This species infects up to a third of the worlds population 3, 4 and represents a serious threat to public health. Plant genome engineering using zinc finger nucleases. Zinc finger nucelases genome editing nucleases ii zfn vs. Zinc finger nuclease zfn technology has recently been introduced to the zebrafish system as a complementary reverse genetic strategy to induce targeted mutations. Evaluation of open zinc finger nucleases for direct gene. Gersbach is rooney family associate professor of biomedical engineering at duke university and has worked with several genomeediting platforms.
Recently, zinc finger nucleases zfns, a new type of restriction enzymes, have been developed as a novel tool for genome engineering in living cells 1214. Genome engineering with zfns, talens and crisprcas9. 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. Genome engineering with compozr custom zinc finger nucleases.
Designed nucleases for targeted genome editing lee 2016. Zinc finger arrays can be designed to recognize diverse dna sequences, thereby making it possible modify any chromosomal target. To be useful for genome engineering, an endonuclease must exhibit an extraordinary combination of qualities. Genome editing in plant cells by zinc finger nucleases. Zfns, talens, and the crisprcas9 system on free shipping on qualified orders. A lightactivated zfn will competence the conditional generation of gene. Genome editing often requires cleavage within a narrow sequence window. The classical cys 2 his 2 c 2 h 2 zinc finger design is ideally suited for such purposes. A guide to genome engineering with programmable nucleases.
This combination of simplicity and flexibility has catapulted zinc finger nucleases zfns and transcription activatorlike effector nucleases talens to the forefront of genetic engineering. To better understand the role of the parasites aah2 in hostparasite interactions, we generated an aah2 fluorescent marker strain of t. Highly specific genomic scissors more important was the discovery that the dnabinding properties of zinc fingers could be fused to proteins with dnacutting function. How to achieve precise gene targeting in plants and especially in crops remains a longsought goal for elucidating gene function and advancing molecular breeding. Zfns are chimeric dna restriction enzymes that consist of a customdesigned zinc finger zf dnabinding domain and the nonspecific nuclease. Buy targeted genome editing using sitespecific nucleases. 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. The finger is a selfcontained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines. Genome editing with engineered zinc finger nucleases. All members have committed to making all protocols, software, dna sequences, and reagents they publish available to the academic scientific community.
In this work, we have designed various was genespecific crisprcas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric wasspecific zinc finger nucleases zfns, using k562 cells as a cellular model and plasmid nucleofection or integrationdeficient lentiviral vectors idlvs for delivery. This new technology opens up revolutionary possibilities in agriculture, animal husbandry and molecular medicine. The discovery of zinc fingers and their development for. How does one achieve targeted genome engineering of plant and mammalian cells. We generated an aah2 fluorescent marker strain of t. Therefore the zinc finger dna motifs are designed based on the site to be targeted. Plant genome engineering with sequencespecific nucleases. The plant genome original research genome engineering of. Supplementary figure 1 and supplementary table 1 pdf 236 kb. Zfn, talen, and crisprcasbased methods for genome engineering thomas gaj1,2,3, charles a. This video outlines these four basic genome editing. Over the past two decades, research groups in both academia and private industry have developed key technologies, including viral delivery vectors and engineered transposonbased or zinc finger proteinbased nucleases, towards achieving the longsought goal of therapeutic genome editing in humans.
Most healthy adults usually show symptoms of latent infection after. Sitedirected cleavage of target genomes by designer nucleases is fundamentally changing genome engineering. Zinc finger nucleases zfns enable precise genome modification in a variety of organisms and cell types. Toxoplasma gondii is an obligate intracellular protozoan parasite and one of the most widespread zoonotic parasites, and it can infect most warmblooded animals and humans 1, 2. Targeted genome engineering via zinc finger nucleases springerlink. In a correspondence in nature methods, some members of the zinc finger consortium reported. A new delivery method that can improve the utility of these. Recently, targeted cleavage of the genome using engineered dna scissors called zinc finger nucleases zfns has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. Gene targeting is a process in which a dna molecule introduced into a cell replaces. Genome editing with engineered zinc finger nucleases nature. Several technologies for genome engineering have been demon. Pioneering work with rare cutting endonucleases and zinc finger nucleases indicated the substantial benefits of targeted nuclease cleavage for genome editing and engineering 2,3.
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. This is a pdf file of an unedited manuscript that has been accepted for publication. Commercial zfns were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. N2 recent advances in genome engineering provide newfound control over a plants genetic material. Unlimited genomic engineering possibilities in any species or cell line. To date, zinc finger nucleases zfns have been the most promising reagents for potential.
Precise engineering of plant genomes using zinc finger nucleases. An unbiased genome wide analysis of zinc finger nuclease specificity. This progress has been facilitated by advances in engineering targeted nucleases with programmable, sitespecific dnabinding domains, including zinc finger proteins and transcription activator. Zfn, talen and crisprcasbased methods for genome engineering.
Comparison of zinc finger nucleases versus crisprspecific. Recently, a new technology based on the use of gene targeted zincfinger nucleases zfns was developed and used for the generation of. Studies within this dissertation seek to develop novel methods for delivering genome engineering reagents to whole plants. However, the deficiencies of current genebased delivery systems may hamper the clinical application of these nucleases. Efficient genome engineering of toxoplasma gondii using the. Zfninduced doublestrand breaks are subject to cellular dna repair processes that lead to both targeted mutagenesis and targeted gene replacement at remarkably high frequencies. Applications of genome editing tools are then described in detail, in ips cells and diverse organisms such as mice, rats, marine invertebrates, fish, frogs, and plants. Genome editing with engineered nucleases in plants plant. Zincfinger nucleases zfns are artificial restriction enzymes generated by fusing a zincfinger dnabinding domain to a dna cleavage domain porteus and carroll, 2005. Genome engineering with zinc finger nuclease and talens. Mar 15, 2019 aromatic amino acid hydroxylase 2 aah2 is a bradyzoitespecific upregulated protein that may alter host behaviour by altering the host dopaminergic pathway. Critical parameters for genome editing using zinc finger. 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.
