer 6 bp sequence of dna. Highfrequency modification of plant genes using engineered. Zinc finger nuclease an overview sciencedirect topics. Each zinc finger nuclease zfn consists of two functional domains.
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. 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. Two histidines from the helix and two cysteines from the beta sheet coordinately bind a zinc. 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. This modification of the cellular dna is permanent and can fully block gene function. Compozr custom zfn compozr zinc finger nucleases service. Gene targeting by homologous recombination in mouse. Knockoutratsviaembryomicroinjection of zincfinger nucleases. Pdf methods of modifying the human genome precisely and efficiently hold great. 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. Highly efficient homologydriven genome editing in human t. Genome editing with engineered zinc finger nucleases.
Your guide to investing in zincfinger zfn gene editing. Gene editing strategies, such as zinc finger nucleases zfns, transcription activatorlike effector nucleases talens, and clustered regularly interspaced short palindromic repeatcas9. Since other nucleases with a similar function to zfn are considered in this opinion the term sitedirected nuclease. Design, construction, and analysis of specific zinc finger nucleases. 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. Aavmediated delivery of zinc finger nucleases targeting hepatitis b virus inhibits active replication nicholas d. Knockout rats via embryo microinjection of zincfinger. Zfn design made use of an archive of prevalidated 2 finger and 1 finger. The zfns have a dna recognition domain composed of three cys 2 his 2 zinc. Dnabinding zinc finger proteins zfps and a nuclease. A toolbox and procedural notes for characterizing novel zinc. By taking advantage of endogenous dna repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Scientific opinion addressing the safety assessment of.
Custom zinc finger nucleases for use in human cells stephen alwin,1 maja b. Donald kohn, director of the human gene medicine program. Zinc finger nucleases zfns are versatile reagents that have redefined genome engineering. Weber1,2, daniel stone1, ruth hall sedlak2, harshana s. Zinc finger engineering the zinc finger consortium. Only a few percent of the injected eggs giving rise totransgenic animals. 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. Customdesigned zinc finger nucleases zfns are becoming powerful tools in gene targetingthe process of replacing a gene within a genome by homologous recombination. 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. Pdf gene editing in human stem cells using zinc finger. Probing isoformspecific functions of polypeptide galnac. Here, we have studied the dna cleavage by one such zfn.
Dnaworks software was used instead of manual design to obtain the. When a dna vector with homology to both sides of the dna double strand break is added, the genomic. Induction and repair of zinc finger nuclease targeted doublestrand breaks in caenorhabditis elegans somatic cells jason morton, m. Oct 19, 2014 in this video we discuss zinc finger nucleases, which are an important tool for cutting dna. Use of zinc finger nuclease technology to knock out efflux. Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins. In principle these technologies are capable of targeting virtually any site in the genome and editing the dna that is there. In addition, zinc fingers have become extremely useful in various therapeutic and research capacities.
The zinc atom binds the proteins and makes them more stable. 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. 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. Engineering zinc fingers to have an affinity for a specific sequence is an area of active research, and zinc finger nucleases and zinc 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. 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. Binding of two zinc finger nuclease monomers to two specific.
Genome editing in plant cells by zinc finger nucleases. Here we describe contextdependent assembly coda, a platform for engineering zfns using only standard cloning. Each subunit encompasses three zincfingers orange, 123, which recognize 9 base pairs within the full target site, and the foki endonuclease domain green. 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. Identity of zinc finger nucleases with specificity to herpes. 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. A toolbox and procedural notes for characterizing novel.
Genome engineering with zincfinger nucleases genetics. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain. Zinc finger nucleases are useful to manipulate the genomes of many plants and animals. One zinc finger can bind about 3 base pairs by itself.
Zinc finger nuclease dimers are more difficult to engineer than single dnabinding domains, but the development of new methods could help. After dimerization the nuclease is activated and cuts the dna in the spacer sequence. 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. The structure shown here on the left, from pdb entry 1znf, shows a classic zinc finger.
Custommade zinc finger nucleases zfns can induce targeted genome modifications with high efficiency in cell types including drosophila, c. The binding specificity of the designed zinc finger. 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. Enhancing zincfingernuclease activity with improved. Zincfinger nuclease enhanced gene targeting in human. Zinc finger nucleases for precise editing of the cho cell. Zinc finger nuclease zfn has become a new tool for gene knockout in other. Figure 1zincfinger nuclease zfnmediated genome editing. Targeted genome modification in mice using zincfinger nucleases. 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. The binding specificity of the designed zincfinger domain directs the zfn to a specific genomic site. Zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. Induction and repair of zincfinger nucleasetargeted. It seems timely to consider the adoption of terminology that more accurately reflects what is being done.
The world now has two major technologies for targeted editing of the genome. Qnkfn, in order to gain insight into how zfns cleave dna and how two inverted sites promote doublestrand cleavage. 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. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. With the advent of zinc finger nucleases zfns to the world of genetic research, the theoretical ideal became a reality. We performed targeted mutagenesis of a transgene and nine endogenous soybean glycine max genes using zincfinger nucleases zfns. Gene targeting is a process in which a dna molecule introduced into a cell replaces. Zinc finger simple english wikipedia, the free encyclopedia. In addition to this flexibility, gene therapy by zinc finger nucleases may enable. 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. 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 zfns are artificial restriction enzymes generated by fusing a zinc finger dnabinding domain to a dnacleavage domain.
This strategy has enabled highly efficient gene disruption in numerous cell types and model organisms opening a door for new therapeutic applications. Targeted mutagenesis of duplicated genes in soybean with. The potential of gene targeting with zinc finger nucleases. Zincfinger nuclease dimers are more difficult to engineer than single. In this approach, human c2bbe1 cells a subclone of caco. 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. Improved cellpenetrating zincfinger nuclease proteins. 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 cells normal repair mechanisms can then generate targeted deletions or integrations. A dnabinding domain comprised of a chain of two finger. A bottleneck in the application of zfn technology has been the generation of highly specific engineered 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. 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. Engineered zinc finger nucleases zfns enable targeted genome modification. The nucleases used in our platform provide high specificity and optimized digestion conditions possible. The finger is a selfcontained domain stabilized by a zinc. Diversifying the structure of zinc finger nucleases for highprecision. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement. Microinjection and homologous recombination inembryonic stem es cells, are robust but overallinefficient. We have been developing chimeric zinc finger nucleases zfns as potential stimulators of gene targeting 7, 8. Weitzman,4 and toni cathomen1, 1institute of virology, charite. Enhancing gene targeting with designed zinc finger nucleases.
These chimeric nucleases are composed of programmable, sequencespecific. Apr 29, 2009 an efficient method for gene targeting in plants has been lacking until now, frustrating efforts to engineer crop plants. The oglycoproteome is differentially regulated in cells by dynamic expression of a subset of. 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. Zinc finger nucleases are made with 36 zinc fingers. Zfn dimers induce targeted dna dsbs that stimulate dna damage response pathways. Aavmediated delivery of zinc finger nucleases targeting. 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. Medical school, campus benjamin franklin, hindenburgdamm 27, 12203 berlin, germany 2skaggs institute for chemical biology and department of molecular biology, the. 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. The zinc finger zf consortium was established to ensure and to promote continued research and development of engineered zinc finger technology. Zinc finger nucleases zfns drive efficient genome editing by introducing a doublestrand break into the targeted gene. Zfninduced doublestrand breaks are subject to cellular dna repair.
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