Targeted genome engineering via zinc finger nucleases. Critical parameters for genome editing using zinc finger nucleases. Genome engineering with zfns, talens and crisprcas9 designer endonucleases zfns zinc finger nucleases, talens transcription activatorlike effector nucleases and crisprcas9 clustered regularly interspaced short palindromic repeatscrisprassociated system 9 are all engineered endonucleases. Genome engineering with zincfinger nucleases dana carroll1 department of biochemistry, university of utah school of medicine, salt lake city, utah 841125650 abstract zinc. Progress in zinc finger nuclease engineering for targeted genome modification. Sep 15, 2015 three types of designed nucleases zinc. Zfns are also used to create a new generation of genetic disease models called isogenic human disease models. The discovery of zinc fingers and their applications in. Customdesigned zinc finger nucleases zfns, proteins designed to cut at specific dna sequences, are becoming. Pdf genome targeting engineering is one of the most important advances in genetic engineering in the third millennium. 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.
Here the authors develop an expanded set of zinc finger nuclease. Targeted genome editing using sitespecific nucleases. Genome editing uses engineered nucleases as powerful tools to target specific dna sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. From meganucleases, to zinc fingers, to talens, and now crisprcas, the use of engineered nucleases for genome editing has been gathering pace. Genome engineering of crops with designer nucleases shaun j. Targeted genome engineering in human induced pluripotent. 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. Therefore, regardless of which protein you use to edit the human genome, it must be able to bind specifically to a unique 16 bp sequence of dna. This book serves as an introduction to targeted genome editing, beginning with the background of this rapidly developing field and methods for generation of engineered nucleases.
Diversifying the structure of zinc finger nucleases for highprecision. This combination of simplicity and flexibility has catapulted zincfinger nucleases zfns and. Zincfinger nucleases zfns are emerging as very powerful tools for directed genome modifications. Zinc finger nucleases and meganucleases are well established as dna targeting reagents. Targeted genome editing using engineered zinc finger nucleases.
Zfns provide an excellent alternative to the tedious and potentially impractical reengineering of existing meganucleases, because they can be engineered to digest virtually any long. Zinc finger nucleases zfns are targetable dna cleavage reagents that have been adopted as genetargeting tools. As a service to our customers we are providing this early version of the manuscript. Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. The development, engineering and application of tal.
Genome engineering with programmable nucleases depends on cellular responses to a targeted doublestrand break dsb. Zfns are artificial restriction enzymes that cause doublestranded breaks at defined points within the genome. Zinc finger proteins and zinc finger nucleases zinc finger nucleases are sequence specific dna binding proteins. The development, engineering and application of tal effector nucleases for targeted genome modification. Pdf mouse genome engineering using designer nucleases. Origins of programmable nucleases for genome engineering. This progress has been facilitated by advances in engineering targeted nucleases with programmable, sitespecific dnabinding domains, including. An enzyme called a zinc finger nuclease zfn is used to generate the chromosome break. Dnabinding domain of a versatile class of eukaryo tic transcription factors zinc finger proteins zfps. To address this issue, zinc finger nuclease zfnbased. 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.
Increasing cloning possibilities using artificial zinc. Zinc finger nucleases to be useful for genome engineering, an endonuclease must exhibit an extraordinary combination of qualities. 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. Pdf target zinc finger nucleases in genome engineering. Programmable nucleases including zfns, talens and rgens derived from the prokaryotic crisprcas system enable targeted highprecision genome engineering. Genome editing in plant cells by zinc finger nucleases uf plant. Genome engineering of crops with designer nucleases. Genome editing with modularly assembled zincfinger nucleases. Compozr custom zfn compozr zinc finger nucleases service. 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. 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. Genome engineering with targetable nucleases dana carroll department of biochemistry, university of utah school of medicine, salt lake city, utah 84112. Discriminating between closely related dna sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins zfps to target genes specifically.
Nonviral delivery of zinc finger nuclease mrna enables. Efficient genome engineering of toxoplasma gondii using. Zinc finger nuclease an overview sciencedirect topics. Introduction genome editing using engineered nucleases geen is an effective genetic engineering method that uses molecular scissors, or arti. We engineer zfns for precision, efficiency, and specificity, which we believe are the critical. Numerous examples of successful genome editing now exist. Conway and colleagues demonstrate for the first time nonviral in vivo genome editing using zinc finger nucleases, achieving therapeutically relevant levels of targeted gene knockout as well as targeted integration of therapeutic transgenes within murine liver at mrna doses 10fold lower than has ever been reported. The field of genome engineering switched into high gear in 2001, when bibikova and carroll used zinc finger nucleases zfns 3, 4, first described by chandrashekaran in 1996 5, to induce a doublestranded breaks dsb into dna in drosophila, and. To date, zinc finger nucleases zfns have been the most. Each zinc finger nuclease zfn consists of two functional domains. 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. The specificity of zincfinger nucleases lays in the zinc finger dna binding motifs. For instance, four zinc finger units can be assembled into a zinc finger chain that recognizes a 12 base pair dna sequence.